Macro TSH in patients with subclinical hypothyroidism.

PubMed

Hattori, Naoki; Ishihara, Takashi; Yamagami, Keiko; Shimatsu, Akira

2015-12-01

TSH is a sensitive indicator of thyroid function. In subclinical hypothyroidism, however, serum TSH concentrations are elevated despite normal thyroid hormone levels, and macro TSH is one of the causes. This study aimed to clarify the prevalence and nature of macro TSH in patients with subclinical hypothyroidism. We conducted a 2-year cross-sectional observational study. We included 681 patients with subclinical hypothyroidism and 38 patients with overt hypothyroidism (controls). Macro TSH was screened by polyethylene glycol (PEG) method and analysed by gel filtration chromatography and bioassays. Among 681 serum samples, 117 exhibited PEG-precipitable TSH ratios greater than 75% (mean + 1·5 SD in controls) and were subjected to gel filtration chromatography. TSH was eluted at a position greater than 100 kDa in 11 patients with subclinical hypothyroidism (1·62%); these patients were diagnosed with macro TSH. The nature of macro TSH included eight anti-TSH autoantibodies of IgG class, two non-IgG-associated and one human anti-mouse antibody (HAMA). Macro TSH showed low bioactivity. Macro TSH was heterogeneous, but it is mostly comprised of TSH and anti-TSH autoantibodies. When PEG-precipitable TSH exceeds 90% in serum samples with TSH above 10 mU/l, clinicians should strongly suspect the presence of macro TSH and confirm it by gel chromatography. Because macro TSH exhibited low bioactivity, thyroid hormone replacement therapy may not be required in patients with subclinical hypothyroidism due to macro TSH except for those with high serum free TSH levels. © 2014 John Wiley & Sons Ltd.

Cost-effectiveness of using recombinant human thyroid-stimulating hormone before radioiodine ablation for thyroid cancer treatment in Spanish hospitals.

PubMed

Vallejo, J A; Muros, M A

In thyroid cancer treatment, the thyroid-stimulating hormone (TSH) must be elevated before radioiodine ablation, either by exogenous (with recombinant human thyrotropin [rhTSH]) or endogenous stimulation by thyroid hormone withdrawal (THW). The use of rhTSH avoids hypothyroidism and favours the subsequent elimination of radioiodine, but involves the cost of the product. For this reason, a cost-effectiveness analysis was performed, taking into account all costs involved and the benefits associated with the use of this therapy. Using a Markov modelling with two analysis arms (rhTSH and THW), stratified into high (100mCi/3700 MBq) and low (30mCi/1110 MBq) radioiodine doses, and using 17 weekly cycles, the incremental cost per quality-adjusted life-year (QALY) related to the use of rhTSH was determined. The clinical inputs included in the model were based on published studies and in a treatment survey conducted in Spain. Radioablation preparation with rhTSH is superior to THW, showing additional benefits (0.048 AVAC), as well as cost savings (-€614.16), with an incremental cost-effectiveness rate (ICER) of -€12,795/QALY. The univariate and multivariate sensitivity analyses showed the result to be robust. The use of rhTSH previous to radioablation in Spain has cost savings, as well as a series of health benefits for the patient, making it highly cost-effective. Copyright © 2017 Elsevier España, S.L.U. y SEMNIM. All rights reserved.

Day 3 thyroglobulin ≤ 1 ng/ml after recombinant human TSH just prior to radioactive iodine is predictive of low risk for persistent/recurrent disease in patients with papillary thyroid carcinoma.

PubMed

Rosario, Pedro W; Siman, Thássio Leonardo; Calsolari, Maria R

2015-05-01

We evaluated the negative predictive value (NPV) of thyroglobulin obtained 24 h after the second recombinant human TSH (rhTSH) ampoule (Tg-D3), before ablation with (131)I, for persistent/recurrent disease (PRD) in low/intermediate risk patients with papillary thyroid carcinoma. One hundred and one patients with Tg-D3 ≤ 1 ng/ml without anti-Tg antibodies (TgAb) were selected. Post-therapy whole-body scanning was negative for metastases in 98 (97 %) patients, and three patients showed discrete ectopic cervical uptake, but no corresponding disease was detected by neck ultrasound or computed tomography. One year after ablation, 98 (97 %) patients were free of the disease. Three patients had stimulated Tg >1 ng/ml, but no metastases were detected by the imaging methods. During follow-up (median 50 months), tumor recurrence was observed in only one patient. Thus, the NPV of Tg-D3 ≤ 1 ng/ml for PRD was 99 %. Among the 101 patients with Tg-D3 ≤ 1 ng/ml, Tg obtained 48 h after ablation (Tg-D5) continued to be ≤ 1 ng/ml in 56, and 45 had Tg-D5 >1 ng/ml. None of these 45 patients had PRD. In conclusion, Tg-D3 ≤ 1 ng/ml had a high NPV for PRD in patients without TgAb or known persistent disease and who are not at high risk. In these patients, Tg-D5 >1 ng/ml is more likely to reflect actinic damage to the remnant thyroid tissue rather than persistence of significant normal or tumor tissue.

TSH test

MedlinePlus

Thyrotropin; Thyroid stimulating hormone; Hypothyroidism - TSH; Hyperthyroidism - TSH; Goiter - TSH ... most often due to an underactive thyroid gland ( hypothyroidism ). There are many causes of this problem. A ...

Thyroid stimulation with recombinant human thyrotropin in healthy cats, cats with non-thyroidal illness and in cats with low serum thyroxin and azotaemia after treatment of hyperthyroidism.

PubMed

van Hoek, Ingrid M; Vandermeulen, Eva; Peremans, Kathelijne; Daminet, Sylvie

2010-02-01

This study investigated the recombinant human thyrotropin (rhTSH) stimulation test in healthy cats (group 1), cats with non-thyroidal illness (group 2) and cats with low serum total T(4) (TT(4)) and azotaemia after (131)I treatment (group 3). Serum TT(4) responses and thyroidal pertechnetate uptake after administration of 25 microg rhTSH IV were assessed. Baseline serum TT(4) was significantly lower in group 3 compared with group 1, but not between other group pairs. Serum TT(4) increased significantly in groups 1 and 2 but not in group 3 after rhTSH administration. Post-rhTSH serum TT(4) concentrations differed significantly between groups 1 and 3 and groups 2 and 3, but not between groups 1 and 2. Thyroid/salivary gland uptake ratio (T/S uptake ratio) differed only significantly between groups 1 and 3. Stimulation with rhTSH is valuable to differentiate euthyroidism from iatrogenic hypothyroidism in cats. Copyright 2009 ESFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Pharmacology of bovine and human thyrotropin: an historical perspective.

PubMed

Robbins, J

1999-05-01

Before the induction of a brief period of hypothyroidism became the standard method for inducing 131I uptake in thyroid cancer diagnosis and therapy, several other methods were explored and used. At the dawn of this new era of recombinant human thyrotropin (TSH) it is of interest to reflect briefly on some of this work. Partially purified bovine TSH (bTSH) was supplied for many years by the Armour Company as Thytropar for intramuscular injection and was first used in thyroid cancer 50 years ago at the Montefiore Hospital and at the Memorial Sloan Kettering Cancer Center in New York City. Most of the patients were already hypothyroid and bTSH induced further 131I uptake in only a few. Experience over the next 30 years revealed frequent allergic reactions, occasionally serious ones, and in 1961 it was shown that prolonged use could result in resistance to both bTSH and human TSH. bTSH was, therefore, reserved for thyroid cancer patients unable to increase endogenous TSH when hypothyroid. bTSH also was used widely as a test to distinguish between hypothyroidism caused by thyroid or pituitary failure until it was replaced by thyrotropin-releasing hormone (TRH). In a few studies, TRH was also tested as an adjuvant to increase endogenous TSH and thus help to stimulate function in thyroid cancer, but this attracted little interest. Prolonged hypothyroidism, enhanced by antithyroid drugs, was used early on, but this very effective stimulant of thyroid cancer function was, for multiple reasons, discarded. Beginning interest 15 to 25 years ago in obtaining TSH from human pituitary glands, a byproduct of growth hormone production, was interrupted when this product was found to risk development of Creutzfeld-Jakob disease. Recombinant human TSH, a safe and effective substitute, is now ready for widespread use and development in thyroid cancer management.

The AID enzyme induces class switch recombination in fibroblasts.

PubMed

Okazaki, Il-mi; Kinoshita, Kazuo; Muramatsu, Masamichi; Yoshikawa, Kiyotsugu; Honjo, Tasuku

2002-03-21

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from Cmu to one of the other CH genes. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

Do TSH, FT3, and FT4 Impact BAT Visualization of Clinical FDG-PET/CT Images?

PubMed

Nishii, Ryuichi; Nagamachi, Shigeki; Mizutani, Youichi; Terada, Tamasa; Kiyohara, Syogo; Wakamatsu, Hideyuki; Fujita, Seigo; Higashi, Tatsuya; Yoshinaga, Keiichiro; Saga, Tsuneo; Hirai, Toshinori

2018-01-01

We retrospectively analyzed activated BAT visualization on FDG-PET/CT in patients with various conditions and TH levels to clarify the relationships between visualization of BAT on FDG-PET/CT and the effect of TH. Patients who underwent clinical FDG-PET/CT were reviewed and we categorized patients into 5 groups: (i) thyroid hormone withdrawal (THW) group; (ii) recombinant human thyrotropin (rhTSH) group; (iii) hypothyroidism group; (iv) hyperthyroidism group; and (v) BAT group. A total of sixty-two FDG-PET/CT imaging studies in fifty-nine patients were performed. To compare each group, gender; age; body weight; serum TSH, FT3, and FT4 levels; and outside temperature were evaluated. No significant visualization of BAT was noted in any of the images in the THW, rhTSH, hypothyroidism, and hyperthyroidism groups. All patients in the BAT group were in a euthyroid state. When the BAT-negative and BAT-positive patient groups were compared, it was noted that the minimum and maximum temperature on the day of the PET study and maximum temperature of the one day before the PET study were significantly lower in BAT-positive group than in all those of other groups. Elevated TSH condition before RIT, hyperthyroidism, or hypothyroidism did not significantly impact BAT visualization of clinical FDG-PET/CT images.

Associations between brominated flame retardants in human milk and Thyroid-Stimulating Hormone (TSH) in neonates

PubMed Central

Eggesbø, Merete; Thomsen, Cathrine; Jørgensen, Jens V.; Becher, Georg; Odland, Jon Øyvind; Longnecker, Matthew P.

2011-01-01

Background Brominated flame retardants (BFRs) have been in widespread use in a vast array of consumer products since the 1970s. The metabolites of some BFRs show a structural similarity to thyroid hormones and experimental animal studies have confirmed that they may interfere with thyroid hormone homeostasis. A major concern has been whether intrauterine exposure to BFRs may disturb thyroid homeostasis since the fetal brain is particularly susceptible to alterations in thyroid hormones. However, few reports on newborns have been published to date. Objectives To evaluate the association between BFRs and neonatal thyroid-stimulating hormone (TSH). Methods We studied six polybrominated diphenyl ethers (PBDEs) measured in milk samples from 239 women who were part of the “Norwegian Human Milk Study” (HUMIS), 2003–2006. Hexabromocyclododecane (HBCD) and BDE-209 were measured in a subset of the women (193 and 46 milk samples, respectively). The milk was sampled at a median of 33 days after delivery. TSH was measured in babies three days after delivery as part of the routine national screening program for early detection of congenital hypothyroidism. Additional information was obtained through the Medical Birth Registry and questionnaires to the mothers. Results The PBDE concentrations in human milk in Norway were comparable to concentrations reported from other European countries and Asia, but not the US and Canada where levels are approximately one order of magnitude higher. We observed no statistically significant associations between BDE-47, 99, 153, 154, 209 and HBCD in human milk and TSH in models adjusted for possible confounders and other environmental toxicants including polychlorinated biphenyls (PCBs). Conclusions We did not observe an association between TSH and exposure to HBCD and PBDEs within the exposure levels observed. PMID:21601188

Do TSH, FT3, and FT4 Impact BAT Visualization of Clinical FDG-PET/CT Images?

PubMed Central

Nagamachi, Shigeki; Mizutani, Youichi; Terada, Tamasa; Kiyohara, Syogo; Wakamatsu, Hideyuki; Fujita, Seigo; Higashi, Tatsuya; Yoshinaga, Keiichiro; Saga, Tsuneo; Hirai, Toshinori

2018-01-01

Objective We retrospectively analyzed activated BAT visualization on FDG-PET/CT in patients with various conditions and TH levels to clarify the relationships between visualization of BAT on FDG-PET/CT and the effect of TH. Methods Patients who underwent clinical FDG-PET/CT were reviewed and we categorized patients into 5 groups: (i) thyroid hormone withdrawal (THW) group; (ii) recombinant human thyrotropin (rhTSH) group; (iii) hypothyroidism group; (iv) hyperthyroidism group; and (v) BAT group. A total of sixty-two FDG-PET/CT imaging studies in fifty-nine patients were performed. To compare each group, gender; age; body weight; serum TSH, FT3, and FT4 levels; and outside temperature were evaluated. Results No significant visualization of BAT was noted in any of the images in the THW, rhTSH, hypothyroidism, and hyperthyroidism groups. All patients in the BAT group were in a euthyroid state. When the BAT-negative and BAT-positive patient groups were compared, it was noted that the minimum and maximum temperature on the day of the PET study and maximum temperature of the one day before the PET study were significantly lower in BAT-positive group than in all those of other groups. Conclusions Elevated TSH condition before RIT, hyperthyroidism, or hypothyroidism did not significantly impact BAT visualization of clinical FDG-PET/CT images. PMID:29666563

Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

PubMed Central

Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

2015-01-01

SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

Pretreatment with a single, low dose of recombinant human thyrotropin allows dose reduction of radioiodine therapy in patients with nodular goiter.

PubMed

Nieuwlaat, Willy-Anne; Huysmans, Dyde A; van den Bosch, Harrie C; Sweep, C G Fred; Ross, H Alec; Corstens, Frans H; Hermus, Ad R

2003-07-01

In patients with nodular goiter, radioiodine ((131)I) therapy results in a mean reduction in thyroid volume (TV) of approximately 40% after 1 yr. We have demonstrated that pretreatment with a single, low dose of recombinant human TSH (rhTSH) doubles 24-h radioactive iodine uptake (RAIU) in these patients. We have now studied the safety and efficacy of therapy with a reduced dose of (131)I after pretreatment with rhTSH. Twenty-two patients with nodular goiter received (131)I therapy, 24 h after im administration of 0.01 (n = 12) or 0.03 (n = 10) mg rhTSH. In preceding diagnostic studies using tracer doses of (131)I, 24-h RAIU without and with rhTSH pretreatment (either 0.01 or 0.03 mg) were compared. Therapeutic doses of (131)I were adjusted to the rhTSH-induced increases in 24-h RAIU and were aimed at 100 micro Ci/g thyroid tissue retained at 24 h. Pretreatment with rhTSH allowed dose reduction of (131)I therapy by a factor of 1.9 +/- 0.5 in the 0.01-mg and by a factor of 2.4 +/- 0.4 in the 0.03-mg rhTSH group (P < 0.05, 0.01 vs. 0.03 mg rhTSH). Before and 1 yr after therapy, TV and the smallest cross-sectional area of the tracheal lumen were measured with magnetic resonance imaging. During the year of follow-up, serum TSH, free T(4) (FT(4)), T(3), and TSH receptor antibodies were measured at regular intervals. TV before therapy was 143 +/- 54 ml in the 0.01-mg group and 103 +/- 44 ml in the 0.03-mg rhTSH group. One year after treatment, TV reduction was 35 +/- 14% (0.01 mg rhTSH) and 41 +/- 12% (0.03 mg rhTSH). In both groups, smallest cross-sectional area of the tracheal lumen increased significantly. In the 0.01-mg rhTSH group, serum FT(4) rose, after (131)I treatment, from 15.8 +/- 2.8 to 23.2 +/- 4.4 pM. In the 0.03-mg rhTSH group, serum FT(4) rose from 15.5 +/- 2.5 to 23.5 +/- 5.1 pM. Individual peak FT(4) levels, reached between 1 and 28 d after (131)I treatment, were above the normal range in 12 patients. TSH receptor antibodies were negative in all

Production of recombinant adenovirus containing human interlukin-4 gene.

PubMed

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-11-01

Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

Ability of the rhTSH stimulation test to predict relapse in patients with differentiated thyroid carcinoma, after long-term follow-up

PubMed Central

MARCELINO, MAFALDA; LOPES, ANA FILIPA; MADUREIRA, DEOLINDA; FERREIRA, TERESA C.; LIMBERT, EDWARD; LEITE, VALERIANO

2015-01-01

The analysis of serum thyroglobulin (Tg) following thyroid-stimulating hormone (TSH) stimulation (sTg) has been recommended in the follow-up of differentiated thyroid carcinoma (DTC) patients, however, its routine use remains controversial. The aim of the current study was to evaluate the accuracy of sTg testing following recombinant human (rh) TSH stimulation in DTC patients, with a follow-up of 12.4 years. Retrospective studies were conducted of 125 DTC patients, who underwent rhTSH stimulation testing between 1999 and 2002. The exclusion criteria were: Patients with anti-Tg antibodies, Tg levels >1 ng/ml under TSH suppression and the absence of radioactive iodine (RAI) ablation therapy following surgery. In total, 49 patients were included in the study and all had been previously treated with total or near total thyroidectomy (with or without central neck dissection) and RAI, postoperatively. The Tg functional sensitivity was 1.0 ng/ml. The follow-up for patients was performed annually. During the median follow-up of 12.4 years after the rhTSH stimulation test, nine patients exhibited recurrence (18.4%). Of the nine patients, six exhibited sTg levels >2 ng/ml (positive result) and three exhibited levels <2 ng/ml (negative result). Relapse occurred at a mean of 5.9 years following the rhTSH stimulation test. The positive predictive value and negative predictive value (NPV) of positive sTg were 50 and 91.9%, respectively, with a sensitivity of 66.6% and a specificity of 85.0%. The rhTSH-stimulated Tg levels have a high NPV, allowing the identification of the patients who are free of the tumour. These results are consistent with the previously published data; however, to the best of our knowledge, this is the study with the longest follow-up duration after rhTSH stimulation. PMID:25663898

[Harmonization of TSH Measurements.

PubMed

Takeoka, Keiko; Hidaka, Yoh; Hishinuma, Akira; Ikeda, Katsuyoshi; Okubo, Shigeo; Tsuchiya, Tatsuyuki; Hashiguchi, Teruto; Furuta, Koh; Hotta, Taeko; Matsushita, Kazuyuki; Matsumoto, Hiroyuki; Murakami, Masami; Maekawa, Masato

2016-05-01

The measured concentration of thyroid stimulating hormone (TSH) differs depending on the reagents used. Harmonization of TSH is crucial because the decision limits are described in current clinical practice guide- lines as absolute values, e.g. 2.5 mIU/L in early pregnancy. In this study, we tried to harmonize the report- ed concentrations of TSH using the all-procedure trimmed mean. TSH was measured in 146 serum samples, with values ranging from 0.01 to 18.8 mIU/L, using 4 immunoassays. The concentration of TSH was highest with E test TOSOH and lowest with LUMIPULSE. The concentrations with each reagent were recalculated with the following formulas: E test TOSOH 0.855x-0.014; ECLusys 0.993x+0.079; ARCHITECT 1.041x- 0.010; and LUMIPULSE 1.096x-0.015. Recalculation eliminated the between-assay discrepancy. These formulas may be used until harmonization of TSH is achieved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).

An Enantiomer of an Oral Small-Molecule TSH Receptor Agonist Exhibits Improved Pharmacologic Properties.

PubMed

Neumann, Susanne; Padia, Umesh; Cullen, Mary Jane; Eliseeva, Elena; Nir, Eshel A; Place, Robert F; Morgan, Sarah J; Gershengorn, Marvin C

2016-01-01

We are developing an orally available small-molecule, allosteric TSH receptor (TSHR) agonist for follow-up diagnostics of patients with thyroid cancer. The agonist C2 (NCGC00161870) that we have studied so far is a racemic mixture containing equal amounts of two enantiomers, E1 and E2. As enantiomers of many drugs exhibit different pharmacologic properties, we assessed the properties of E1 and E2. We separated the two enantiomers by chiral chromatography and determined E2 as the (S)-(+) isomer via crystal structure analysis. E1 and E2 were shown to bind differently to a homology model of the transmembrane domain of TSHR in which E2 was calculated to exhibit lower binding energy than E1 and was, therefore, predicted to be more potent than E1. In HEK293 cells expressing human TSHRs, C2, E1, and E2 were equally efficacious in stimulating cAMP production, but their potencies were different. E2 was more potent (EC50 = 18 nM) than C2 (EC50 = 46 nM), which was more potent than E1 (EC50 = 217 nM). In primary cultures of human thyrocytes, C2, E1, and E2 stimulated increases in thyroperoxidase mRNA of 92-, 55-, and 137-fold and in sodium-iodide symporter mRNA of 20-, 4-, and 121-fold above basal levels, respectively. In mice, C2 stimulated an increase in radioactive iodine uptake of 1.5-fold and E2 of 2.8-fold above basal level, whereas E1 did not have an effect. C2 stimulated an increase in serum T4 of 2.4-fold, E1 of 1.9-fold, and E2 of 5.6-fold above basal levels, and a 5-day oral dosing regimen of E2 increased serum T4 levels comparable to recombinant human TSH (rhTSH, Thyrogen(®)). Thus, E2 is more effective than either C2 or E1 in stimulating thyroid function and as efficacious as rhTSH in vivo. E2 represents the next step toward developing an oral drug for patients with thyroid cancer.

Diural TSH variations in hypothyroidism.

PubMed

Weeke, J; Laurberg, P

1976-07-01

There is a circadian variation in serum TSH in euthyroid subjects. A similar diurnal variation has been demonstrated in patients with hypothyroidism. In the present study the 24-hour pattern of serum TSH was investigated in eight patients with hypothyroidism of varying severity and in five hypothyroid patients treated with thyroxine (T4). There was a circadian variation in serum TSH in patients with hypothyroidism of moderate degree, and in patients treated for severe hypothyrodism with thyroxine. The pattern was similar to that found in normal subjects, i.e., low TSH levels in the daytime and higher levels at night. In severely hypothyroid patients, no diurnal variation in serum TSH was observed. A practical consequence is that blood samples for TSH measurements in patients with moderately elevated TSH levels are best taken after 1100 h, when the low day levels are reached.

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

PubMed Central

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. Materials and Methods In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Results Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. Conclusion This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics. PMID:23493491

Regulating infidelity: RNA-mediated recruitment of AID to DNA during class switch recombination.

PubMed

DiMenna, Lauren J; Chaudhuri, Jayanta

2016-03-01

The mechanism by which the DNA deaminase activation-induced cytidine deaminase (AID) is specifically recruited to repetitive switch region DNA during class switch recombination is still poorly understood. Work over the past decade has revealed a strong link between transcription and RNA polymerase-associated factors in AID recruitment, yet none of these processes satisfactorily explain how AID specificity is affected. Here, we review a recent finding wherein AID is guided to switch regions not by a protein factor but by an RNA moiety, and especially one associated with a noncoding RNA that has been long thought of as being inert. This work explains the long-standing requirement of splicing of noncoding transcripts during class switching, and has implications in both B cell-mediated immunity as well as the underlying pathological syndromes associated with the recombination reaction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Age-Dependent Recombination Rates in Human Pedigrees

PubMed Central

Hussin, Julie; Roy-Gagnon, Marie-Hélène; Gendron, Roxanne; Andelfinger, Gregor; Awadalla, Philip

2011-01-01

In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies. PMID:21912527

Thyrotropic action of human chorionic gonadotropin.

PubMed

Yoshimura, M; Hershman, J M

1995-10-01

Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent

TSH/IGF-1 Receptor Cross-Talk Rapidly Activates Extracellular Signal-Regulated Kinases in Multiple Cell Types.

PubMed

Krieger, Christine C; Perry, Joseph D; Morgan, Sarah J; Kahaly, George J; Gershengorn, Marvin C

2017-10-01

We previously showed that thyrotropin (TSH)/insulinlike growth factor (IGF)-1 receptor cross-talk appears to be involved in Graves' orbitopathy (GO) pathogenesis and upregulation of thyroid-specific genes in human thyrocytes. In orbital fibroblasts from GO patients, coadministration of TSH and IGF-1 induces synergistic increases in hyaluronan secretion. In human thyrocytes, TSH plus IGF-1 synergistically increased expression of the sodium-iodide symporter that appeared to involve ERK1/2 activation. However, the details of ERK1/2 activation were not known, nor was whether ERK1/2 was involved in this synergism in other cell types. Using primary cultures of GO fibroblasts (GOFs) and human thyrocytes, as well as human embryonic kidney (HEK) 293 cells overexpressing TSH receptors (HEK-TSHRs), we show that simultaneous activation of TSHRs and IGF-1 receptors (IGF-1Rs) causes rapid, synergistic phosphorylation/activation of ERK1 and ERK2 in all three cell types. This effect is partially inhibited by pertussis toxin, an inhibitor of TSHR coupling to Gi/Go proteins. In support of a role for Gi/Go proteins in ERK1/2 phosphorylation, we found that knockdown of Gi(1-3) and Go in HEK-TSHRs inhibited ERK1/2 phosphorylation stimulated by TSH and TSH plus IGF-1. These data demonstrate that the synergistic effects of TSH plus IGF-1 occur early in the TSHR signaling cascade and further support the idea that TSHR/IGF-1R cross-talk is an important mechanism for regulation of human GOFs and thyrocytes.

Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

NASA Astrophysics Data System (ADS)

Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

1992-06-01

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Long-term outcome after radioiodine therapy with adjuvant rhTSH treatment: comparison between patients with non-toxic and pre-toxic large multinodular goitre.

PubMed

Giusti, M; Caorsi, V; Mortara, L; Caputo, M; Monti, E; Schiavo, M; Bagnara, M C; Minuto, F; Bagnasco, M

2014-03-01

In multinodular goitre (MNG), low radioiodine (RAI) activity after recombinant human (rh) TSH is able to reduce thyroid volume (TV) and improve symptoms. Our aim was to evaluate the long-term outcome of RAI after rhTSH treatment in patients who were divided according to their baseline TSH levels. Eighteen patients (69.2 ± 6.1 year) presented non-toxic (TSH >0.3 mIU/l) MNG (TV: 61.0 ± 3.8 ml; group 1), while 13 patients (74.1 ± 7.9 year) had non-autoimmune pre-toxic (TSH <0.3 mIU/l) MNG (TV: 82.6 ± 14.4 ml; group 2). TSH, thyroid hormones, TV (by ultrasonography), body mass index (BMI), symptoms and quality of life (QoL) were evaluated. Treatment induced short-term thyrotoxicosis in both groups, but this was slightly more marked in group 2 than in group 1. The number and severity of adverse events were similar. The follow-up period was 55.3 ± 4.1 months in group 1 and 57.2 ± 5.1 months in group 2. The final TV reduction was similar in groups 1 (63.4 ± 3.6%) and 2 (57.2 ± 4.6%) and TV reduction positively correlated only with initial TV. At the last examination, 14 group-1 subjects were on L-T4 therapy, while 2 group-2 subjects were on methimazole. An increase in BMI was noted only in group 2. MNG-related symptoms were significantly reduced in both groups. Symptoms related to sub-clinical hyperthyroidism improved in group 2, while no significant changes in QoL were noted in either group. This study confirms the effectiveness of rhTSH adjuvant treatment in reducing TV after low RAI activities, irrespective of baseline thyroid status. TSH levels <0.3 mIU/l proved to be predictive of a more severe thyrotoxic phase after rhTSH and RAI, while initial TSH levels >0.3 mIU/l were more frequently followed by a need for L-T4 therapy. Compressive symptoms improved in the majority of subjects.

Heterologous mitochondrial DNA recombination in human cells.

PubMed

D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

2004-12-15

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Bayesian Inference of Shared Recombination Hotspots Between Humans and Chimpanzees

PubMed Central

Wang, Ying; Rannala, Bruce

2014-01-01

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. PMID:25261696

Bayesian inference of shared recombination hotspots between humans and chimpanzees.

PubMed

Wang, Ying; Rannala, Bruce

2014-12-01

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. Copyright © 2014 by the Genetics Society of America.

A New Small-Molecule Antagonist Inhibits Graves' Disease Antibody Activation of the TSH Receptor

PubMed Central

Eliseeva, Elena; McCoy, Joshua G.; Napolitano, Giorgio; Giuliani, Cesidio; Monaco, Fabrizio; Huang, Wenwei; Gershengorn, Marvin C.

2011-01-01

Context: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. Objective: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. Design: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. Results: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. Conclusion: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera. PMID:21123444

A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor.

PubMed

Neumann, Susanne; Eliseeva, Elena; McCoy, Joshua G; Napolitano, Giorgio; Giuliani, Cesidio; Monaco, Fabrizio; Huang, Wenwei; Gershengorn, Marvin C

2011-02-01

Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Diagnostic methods of TSH in thyroid screening tests.

PubMed

Matyjaszek-Matuszek, Beata; Pyzik, Aleksandra; Nowakowski, Andrzej; Jarosz, Mirosław J

2013-01-01

Reliable and quick thyreologic diagnostics, as well as verification of the effectiveness of the therapy undertaken, is of great importance for the state of health of society. The measurement of plasma TSH is the commonly accepted and most sensitive screening test for primary thyroid disorders, which are the most frequent diseases related to the endocrine glands. At present, the available methods for the determination of TSH are characterized by high sensitivity ≤0.01 µIU/ml and lack of cross-reactivity. However, many drugs and substances, as well as pathological conditions, may affect the TSH level. evaluation of contemporary laboratory methods for the determination of TSH and the principles of interpretation of screening tests. In many countries, the TSH test is the only test performed in the diagnostics of thyroid function; nevertheless, it seems that for genuine and objective assessment of thyroid status the TSH level, together with FT4 level, should be absolutely determined, which allows the differentiation and assessment of the intensity of thyroid function disorders and foresee its consequences. The interpretation of TSH results in screening tests is different in such population groups as: children aged under 14, pregnant women, the elderly, and patients with non-thyroidal illnesses. From among currently used laboratory methods for determination of TSH levels, third generation non-isotopic methods are most frequently recommended, especially the method of immunochemiluminescence.

Falsely undetectable TSH in a cohort of South Asian euthyroid patients.

PubMed

Drees, Julia C; Stone, Judith A; Reamer, C Randy; Arboleda, Victoria E; Huang, Karl; Hrynkow, Jane; Greene, Dina N; Petrie, Matthew S; Hoke, Carolyn; Lorey, Thomas S; Dlott, Richard S

2014-04-01

An index case of a clinically euthyroid woman of South Asian descent was identified with discordant TSH results: undetectable TSH on our routine assay and normal TSH on an alternate assay. Low TSH concentrations due to functionally compromising TSH mutations have been reported. Here we describe a new phenomenon of functional TSH that is undetectable by 4 widely used US Food and Drug Administration (FDA)-approved TSH immunoassays marketed by a single vendor. The purpose of this study was to identify additional cases and investigate the cause of the falsely undetectable TSH. All samples with TSH results of <0.01 μIU/mL were retested with a second TSH assay. Discordant samples were evaluated on up to 8 FDA-approved TSH immunoassays and the TSHβ gene was sequenced. Retrospectively, thyroid function tests, diagnoses, and medications from 1.6 million individuals were analyzed. Out of approximately 2 million individuals, we have identified a cohort of 20 hypothyroid and euthyroid patients of shared ethnicity with falsely undetectable TSH (<0.01 μIU/mL) in 4 of 8 commercially available TSH assays. Half of these individuals were initially treated based on repeated falsely undetectable TSH values (7 euthyroid patients were treated with methimazole and 2 hypothyroid patients had doses of levothyroxine decreased). In all cases, a retrospective chart review revealed that clinical assessments and free T4 and total T3 results were inconsistent with the undetectable TSH results. Specific antibodies failing to detect TSH in these cases were identified in the 4 affected assays. A novel TSHβ point mutation was identified. Our data suggest that these individuals have a previously unrecognized, functionally normal, TSH variant to which some monoclonal antibodies fail to bind. To assure appropriate patient management, clinicians and laboratorians need to be aware that certain TSH variants may be undetectable in some hyperselective TSH assays.

[Clinical studies on regulatory system of thyroid hormone secretion and serum triiodothyronine. Part. I. Solid-state radioimmunoassy for human serum TSH and its clinical application (author's transl)].

PubMed

Takeda, Y

1975-01-20

A solid-state RIA method using a plastic microtiter plate for human TSH was developed: 1) The choice of carrier protein for standard TSH was critical in this method and pooled sera from untreated Graves patients was found to be suitable for this purpose. The mean lowest detectable TSH level was 0.2 muU/assay, which was almost equal to those reported by other methods. This method is superior in simple assay procedure, especially in the separation of bound and free TSH and in the shorter incubation time required in the double antibody method. 2) Serum TSH concentration in 22 normal subjects, 17 patients with Graves' disease, 35 Hashimoto's thyroiditis, 18 primary hypothyrodism, 16 simple goiter, 4 nodular goiter and 7 secondary hypothyroidism was estimated as 4.7 +/- 2.0 muU/ml (mean +/- s.d.), 2.1 +/- 0.2 mu/U/ml, 14.1 +/- 26.5 muU/ml, 211 +/- 177 muU/ml, 3.6 +/- 2.4 muU/ml, 3.2 +/- 2.4 muU/ml and 2.6 +/- 1.0 muU/ml, respectively. 3) A statistically significant and hyperbolic inverse correlation (r= --0.37, N=90) was found between TSH and T4 levels. Some cases with normal T4 level were found to be high in TSH levels. It was also noted that 36 of 65 euthyroid cases (55.4%) who had been treated with 131I for Graves' disease showed elevated TSH levels. 4) After intravenous injection of 500 mug TRH, TSH level reached its peak value of 8 to 32 muU/ml at 15 to 45 minutes in normal subjects. Low to no response was found in patients with Graves' disease. An exaggerated response in patients with primary hypothyroidism to TRH was observed and an inhibitory process in TSH production at the pituitary level was suggested in patients with Cushing syndrome. Hypothyroid patients with pituitary lesion showed low or no response, on the other hand some hypothyroid patients with lesions around the pituitary and hypothalamus showed high basal TSH and exaggerated response to TRH.

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

PubMed

Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

2002-07-01

Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

Multiple metals predict prolactin and thyrotropin (TSH) levels in men

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meeker, John D., E-mail: meekerj@umich.edu; Rossano, Mary G.; Protas, Bridget

2009-10-15

Exposure to a number of metals can affect neuroendocrine and thyroid signaling, which can result in adverse effects on development, behavior, metabolism, reproduction, and other functions. The present study assessed the relationship between metal concentrations in blood and serum prolactin (PRL) and thyrotropin (TSH) levels, markers of dopaminergic, and thyroid function, respectively, among men participating in a study of environmental influences on male reproductive health. Blood samples from 219 men were analyzed for concentrations of 11 metals and serum levels of PRL and TSH. In multiple linear regression models adjusted for age, BMI and smoking, PRL was inversely associated withmore » arsenic, cadmium, copper, lead, manganese, molybdenum, and zinc, but positively associated with chromium. Several of these associations (Cd, Pb, Mo) are consistent with limited studies in humans or animals, and a number of the relationships (Cr, Cu, Pb, Mo) remained when additionally considering multiple metals in the model. Lead and copper were associated with non-monotonic decrease in TSH, while arsenic was associated with a dose-dependent increase in TSH. For arsenic these findings were consistent with recent experimental studies where arsenic inhibited enzymes involved in thyroid hormone synthesis and signaling. More research is needed for a better understanding of the role of metals in neuroendocrine and thyroid function and related health implications.« less

Treatment room length-of-stay and patient throughput with radioiodine thyroid remnant ablation in differentiated thyroid cancer: comparison of thyroid-stimulating hormone stimulation methods.

PubMed

Vallejo Casas, Juan Antonio; Mena Bares, Luisa M; Gálvez, María Angeles; Marlowe, Robert J; Latre Romero, José M; Martínez-Paredes, María

2011-09-01

We sought to empirically compare treatment room length-of-stay and patient throughput for recombinant human thyroid-stimulating hormone (rhTSH)-aided thyroid remnant ablation with thyroid hormone withdrawal (THW)-aided ablation in patients with differentiated thyroid carcinoma (DTC). We retrospectively reviewed charts of all eligible (near) totally thyroidectomized patients with DTC undergoing ablation and 1-year ablation success evaluation at our tertiary referral centre from January 2003 to February 2009 (N=274). M1 disease caused exclusion unless discovered by a postablation scan or present when rhTSH was the only tolerable stimulation method. We extracted data on the length-of-stay, defined as the time between treatment room admission and discharge, and patient throughput, defined as patients ablated per treatment room per week. The treatment room discharge criterion was a whole-body dose rate of less than 60 μSv/h at 50 cm. The treatment groups (rhTSH, n=187; THW, n=87) had mostly statistically similar characteristics, but differed in primary tumour status distribution. In addition, at ablation, the rhTSH patients had a greater prevalence of prior diagnostic scintigraphy, higher mean serum TSH, and shorter interval since surgery, and received a 5.6% larger mean ablation activity. On average, rhTSH patients had a significantly lower peak whole-body dose rate (57.1 vs. 83.4 μSv/h at 50 cm; P<0.0001) and a significantly shorter treatment room stay than did the THW patients (1.41 vs. 2.02 days; P<0.001). rhTSH use allowed significantly more patients to be ablated per room per week (2.7 vs. 1.2; P<0.001). Relative to THW, rhTSH use to aid ablation reduced mean treatment room length-of-stay by almost one-third and more than doubled the average weekly patient throughput, both of which were significant differences.

Cell-phone-based measurement of TSH using Mie scatter optimized lateral flow assays.

PubMed

You, David J; Park, Tu San; Yoon, Jeong-Yeol

2013-02-15

OS application directly on the phone and verified using the code written in MATLAB. The limit of detection of the system was determined to be 0.31 mIU L(-1) (never achieved before on an LFA format), below the commonly accepted minimum concentration of 0.4 mIU L(-1) indicating clinical significance of hyperthyroidism. The system was further evaluated using human serum showing an accurate and reproducible platform for rapid and point-of-care quantification of TSH using a cell phone. Copyright © 2012 Elsevier B.V. All rights reserved.

Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice

PubMed Central

Neumann, Susanne; Huang, Wenwei; Titus, Steve; Krause, Gerd; Kleinau, Gunnar; Alberobello, Anna Teresa; Zheng, Wei; Southall, Noel T.; Inglese, James; Austin, Christopher P.; Celi, Francesco S.; Gavrilova, Oksana; Thomas, Craig J.; Raaka, Bruce M.; Gershengorn, Marvin C.

2009-01-01

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer. PMID:19592511

A comparison of 1850 (50 mCi) and 3700 MBq (100 mCi) 131-iodine administered doses for recombinant thyrotropin-stimulated postoperative thyroid remnant ablation in differentiated thyroid cancer.

PubMed

Pilli, Tania; Brianzoni, Ernesto; Capoccetti, Francesca; Castagna, Maria Grazia; Fattori, Sara; Poggiu, Angela; Rossi, Gloria; Ferretti, Francesca; Guarino, Elisa; Burroni, Luca; Vattimo, Angelo; Cipri, Claudia; Pacini, Furio

2007-09-01

Recently, a multicenter study in differentiated thyroid cancer (DTC) patients showed that 3700 MBq 131-iodine ((131)I) after recombinant human TSH (rhTSH) had a successful thyroid ablation rate similar to that obtained after thyroid hormone withdrawal. We investigated whether 1850 MBq (131)I had a similar successful rate to 3700 MBq in patients prepared with rhTSH. A total of 72 patients with DTC were randomly assigned to receive 1850 (group A, n = 36) or 3700 MBq (group B, n = 36) (131)I after rhTSH. One injection of 0.9 mg rhTSH was administered for 2 consecutive days; (131)I therapy was delivered 24 h after the last injection, followed by a posttherapy whole-body scan. Successful ablation was assessed 6-8 months later. Successful ablation (no visible uptake in the diagnostic whole-body scan after rhTSH stimulation) was achieved in 88.9% of group A and B patients. Basal and rhTSH-stimulated serum thyroglobulin was undetectable (<1 ng/ml) in 78.9% of group A and 66.6% of group B patients (P = 0.46). Similar rates of ablation were obtained in both groups also in patients with node metastases. Therapeutic (131)I activities of 1850 MBq are equally effective as 3700 MBq for thyroid ablation in DTC patients prepared with rhTSH, even in the presence of node metastases.

Hyperthyroid-associated osteoporosis is exacerbated by the loss of TSH signaling

USDA-ARS?s Scientific Manuscript database

The osteoporosis associated with human hyperthyroidism has traditionally been attributed to elevated thyroid hormone levels. There is evidence, however, that thyroid-stimulating hormone (TSH), which is low in most hyperthyroid states, directly affects the skeleton. Importantly, Tshr-knockout mice ar...

Blocking mitochondrial cyclophilin D ameliorates TSH-impaired defensive barrier of artery.

PubMed

Liu, Xiaojing; Du, Heng; Chai, Qiang; Jia, Qing; Liu, Lu; Zhao, Meng; Li, Jun; Tang, Hui; Chen, Wenbin; Zhao, Lifang; Fang, Li; Gao, Ling; Zhao, Jiajun

2018-05-01

Endothelial cells (ECs) constitute the defensive barrier of vasculature, which maintains the vascular homeostasis. Mitochondrial oxidative stress (mitoOS) in ECs significantly affects the initiation and progression of vascular diseases. The higher serum thyroid stimulating hormone (TSH) level is being recognized as a nonconventional risk factor responsible for the increased risk of cardiovascular diseases in subclinical hypothyroidism (SCH). However, effects and underlying mechanisms of elevated TSH on ECs are still ambiguous. We sought to investigate whether cyclophilin D (CypD), emerging as a crucial mediator in mitoOS, regulates effects of TSH on ECs. SCH patients with TSH > = 10mIU/L showed a positive correlation between serum TSH and endothelin-1 levels. When TSH levels declined to normal in these subjects after levothyroxine therapy, serum endothelin-1 levels were significantly reduced. Supplemented with exogenous thyroxine to keep normal thyroid hormones, thyroid-specific TSH receptor (TSHR)-knockout mice with injection of exogenous TSH exhibited elevated serum TSH levels, significant endothelial oxidative injuries and disturbed endothelium-dependent vasodilation. However, Tshr -/- mice resisted to TSH-impaired vasotonia. We further confirmed that elevated TSH triggered excessive mitochondrial permeability transition pore (mPTP) opening and mitochondrial oxidative damages in mouse aorta, as well as in cultured ECs. Genetic or pharmacological inhibition of CypD (the key regulator for mPTP opening) attenuated TSH-induced mitochondrial oxidative damages and further rescued endothelial functions. Finally, we confirmed that elevated TSH could activate CypD by enhancing CypD acetylation via inhibiting adenosine monophosphate-activated protein kinase/sirtuin-3 signaling pathway in ECs. These findings reveal that elevated TSH triggers mitochondrial perturbations in ECs and provide insights that blocking mitochondrial CypD enhances the defensive ability of ECs under

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

[A case of GH and TSH secreting pituitary macroadenoma].

PubMed

Gołkowski, Filip; Buziak-Bereza, Monika; Stefańska, Agnieszka; Trofimiuk, Małgorzata; Pantofliński, Jacek; Huszno, Bohdan; Czepko, Ryszard; Adamek, Dariusz

2006-01-01

A case of GH and TSH secreting pituitary macroadenoma is reported. A 45-year-old female presented clinical features of acromegaly (the abnormal growth of the hands and feet, with lower jaw protrusion), diabetes mellitus, hypertension, nodular goiter and hyperthyroidism of unclear origin. NMR pituitary imaging revealed intra and extrasellar tumor. The laboratory examinations showed very high plasma levels of GH and IGF-1 and normal level of TSH coexisting with high plasma levels of free thyroid hormones. Pharmacological pretreatment with somatostatin analogues caused the substantial reduction of GH and TSH plasma levels. Histological and immunohistochemical examination of the tissue obtained at transsphenoidal surgery showed GH and TSH secreting adenoma. The laboratory examinations after surgery showed normal GH and IGF-1 plasma levels and reduced insulin requirement, what indicates radical operation. The very low plasma levels of TSH and free thyroid hormones after surgery and immunohistochemical examination suggest central hyperthyroidism due to TSH secreting pituitary tumor (thyrotropinoma).

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure

PubMed Central

Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-01-01

Objectives In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. Methods This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Results Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. Conclusion This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula. PMID:26622960

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure.

PubMed

Kucuk, Nurten; Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-12-01

In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula.

Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen

PubMed Central

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-01-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT. PMID:25816228

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

PubMed

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-03-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Human cells: new platform for recombinant therapeutic protein production.

PubMed

Swiech, Kamilla; Picanço-Castro, Virgínia; Covas, Dimas Tadeu

2012-07-01

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production. Copyright © 2012 Elsevier Inc. All rights reserved.

Population-specific recombination sites within the human MHC region.

PubMed

Lam, T H; Shen, M; Chia, J-M; Chan, S H; Ren, E C

2013-08-01

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

AIDing Chromatin and Transcription-Coupled Orchestration of Immunoglobulin Class-Switch Recombination

PubMed Central

Vaidyanathan, Bharat; Yen, Wei-Feng; Pucella, Joseph N.; Chaudhuri, Jayanta

2014-01-01

Secondary diversification of the antibody repertoire upon antigenic challenge, in the form of immunoglobulin heavy chain (IgH) class-switch recombination (CSR) endows mature, naïve B cells in peripheral lymphoid organs with a limitless ability to mount an optimal humoral immune response, thus expediting pathogen elimination. CSR replaces the default constant (CH) region exons (Cμ) of IgH with any of the downstream CH exons (Cγ, Cε, or Cα), thereby altering effector functions of the antibody molecule. This process depends on, and is orchestrated by, activation-induced deaminase (AID), a DNA cytidine deaminase that acts on single-stranded DNA exposed during transcription of switch (S) region sequences at the IgH locus. DNA lesions thus generated are processed by components of several general DNA repair pathways to drive CSR. Given that AID can instigate DNA lesions and genomic instability, stringent checks are imposed that constrain and restrict its mutagenic potential. In this review, we will discuss how AID expression and substrate specificity and activity is rigorously enforced at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and how the DNA-damage response is choreographed with precision to permit targeted activity while limiting bystander catastrophe. PMID:24734031

Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Human Factors Job Aid

DOT National Transportation Integrated Search

1996-12-09

The purpose of this Human Factors Job Aid is to serve as a desk reference for : human factors integration during system acquisition. The first chapter contains : an overview of the FAA human factors process in system acquisitions. The : remaining eig...

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Effect of Recombination in the Evolutionary Dynamics of HIV under the Surveillance of Immune System

NASA Astrophysics Data System (ADS)

Peng, Weiqun; Yang, Wenjing; Wang, Guanyu

2009-03-01

Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS), which has become one of the most destructive pandemics in history. The fact that HIV virus evolves very fast plays a central role in AIDS immunopathogenesis and the difficulty we face in finding a cure or a vaccine for AIDS. A distinguishing feature of HIV is its high frequency of recombination. The effect of recombination in the HIV evolution is not clear. We establish a mathematical model of the evolutionary dynamics. This model incorporates both point mutation and recombination for genetic diversity, and employs a fitness function developed by Wang and Deem (PRL 97, 188106, 2006) that accounts for the effect of immune system. Using this model, we explore the role of recombination in the battle between the virus population and the immune system, with a special focus on the condition under which recombination helps the virus population to escape from the immune system.

Epitope mapping of tsh receptor-blocking antibodies in Graves' disease that appear during pregnancy.

PubMed

Kung, A W; Lau, K S; Kohn, L D

2001-08-01

Spontaneous remission of Graves' disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves' disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-LH/hCG receptor chimeras: Mc1+2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9-165, 90-165, and 261-370 substituted with equivalent residues of the LH/hCG receptor. Overall thyroid-stimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues 9-165 of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues 261-370 of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9-89 or 90-165 as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy

Are lower TSH cutoffs in neonatal screening for congenital hypothyroidism warranted?

PubMed Central

Lain, Samantha; Trumpff, Caroline; Grosse, Scott D; Olivieri, Antonella; Van Vliet, Guy

2018-01-01

When newborn screening (NBS) for congenital hypothyroidism (CH) using thyroid-stimulating hormone (TSH) as a primary screening test was introduced, typical TSH screening cutoffs were 20–50 U/L of whole blood. Over the years, lowering of TSH cutoffs has contributed to an increased prevalence of detected CH. However, a consensus on the benefit deriving from lowering TSH cutoffs at screening is lacking. The present paper outlines arguments both for and against the lowering of TSH cutoffs at NBS. It includes a review of recently published evidence from Australia, Belgium and Italy. A section focused on economic implications of lowering TSH cutoffs is also provided. One issue that bears further examination is the extent to which mild iodine deficiency at the population level might affect the association of neonatal TSH values with cognitive and developmental outcomes. A debate on TSH cutoffs provides the opportunity to reflect on how to make NBS for CH more effective and to guarantee optimum neurocognitive development and a good quality of life to babies with mild as well as with severe CH. All authors of this debate article agree on the need to establish optimal TSH cutoffs for screening programs in various settings and to ensure the benefits of screening and access to care for newborns worldwide. PMID:28694389

Termitarium-inhabiting Bacillus endophyticus TSH42 and Bacillus cereus TSH77 colonizing Curcuma longa L.: isolation, characterization, and evaluation of their biocontrol and plant-growth-promoting activities.

PubMed

Chauhan, Ankit Kumar; Maheshwari, Dinesh Kumar; Kim, Kangmin; Bajpai, Vivek K

2016-10-01

Bacillus strains were isolated from termitarium soil and screened for their antifungal activity through the production of diffusible and volatile metabolites. Further, the bacterial strains that showed antifungal activity were evaluated for their biocontrol potential on the basis of their plant-growth-promoting attributes. Termitarium-inhabiting Bacillus strains TSH42 and TSH77 significantly reduced the growth of pathogenic fungus Fusarium solani, controlled the symptoms of rhizome rot in turmeric (Curcuma longa L.), and demonstrated various plant-growth-promoting traits in different in vitro assays. On the basis of morphological, physiological, biochemical, and 16S rDNA characteristics, isolates TSH42 and TSH77 were identified as Bacillus endophyticus (KT379993) and Bacillus cereus (KT379994), respectively. Through liquid chromatography - mass spectrometry analysis, acidified cell-free culture filtrate (CFCF) of B. cereus TSH77 was shown to contain surfactin and fengycin, while CFCF of B. endophyticus TSH42 contained iturin in addition to surfactin and fengycin. Treatment of the turmeric (C. longa L.) plants with TSH42 and TSH77 significantly reduced the percentage incidence of rhizome rot disease caused by F. solani. The same treatment also increased the fresh rhizome biomass and plant growth in greenhouse conditions.

Human recombinant lysosomal enzymes produced in microorganisms.

PubMed

Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

2015-01-01

Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. Copyright © 2015 Elsevier Inc. All rights reserved.

Producing recombinant human milk proteins in the milk of livestock species.

PubMed

Bösze, Zsuzsanna; Baranyi, Mária; Whitelaw, C Bruce A

2008-01-01

Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.

A transcriptional serenAID: the role of noncoding RNAs in class switch recombination

PubMed Central

Yewdell, William T.; Chaudhuri, Jayanta

2017-01-01

Abstract During an immune response, activated B cells may undergo class switch recombination (CSR), a molecular rearrangement that allows B cells to switch from expressing IgM and IgD to a secondary antibody heavy chain isotype such as IgG, IgA or IgE. Secondary antibody isotypes provide the adaptive immune system with distinct effector functions to optimally combat various pathogens. CSR occurs between repetitive DNA elements within the immunoglobulin heavy chain (Igh) locus, termed switch (S) regions and requires the DNA-modifying enzyme activation-induced cytidine deaminase (AID). AID-mediated DNA deamination within S regions initiates the formation of DNA double-strand breaks, which serve as biochemical beacons for downstream DNA repair pathways that coordinate the ligation of DNA breaks. Myriad factors contribute to optimal AID targeting; however, many of these factors also localize to genomic regions outside of the Igh locus. Thus, a current challenge is to explain the specific targeting of AID to the Igh locus. Recent studies have implicated noncoding RNAs in CSR, suggesting a provocative mechanism that incorporates Igh-specific factors to enable precise AID targeting. Here, we chronologically recount the rich history of noncoding RNAs functioning in CSR to provide a comprehensive context for recent and future discoveries. We present a model for the RNA-guided targeting of AID that attempts to integrate historical and recent findings, and highlight potential caveats. Lastly, we discuss testable hypotheses ripe for current experimentation, and explore promising ideas for future investigations. PMID:28535205

GLIS3 is indispensable for TSH/TSHR-dependent thyroid hormone biosynthesis and follicular cell proliferation

PubMed Central

Kang, Hong Soon; Kumar, Dhirendra; Liao, Grace; Lichti-Kaiser, Kristin; Gerrish, Kevin; Liao, Xiao-Hui; Refetoff, Samuel; Jothi, Raja; Jetten, Anton M.

2017-01-01

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division–related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development. PMID:29083325

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

PubMed

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

Molecular epidemiology of HIV: tracking AIDS pandemic.

PubMed

TakebE, Yutaka; Kusagawa, Shigeru; Motomura, Kazushi

2004-04-01

Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) epidemic is a global threat to maternal and child health, especially in developing countries. It is estimated that 800 000 children are infected and 580 000 children die of AIDS-related illnesses every year. Molecular epidemiology has been a useful tool in analyzing the origin of HIV and tracking the course of global HIV spread. This article provides an overview of recent advances in the field of molecular epidemiology of HIV across the world, and discuss the biological implications. Based on the near full-length or partial nucleotide sequence information, the phylogeny and recombinant structure of HIV strains are analyzed. Using genotype classification of HIV as a molecular marker, the origin and the genesis of HIV epidemic are investigated. The HIV-1 group M, a major HIV group responsible for current AIDS pandemic, began its expansion in human population approximately 70 years ago and diversified rapidly over time, now comprising a number of different subtypes and circulating recombinant forms (CRF). Of note, recent studies revealed that new recombinant strains are arising continually, becoming a powerful force in the spread of HIV-1 across the globe. Global dissemination of HIV is a dramatic and deadly example of recent genome emergence and expansion. Molecular epidemiological investigation is expected to provide information critical for prevention and future vaccine strategies.

High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

PubMed Central

Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

2002-01-01

Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

Variation in recombination rate may bias human genetic disease mapping studies.

PubMed

Boyle, A Susannah; Noor, Mohamed A F

2004-11-01

The availability of the human genome sequence and variability information (as from the International HapMap project) will enhance our ability to map genetic disorders and choose targets for therapeutic intervention. However, several factors, such as regional variation in recombination rate, can bias conclusions from genetic mapping studies. Here, we examine the impact of regional variation in recombination rate across the human genome. Through computer simulations and literature surveys, we conclude that genetic disorders have been mapped to regions of low recombination more often than expected if such diseases were randomly distributed across the genome. This concentration in low recombination regions may be an artifact, and disorders appearing to be caused by a few genes of large effect may be polygenic. Future genetic mapping studies should be conscious of this potential complication by noting the regional recombination rate of regions implicated in diseases.

Transient neonatal hypothyroidism due to transplacental transfer of maternal immunoglobulins that inhibit TSH binding, TSH-induced cAMP increase and cell growth.

PubMed

Cho, B Y; Shong, Y K; Lee, H K; Koh, C S; Min, H K; Lee, M

1988-12-01

Transient neonatal hypothyroidism due to transplacental transfer of maternal blocking type TSH receptor antibodies (TRAb) was found in a baby born to a 27-yr-old mother, who had been receiving thyroxine medication for primary myxedema. Maternal IgG inhibited radiolabelled TSH binding to its receptor (TBII), TSH-stimulated thyroid adenylate cyclase (AC) activation (TSII) and TSH-stimulated 3H-thymidine uptake (TGII) in cultured rat thyroid cells (FRTL-5). At birth, the baby's IgG showed similar activities to maternal IgG but all these activities decreased gradually, and disappeared from her serum within 12 weeks of age. In the baby, initially nonvisualized thyroid was clearly visualized on 99 m-Tc thyroid scintigraphy when all these blocking activities disappeared, TSII and TGII being decreased more slowly than TBII, and the baby remained euthyroid after discontinuation of thyroxine. This study suggests that such IgGs induced hypothyroidism and thyroid atrophy in the mother and were responsible for transient neonatal hypothyroidism in the baby.

Antibody protection reveals extended epitopes on the human TSH receptor.

PubMed

Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A; Davies, Terry F

2012-01-01

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

PubMed Central

Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M. Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A.; Davies, Terry F.

2012-01-01

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1–412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. PMID:22957097

Targeting thyroid diseases with TSH receptor analogs.

PubMed

Galofré, Juan C; Chacón, Ana M; Latif, Rauf

2013-12-01

The thyroid-stimulating hormone (TSH) receptor (TSHR) is a major regulator of thyroid function and growth, and is the key antigen in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Various effective treatment strategies are currently available for many of these clinical conditions such as antithyroid drugs or radioiodine therapy, but they are not devoid of side effects. In addition, treatment of complications of Graves' disease such as Graves' ophthalmopathy is often difficult and unsatisfactory using current methods. Recent advances in basic research on both in vitro and in vivo models have suggested that TSH analogs could be used for diagnosis and treatment of some of the thyroid diseases. The advent of high-throughput screening methods has resulted in a group of TSH analogs called small molecules, which have the potential to be developed as promising drugs. Small molecules are low molecular weight compounds with agonist, antagonist and, in some cases, inverse agonist activity on TSHR. This short review will focus on current advances in development of TSH analogs and their potential clinical applications. Rapid advances in this field may lead to the conduct of clinical trials of small molecules related to TSHR for the management of Graves' disease, thyroid cancer, and thyroid-related osteoporosis in the coming years. Copyright © 2012 SEEN. Published by Elsevier Espana. All rights reserved.

Validation of biological activity testing procedure of recombinant human interleukin-7.

PubMed

Lutsenko, T N; Kovalenko, M V; Galkin, O Yu

2017-01-01

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation charac­teristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Construction of human artificial chromosome vectors by recombineering.

PubMed

Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

2005-05-23

Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

PubMed

Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

2017-10-01

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Thyroid hormone independent associations between serum TSH levels and indicators of bone turnover in cured patients with differentiated thyroid carcinoma.

PubMed

Heemstra, Karen A; van der Deure, Wendy M; Peeters, Robin P; Hamdy, Neveen A; Stokkel, Marcel P; Corssmit, Eleonora P; Romijn, Johannes A; Visser, Theo J; Smit, Johannes W

2008-07-01

It has been proposed that TSH has thyroid hormone-independent effects on bone mineral density (BMD) and bone metabolism. This concept is still controversial and has not been studied in human subjects in detail. We addressed this question by studying relationships between serum TSH concentration and indicators of bone turnover, after controlling for triiodothyronine (T(3)), free thyroxine (FT(4)), and non-thyroid factors relevant to BMD and bone metabolism. We also studied the contribution of the TSH receptor (TSHR)-Asp727Glu polymorphism to these relationships. We performed a cross-sectional study with 148 patients, who had been thyroidectomized for differentiated thyroid carcinoma. We measured BMD of the femoral neck and lumbar spine. FT(4), T(3), TSH, bone-specific alkaline phosphatase, procollagen type 1 aminoterminal propeptide levels, C-cross-linking terminal telopeptide of type I collagen, and urinary N-telopeptide of collagen cross-links were measured. Genotypes of the TSHR-Asp727Glu polymorphism were determined by Taqman assay. We found a significant, inverse correlation between serum TSH levels and indicators of bone turnover, which was independent of serum FT(4) and T(3) levels as well as other parameters influencing bone metabolism. We found that carriers of the TSHR-Asp727Glu polymorphism had an 8.1% higher femoral neck BMD, which was, however, no longer significant after adjusting for body mass index. We conclude that in this group of patients, serum TSH was related to indicators of bone remodeling independently of thyroid hormone levels. This may point to a functional role of the TSHR in bone in humans. Further research into this mechanism needs to be performed.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

PubMed

Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor

Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

PubMed

Qiu, Huawei; Boudanova, Ekaterina; Park, Anna; Bird, Julie J; Honey, Denise M; Zarazinski, Christine; Greene, Ben; Kingsbury, Jonathan S; Boucher, Susan; Pollock, Julie; McPherson, John M; Pan, Clark Q

2013-03-20

Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and β subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H.

PubMed

Fayet, G; Aouani, A; Hovsépian, S

1986-01-06

The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation.

[Monosymptomatic hyperthyroidism and TSH-producing adenoma: successful therapy with octreotide].

PubMed

Mayinger, B; Axelos, D; Pavel, M; Hahn, E G; Hensen, J

1999-01-29

Magnetic resonance imaging (MRI) of the central nervous system was performed on a 72-year-old woman who was hyperthyroid without suppression of the thyroid-stimulating hormone (TSH) and had complained of a recent onset of headaches. MRI demonstrated a space-occupying lesion, 1 cm in diameter, in the anterior pituitary. The clinical symptoms were marked by a long-standing monosymptomatic illness of rapidly changing mood swings with depressive and manic phases. Endocrinological-biochemical tests showed hyperthyroidism (fT3 10.55 pmol/l and fT4 39 pmol/l) but no TSH suppression (TSH: 2.9 microU/ml). Octreotide scintigraphy documented an activity-rich area in the anterior pituitary and the upper anterior mediastinum. Mediastinal MRI revealed a 5 cm space-occupying mass lying on the right atrium. 131I scintigraphy identified the mass as a retrosternal goitre. As an operation on the anterior pituitary would have carried a high risk for the patient who was in a poor general condition and she had refused to be operated, treatment with octreotide, a long-acting somatostatin analogue, was initiated. This achieved a euthyroid state with partly suppressed TSH, and the patient's emotional swings ceased. If hyperthyroidism coexists with non-suppressed TSH levels, a TSH-producing hypophyseal adenoma should be considered in the differential diagnosis despite its rarity. Octreotide administration is an effective and safe treatment and is the method of choice, especially when there are contraindications to surgical resection of the anterior pituitary.

Evidence of reduced recombination rate in human regulatory domains.

PubMed

Liu, Yaping; Sarkar, Abhishek; Kheradpour, Pouya; Ernst, Jason; Kellis, Manolis

2017-10-20

Recombination rate is non-uniformly distributed across the human genome. The variation of recombination rate at both fine and large scales cannot be fully explained by DNA sequences alone. Epigenetic factors, particularly DNA methylation, have recently been proposed to influence the variation in recombination rate. We study the relationship between recombination rate and gene regulatory domains, defined by a gene and its linked control elements. We define these links using expression quantitative trait loci (eQTLs), methylation quantitative trait loci (meQTLs), chromatin conformation from publicly available datasets (Hi-C and ChIA-PET), and correlated activity links that we infer across cell types. Each link type shows a "recombination rate valley" of significantly reduced recombination rate compared to matched control regions. This recombination rate valley is most pronounced for gene regulatory domains of early embryonic development genes, housekeeping genes, and constitutive regulatory elements, which are known to show increased evolutionary constraint across species. Recombination rate valleys show increased DNA methylation, reduced doublestranded break initiation, and increased repair efficiency, specifically in the lineage leading to the germ line. Moreover, by using only the overlap of functional links and DNA methylation in germ cells, we are able to predict the recombination rate with high accuracy. Our results suggest the existence of a recombination rate valley at regulatory domains and provide a potential molecular mechanism to interpret the interplay between genetic and epigenetic variations.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

PubMed

Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

2009-12-29

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

PubMed Central

Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

2016-01-01

Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

Termini of human chromosomes display elevated rates of mitotic recombination.

PubMed

Cornforth, M N; Eberle, R L

2001-01-01

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

Individual Substitution Mutations in the AID C Terminus That Ablate IgH Class Switch Recombination

PubMed Central

Kadungure, Tatenda; Ucher, Anna J.; Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

2015-01-01

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid -/- mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to ΔAID, the substitutions reduce binding of UNG to Ig Sμ regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)μ and acceptor Sα regions from cells expressing ΔAID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the Sμ region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged Sμ regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that Sμ DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of Sμ segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR. PMID:26267846

Targeting the thyroid gland with thyroid-stimulating hormone (TSH)-nanoliposomes.

PubMed

Paolino, Donatella; Cosco, Donato; Gaspari, Marco; Celano, Marilena; Wolfram, Joy; Voce, Pasquale; Puxeddu, Efisio; Filetti, Sebastiano; Celia, Christian; Ferrari, Mauro; Russo, Diego; Fresta, Massimo

2014-08-01

Various tissue-specific antibodies have been attached to nanoparticles to obtain targeted delivery. In particular, nanodelivery systems with selectivity for breast, prostate and cancer tissue have been developed. Here, we have developed a nanodelivery system that targets the thyroid gland. Nanoliposomes have been conjugated to the thyroid-stimulating hormone (TSH), which binds to the TSH receptor (TSHr) on the surface of thyrocytes. The results indicate that the intracellular uptake of TSH-nanoliposomes is increased in cells expressing the TSHr. The accumulation of targeted nanoliposomes in the thyroid gland following intravenous injection was 3.5-fold higher in comparison to untargeted nanoliposomes. Furthermore, TSH-nanoliposomes encapsulated with gemcitabine showed improved anticancer efficacy in vitro and in a tumor model of follicular thyroid carcinoma. This drug delivery system could be used for the treatment of a broad spectrum of thyroid diseases to reduce side effects and improve therapeutic efficacy. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Association of TSH With Cardiovascular Disease Risk in Overweight and Obese Children During Lifestyle Intervention.

PubMed

Rijks, Jesse M; Plat, Jogchum; Dorenbos, Elke; Penders, Bas; Gerver, Willem-Jan M; Vreugdenhil, Anita C E

2017-06-01

Overweight and obese children have an increased risk to develop cardiovascular diseases (CVDs) in which thyroid-stimulating hormone (TSH) has been suggested as an intermediary factor. However, results of cross-sectional studies are inconclusive, and intervention studies investigating changes in TSH concentrations in association with changes in cardiovascular risk parameters in overweight and obese children are scarce. To gain insight in associations of circulating TSH concentrations and cardiovascular risk parameters in overweight and obese children. Nonrandomized lifestyle intervention. Centre for Overweight Adolescent and Children's Healthcare. Three hundred thirty euthyroid overweight and obese children. Long-term lifestyle intervention. TSH concentrations, pituitary TSH release in response to thyrotropin-releasing hormone (TRH), and cardiovascular risk parameters. At baseline, serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triacylglycerol (TAG), and monocyte chemotactic protein 1 concentrations were significantly associated with serum TSH concentrations. TSH release by the pituitary in response to exogenous TRH was not associated with cardiovascular risk parameters. During lifestyle intervention, several cardiovascular risk parameters significantly improved. In children whose body mass index z score improved, changes in TSH concentrations were significantly associated with changes in TC, LDL-C, and TAG concentrations. In euthyroid overweight and obese children, circulating TSH concentrations are positively associated with markers representing increased CVD risk. Changes in TSH concentrations are also associated with changes in lipid concentrations in children with successful weight loss, which is consistent with TSH being an intermediary factor in modulating lipid and lipoprotein metabolism. Copyright © 2017 Endocrine Society

Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

PubMed Central

Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

2014-01-01

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

Elevated TSH in adults treated for hypothyroidism is associated with increased mortality.

PubMed

Akirov, Amit; Gimbel, Hannah; Grossman, Alon; Shochat, Tzipora; Shimon, Ilan

2017-01-01

Numerous studies investigated the link between hypothyroidism and mortality, but a definite conclusion is hard to reach as these were limited by a number of factors, including age of participants, comorbidities and single measurement of thyroid function. To evaluate the association between TSH and fT4 levels and mortality in patients with levothyroxine-treated hypothyroidism. Observational data of hospitalized patients (2011-2014). TSH and fT4 levels obtained between at least 30 days after discharge and until death or end of follow-up were collected. Median TSH and fT4 levels were stratified into categories. In total, 611 patients with treated hypothyroidism, aged 60-80 years (72% females, mean age 71 ± 6 years) were included in the study. All-cause mortality up to 66 months after discharge, by TSH and fT4 categories. During follow-up, the average numbers of TSH and fT4 measurements were 5.5 ± 3.8 and 2.5 ± 4.2 per patient respectively. Mortality rates were 28%, 29% and 54% with median TSH of 0.5-2.5, 2.5-5.0 and 5.0-10.0 IU/L respectively. Adjusted hazard ratios for mortality with median TSH between 5.0 and 10.0 IU/L were 2.3 (95% CI: 1.6-3.4) and 2.2 (95% CI: 1.6-3.2) compared with patients with TSH between 0.5-2.5 IU/L and 2.5-5 IU/L respectively. There was no difference in mortality between patients with median fT4 10-15 or 15-20 pmol/L. In treated hypothyroid adult patients and serial measurements of thyroid function tests, median TSH levels of 5-10 IU/L are associated with increased mortality with no effect of fT4 levels. Treatment should aim at achieving euthyroidism to improve survival. © 2017 European Society of Endocrinology.

Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

PubMed

Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

2009-12-01

Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

Recombination or mutational hot spots in human mtDNA?

PubMed

Innan, Hideki; Nordborg, Magnus

2002-07-01

Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.

The Effect of Differentially Designed Fusion Proteins to Elicit Efficient Anti-human Thyroid Stimulating Hormone Immune Responses.

PubMed

Mard-Soltani, Maysam; Rasaee, Mohamad Javad; Khalili, Saeed; Sheikhi, Abdol-Karim; Hedayati, Mehdi; Ghaderi-Zefrehi, Hossein; Alasvand, Milad

2018-04-01

The production of human thyroid stimulating hormone (hTSH) immunoassays requires specific antibodies against hTSH which is a cumbersome process. Therefore, producing specific polyclonal antibodies against engineered recombinant fusion hTSH antigens would be of great significance. The best immunogenic region of the hTSH was selected based on in silico analyses and equipped with two different fusions. Standard methods were used for protein expression, purification, verification, structural evaluation, and immunizations of the white New Zealand rabbits. Ultimately, immunized serums were used for antibody titration, purification and characterization (specificity, sensitivity and cross reactivity). The desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. Structural analyses indicated that only the bigger antigen has showed changed 2 dimensional (2D) and 3D structural properties in comparison to the smaller antigen. The raised polyclonal antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum and negligible. The fusion which was solely composed of the tetanus toxin epitopes led to better protein folding and was capable of immunizing the host animals resulting into high titer antibody. Therefore, the minimal fusion sequences seem to be more effective in eliciting specific antibody responses.

Serum TSH levels are associated with cardiovascular risk factors in overweight and obese adolescents.

PubMed

Souza, Luciana Lopes de; Guedes, Erika Paniago; Teixeira, Patrícia Fátima Dos Santos; Moreira, Rodrigo Oliveira; Godoy-Matos, Amelio Fernando; Vaisman, Mario

2016-01-01

To investigate the relationship between serum thyrotropin (TSH), insulin resistance (IR), and cardiovascular risk factors (CRF) in a sample of overweight and obese Brazilian adolescents. A retrospective, longitudinal analysis of 199 overweight and obese pubescent adolescents was performed. The TSH and free T4 (fT4) levels, anthropometric measurements, and laboratory test results of these patients were analyzed. 27 individuals (13.56%) presented with TSH levels above the normal level (subclinical hypothyroidism [SCH]). Their waist circumference (WC) was significantly higher than those of euthyroid individuals. Serum TSH was positively correlated with the homeostasis model assessment of insulin resistance (HOMA-IR) index, triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C). Using TSH and BMI as independent variables, TSH levels were shown to be independently related to HOMA-IR (p=0.001) and TG (p=0.007). Among euthyroid subjects, individuals with TSH values <2.5mIU/mL exhibited statistically significant decreases in waist-to-hip ratio, HDL-C levels, and HOMA-IR scores and a tendency toward lower WC values. SCH in overweight and obese adolescents appears to be associated with excess weight, especially visceral weight. In euthyroid adolescents, there appears to be a direct relationship between TSH and some CRF. In conclusion, in the present sample of overweight and obese adolescents, TSH levels appear to be associated with IR and CRF. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

PubMed

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

Thyroid hyperfunctioning adenomas with and without Gsp/TSH receptor mutations show similar clinical features.

PubMed

Arturi, F; Capula, C; Chiefari, E; Filetti, S; Russo, D

1998-01-01

Activating mutations of Gs alpha protein (gsp) and TSH receptor (TSH-R) identified in autonomously hyperfunctioning thyroid adenomas have been proposed as the primary event responsible for this disease. Since mutations have not been detected in 100% (ranging from less than 10% to 90%) of the patients, we evaluated whether the presence of gsp and TSH-R mutations cause differences in the clinical and biochemical parameters of the affected patients. Fifteen consecutive patients (11 women and 4 men) with autonomously hyperfunctioning thyroid adenomas who underwent thyroidectomy, previously examined for the presence of gsp or TSH-R mutations, were investigated. In all of the patients we examined plasma free T3, free T4, TSH levels and ultrasound volume of the nodules. The patients with mutations in gsp or TSH-R were similar to the patients without mutations for clinical presentation, sex distribution and mean age. Furthermore, basal serum FT3, TSH and tumor volume in the patients with mutations were not significantly different from the group without mutations. Our preliminary data demonstrate that no significant differences are present in the two groups of patients examined, suggesting that factors other than gsp or TSH-R mutations play a role in the clinical presentation of the disease.

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

PubMed

Pan, Fan; Yang, Wende; Li, Wei; Yang, Xiao-Yan; Liu, Shuhao; Li, Xin; Zhao, Xiaoxu; Ding, Hui; Qin, Li; Pan, Yunlong

2017-07-01

Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize

Effects of recombinant human growth hormone in the treatment of dwarfism and relationship between IGF-1, IGFBP-3 and thyroid hormone

PubMed Central

Ren, Shanxiang; Nie, Yuxiang; Wang, Aihong

2016-01-01

The effects of recombinant human growth hormone (rhGH) in the treatment of dwarfism and the relationship between insulin-like growth factor (IGF)-1, IGF-binding protein (IGFBP)-3 and thyroid hormone were examined in the present study. For this purpose, 66 patients diagnosed with dwarfism were selected retrospectively, with 36 cases of growth hormone deficiency (GHD) and 30 cases of idiopathic short stature (ISS). The therapeutic dose of GHD 0.10 IU/kg·day and ISS 0.15 IU/kg·day were injected subcutaneously every night before sleep until adulthood. The average follow-up was 5 years, and the results were evaluated and measured every 3 months, including height, BA, secondary test of growth hormone (GH peak), IGF-1, IGFBP-3 and thyroid hormone (FT3, FT4 and TSH). After treatment, the height, BA, GH peak, IGF-A and IGFBP-3 of the GHD group were all increased, and the differences were statistically significant (P<0.05), while FT3, FT4 and TSH had no significant change (P<0.05). The height and BA increased and the differences were statistically significant (P<0.05). The indexes of the ISS group were not statistically significant (P>0.05). The results of the Pearson-related analysis suggested that GH peak of the GHD group, IGF-1 and IGFBP-3 were positively associated with height (P<0.05), and had no relationship with BA (P<0.05). None of the above indexes of the ISS group had an obvious correlation with height and BA (P>0.05). rhGH was effective for GHD and ISS, with the GHD effect being positively associated with the GH peak, IGF-1 and IGFBP-3. ISS had no obvious relationship with GH peak, IGF-1 and IGFBP-3 although other influencing factors may be involved. PMID:28105090

Effects of recombinant human growth hormone in the treatment of dwarfism and relationship between IGF-1, IGFBP-3 and thyroid hormone.

PubMed

Ren, Shanxiang; Nie, Yuxiang; Wang, Aihong

2016-12-01

The effects of recombinant human growth hormone (rhGH) in the treatment of dwarfism and the relationship between insulin-like growth factor (IGF)-1, IGF-binding protein (IGFBP)-3 and thyroid hormone were examined in the present study. For this purpose, 66 patients diagnosed with dwarfism were selected retrospectively, with 36 cases of growth hormone deficiency (GHD) and 30 cases of idiopathic short stature (ISS). The therapeutic dose of GHD 0.10 IU/kg·day and ISS 0.15 IU/kg·day were injected subcutaneously every night before sleep until adulthood. The average follow-up was 5 years, and the results were evaluated and measured every 3 months, including height, BA, secondary test of growth hormone (GH peak), IGF-1, IGFBP-3 and thyroid hormone (FT3, FT4 and TSH). After treatment, the height, BA, GH peak, IGF-A and IGFBP-3 of the GHD group were all increased, and the differences were statistically significant (P<0.05), while FT3, FT4 and TSH had no significant change (P<0.05). The height and BA increased and the differences were statistically significant (P<0.05). The indexes of the ISS group were not statistically significant (P>0.05). The results of the Pearson-related analysis suggested that GH peak of the GHD group, IGF-1 and IGFBP-3 were positively associated with height (P<0.05), and had no relationship with BA (P<0.05). None of the above indexes of the ISS group had an obvious correlation with height and BA (P>0.05). rhGH was effective for GHD and ISS, with the GHD effect being positively associated with the GH peak, IGF-1 and IGFBP-3. ISS had no obvious relationship with GH peak, IGF-1 and IGFBP-3 although other influencing factors may be involved.

Serum TSH reference interval in healthy Finnish adults using the Abbott Architect 2000i Analyzer.

PubMed

Schalin-Jäntti, Camilla; Tanner, Pirjo; Välimäki, Matti J; Hämäläinen, Esa

2011-07-01

Current serum TSH reference intervals have been criticized as they were established from unselected background populations. A special concern is that the upper limit, which defines subclinical hypothyroidism, is too high. The objective was to redefine the TSH reference interval in the adult Finnish population. The current reference interval for the widely used Abbott Architect method in Finland is 0.4-4.0 mU/L. Serum TSH and free T4 concentrations were derived from 606 healthy, non-pregnant, 18-91-year-old Finns from the Nordic Reference Interval Project (NORIP) and the possible effects of age, sex and thyroid peroxidase antibody (TPOAb) status were evaluated. After excluding TPOAb-positive subjects and outliers, a reference population of 511 subjects was obtained. In the reference population, no statistically significant gender- or age-specific differences in mean TSH (1.55 ± 3.30 mU/L) or TSH reference intervals were observed. The new reference interval was 0.5-3.6 mU/L (2.5th-97.5th percentiles). The current upper TSH reference limit is 10% too high. A TSH > 3.6 mU/L, confirmed with a repeat TSH sampling, may indicate subclinical hypothyroidism. Differences in ethnicity, regional iodine-intake and analytical methods underline the need for redefining the TSH reference interval in central laboratories in different countries.

Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells

PubMed Central

2010-01-01

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory. PMID:20680536

Ancient Recombination Events between Human Herpes Simplex Viruses

PubMed Central

Burrel, Sonia; Boutolleau, David; Ryu, Diane; Agut, Henri; Merkel, Kevin; Leendertz, Fabian H.

2017-01-01

Abstract Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are seen as close relatives but also unambiguously considered as evolutionary independent units. Here, we sequenced the genomes of 18 HSV-2 isolates characterized by divergent UL30 gene sequences to further elucidate the evolutionary history of this virus. Surprisingly, genome-wide recombination analyses showed that all HSV-2 genomes sequenced to date contain HSV-1 fragments. Using phylogenomic analyses, we could also show that two main HSV-2 lineages exist. One lineage is mostly restricted to subSaharan Africa whereas the other has reached a global distribution. Interestingly, only the worldwide lineage is characterized by ancient recombination events with HSV-1. Our findings highlight the complexity of HSV-2 evolution, a virus of putative zoonotic origin which later recombined with its human-adapted relative. They also suggest that coinfections with HSV-1 and 2 may have genomic and potentially functional consequences and should therefore be monitored more closely. PMID:28369565

“Humanized mice for HIV and AIDS research”

PubMed Central

Garcia, J. Victor

2016-01-01

HIV has a very limited species tropism that prevents the use of most conventional small animal models for AIDS research. The in vivo analysis of HIV/AIDS has benefited extensively from novel chimeric animal models that accurately recapitulate key aspects of the human condition. Specifically, immunodeficient mice that are systemically repopulated with human hematolymphoid cells offer a viable alternative for the study of a multitude of highly relevant aspects of HIV replication, pathogenesis, therapy, transmission, prevention, and eradication. This article summarizes some of the multiple contributions that humanized mouse models of HIV infection have made to the field of AIDS research. These models have proven to be highly informative and hold great potential for accelerating multiple aspects of HIV research in the future. PMID:27447446

A reanalysis of the indirect evidence for recombination in human mitochondrial DNA.

PubMed

Piganeau, G; Eyre-Walker, A

2004-04-01

In an attempt to resolve the controversy about whether recombination occurs in human mtDNA, we have analysed three recently published data sets of complete mtDNA sequences along with 10 RFLP data sets. We have analysed the relationship between linkage disequilibrium (LD) and distance between sites under a variety of conditions using two measures of LD, r2 and /D'/. We find that there is a negative correlation between r2 and distance in the majority of data sets, but no overall trend for /D'/. Five out of six mtDNA sequence data sets show an excess of homoplasy, but this could be due to either recombination or hypervariable sites. Two additional recombination detection methods used, Geneconv and Maximum Chi-Square, showed nonsignificant results. The overall significance of these findings is hard to quantify because of nonindependence, but our results suggest a lack of evidence for recombination in human mtDNA.

Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

PubMed

Hanada, Katsuhiro; Yamaoka, Yoshio

2014-10-01

Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows.

PubMed

Wang, J; Yang, P; Tang, B; Sun, X; Zhang, R; Guo, C; Gong, G; Liu, Y; Li, R; Zhang, L; Dai, Y; Li, N

2008-12-01

Improvement of the nutritional value of cow milk with transgenic expression of recombinant human alpha-lactalbumin (alpha-LA) has been previously attempted. However, the detailed characterization of the recombinant protein and analysis of the transgenic milk components are not explored yet. Here, we first report production of healthy transgenic cows by somatic cell nuclear transfer, in which expression of up to 1.55 g/L of recombinant human alpha-LA was achieved. The recombinant human alpha-LA was purified from transgenic milk and displayed physicochemical properties similar to its natural counterpart with respect to molecular weight, structure, and regulatory activity for beta-1,4-galactosyltransferase. Additionally, no N-glycosylation was found in the recombinant human alpha-LA, whereas the endogenous bovine alpha-LA was glycosylated at the unusual site (71)Asn-Ile-(73)Cys. Compared with milk from nontransgenic cows, expression of the transgene did not materially alter milk composition, such as fat and protein content. Our research thus provides scientific evidence supporting the feasibility of humanizing cow milk.

Counseling Gay Men with AIDS: Human Dimensions.

ERIC Educational Resources Information Center

Barret, Robert L.

1989-01-01

Provides case material that demonstrates the emotional response of homosexual men to Acquired Immune Deficiency Syndrome (AIDS). Discusses denial, anger, rage, guilt, and shame experienced by these men with an emphasis on the human dimensions of AIDS. Concludes that most powerful contribution of counselors is their willingness to "be with" persons…

Fall in thyroid stimulating hormone (TSH) may be an early marker of ipilimumab-induced hypophysitis.

PubMed

De Sousa, Sunita M C; Sheriff, Nisa; Tran, Chau H; Menzies, Alexander M; Tsang, Venessa H M; Long, Georgina V; Tonks, Katherine T T

2018-06-01

Hypophysitis develops in up to 19% of melanoma patients treated with ipilimumab, a cytotoxic T-lymphocyte antigen-4 antibody. Early detection may avert life-threatening hypopituitarism. We aimed to assess the incidence of ipilimumab-induced hypophysitis (IH) at a quaternary melanoma referral centre, and to determine whether cortisol or thyroid stimulating hormone (TSH) monitoring could predict IH onset. We performed a retrospective cohort study of ipilimumab-treated patients at a quaternary melanoma referral centre in Australia. The inclusion criteria were patients with metastatic or unresectable melanoma treated with ipilimumab monotherapy, and cortisol and TSH measurements prior to ≥ 2 infusions. The main outcomes were IH incidence and TSH and cortisol patterns in patients who did and did not develop IH. Of 78 ipilimumab-treated patients, 46 met the study criteria and 9/46 (20%) developed IH at a median duration of 13.0 weeks (range 7.7-18.1) following ipilimumab initiation. All patients whose TSH fell ≥ 80% compared to baseline developed IH, and, in 5/9 patients with IH, TSH fell prior to cortisol fall and IH diagnosis. Pre-cycle-4 TSH was significantly lower in those who developed IH (0.31 vs. 1.73 mIU/L, P = 0.006). TSH fall was detected at a median time of 9.2 (range 7.7-16.4) weeks after commencing ipilimumab, and a median of 3.6 (range of - 1.4 to 9.7) weeks before IH diagnosis. There was no difference in TSH between the groups before cycles 1-3 or in cortisol before cycles 1-4. TSH fall ≥ 80% may be an early marker of IH. Serial TSH measurement during ipilimumab therapy may be an inexpensive tool to expedite IH diagnosis.

TSH-induced hyperthyroidism caused by a pituitary tumor.

PubMed

Beck-Peccoz, Paolo; Persani, Luca

2006-09-01

A 45-year-old man presented with frontal headache and visual disturbances to our clinic. For the previous 5 years, he had been receiving treatment for long-lasting mild hyperthyroidism with antithyroid therapy, but therapy had not been carefully followed. During the last 2 years he had also complained of erectile dysfunction and loss of libido. On physical examination, he had a small goiter, normal skin, no Graves' ophthalmopathy, normal BMI, and reduced testis volume and pubic hair. Serum levels of free T3 and T4, serum prolactin, testosterone, serum gonadotropins, insulin-like growth factor 1, adrenocorticotropic hormone, and cortisol were measured. MRI scan, TSH-releasing hormone test, and T3 suppression test were carried out. Levels of pituitary glycoprotein hormone alpha-subunit and sex-hormone-binding protein were also measured. Hyperthyroidism caused by a mixed pituitary adenoma that secretes prolactin and TSH. Trans-sphenoidal resection of the pituitary tumor. After surgery, T3 suppression test failed to completely suppress TSH secretion, which suggested a persistence of residual adenomatous cells. Hyperthyroidism and hypogonadism recurred after 5 years, therefore, treatment with lanreotide was initiated, and resulted in complete resolution of signs and symptoms of the disease.

International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

EPA Science Inventory

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

NASA Technical Reports Server (NTRS)

Lee, Y. C.; Martin, E.; Murad, F.

2000-01-01

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Ancient Recombination Events between Human Herpes Simplex Viruses.

PubMed

Burrel, Sonia; Boutolleau, David; Ryu, Diane; Agut, Henri; Merkel, Kevin; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

2017-07-01

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are seen as close relatives but also unambiguously considered as evolutionary independent units. Here, we sequenced the genomes of 18 HSV-2 isolates characterized by divergent UL30 gene sequences to further elucidate the evolutionary history of this virus. Surprisingly, genome-wide recombination analyses showed that all HSV-2 genomes sequenced to date contain HSV-1 fragments. Using phylogenomic analyses, we could also show that two main HSV-2 lineages exist. One lineage is mostly restricted to subSaharan Africa whereas the other has reached a global distribution. Interestingly, only the worldwide lineage is characterized by ancient recombination events with HSV-1. Our findings highlight the complexity of HSV-2 evolution, a virus of putative zoonotic origin which later recombined with its human-adapted relative. They also suggest that coinfections with HSV-1 and 2 may have genomic and potentially functional consequences and should therefore be monitored more closely. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

PubMed

Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

1991-04-01

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

PubMed

Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

2012-09-01

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

The Development of Novel Recombinant Human Gelatins as Replacements for Animal-Derived Gelatin in Pharmaceutical Applications

NASA Astrophysics Data System (ADS)

Olsen, David; Chang, Robert; Williams, Kim E.; Polarek, James W.

We have developed a recombinant expression system to produce a series of novel recombinant human gelatins that can substitute for animal sourced gelatin preparations currently used in pharmaceutical and nutraceutical applications. This system allows the production of human sequence gelatins, or, if desired, gelatins from any other species depending on the availability of the cloned gene. The gelatins produced with this recombinant system are of defined molecular weight, unlike the animal-sourced gelatins, which consist of numerous polypeptides of varying size. The fermentation and purification process used to prepare these recombinant gelatins does not use any human- or animal-derived components and thus this recombinant material should be free from viruses and agents that cause transmissible spongiform encephalopathies. The recombinant gelatins exhibit lot-to-lot reproducibility and we have performed extensive analytical testing on them. We have demonstrated the utility of these novel gelatins as biological stabilizers and plasma expanders, and we have shown they possess qualities that are important in applications where gel formation is critical. Finally, we provide examples of how our system allows the engineering of these recombinant gelatins to optimize the production process.

Health and human rights in today’s fight against HIV/AIDS

PubMed Central

Stemple, Lara

2012-01-01

The development of the health and human rights framework coincided with the beginning of the rapid spread of HIV/AIDS. Since then, the international community has increasingly turned to human rights language and instruments to address the disease. Not only are human rights essential to addressing a disease that impacts marginalized groups most severely, but the spread of HIV/AIDS itself exacerbates inequality and impedes the realization of a range of human rights. Policy developments of the past decade include the United Nations (UN) Committee on Economic, Social and Cultural Rights’ General Comment on the ‘Right to Health’, the UN Declaration of Commitment on HIV/AIDS, and the UN’s International Guidelines on HIV/AIDS and Human Rights, among others. Rights-related setbacks include the failure of the Declaration and its 5-year follow-up specifically to address men who have sex with men, sex workers, and intravenous drug users, political restrictions placed on urgently needed US President’s Emergency Plan for AIDS Relief (PEPFAR) funds, and the failure of many countries to decriminalize same-sex sex and outlaw discrimination against people living with HIV/AIDS. Male circumcision as an HIV prevention measure is a topic around which important debate, touching on gender, informed consent and children’s rights, serves to illustrate the ongoing vitality of the health and human rights dialogue. Mechanisms to increase state accountability for addressing HIV/AIDS should be explored in greater depth. Such measures might include an increase in the use of treaty-based judicial mechanisms, the linking of human rights compliance with preferential trade agreements, and rights requirements tied to HIV/ AIDS funding. PMID:18641463

Changes in thyroid function in Ethiopian and non-Ethiopian Israeli patients with human immunodeficiency virus infection or acquired immunodeficiency syndrome.

PubMed

Cahn, Avivit; Chairsky-Segal, Irena; Olshtain-Pops, Keren; Maayan, Sholomo; Wolf, Dana; Dresner-Pollak, Rivka

2012-01-01

To investigate whether human immunodeficiency virus (HIV) infection or its treatment is a risk factor for thyroid dysfunction and whether thyroid function changes over time in 2 distinct subpopulations with HIV or acquired immunodeficiency syndrome (AIDS) in Israel: Ethiopian immigrants and Israeli patients. Serum thyroid-stimulating hormone (TSH) and free thyroxine levels were determined in HIV carriers undergoing follow-up at the Hadassah-Hebrew University Medical Center HIV clinic in Jerusalem, Israel, and these thyroid measurements were correlated with clinical and laboratory variables pertaining to their disease, including disease duration, drug therapy, viral load, CD4 count, low-density lipoprotein cholesterol, and creatine kinase. Serum samples stored at -20°C from the time of referral were tested as well. We recruited 121 consecutive patients with HIV or AIDS for this study: 60 Ethiopians and 61 Israeli patients. Of the 121 patients, 4 (3%) had abnormal thyroid function-subclinical hypothyroidism in 2, overt hypothyroidism in 1, and overt hyperthyroidism in 1. Previously stored serum samples were available for 60 of the 121 patients and revealed 2 additional patients with subclinical hypothyroidism, whose TSH has normalized in the subsequent test. Throughout the follow-up period of 3.2 ± 1.9 years, the mean TSH level remained unchanged in the Israeli cohort but significantly declined in the Ethiopian cohort. Thyroid function abnormalities were uncommon in these Israeli patients with HIV or AIDS. This finding does not support the need for routine thyroid function tests in this patient population. The decline in TSH level in the Ethiopian population over time probably represents a shift from an iodine-deficient to an iodine-sufficient country.

Radioiodine remnant ablation in differentiated thyroid cancer after combined endogenous and exogenous TSH stimulation.

PubMed

Vrachimis, A; Schober, O; Riemann, B

2012-01-01

Radioiodine remnant ablation (RRA) after (near-)total thyroidectomy (TE) is a key element in patients with differentiated thyroid cancer (DTC). The use of exogenous TSH stimulation (rhTSH) prior to RRA has shown promising results as compared to conventional thyroid hormone withdrawal (THW). As yet, the efficacy of RRA after brief THW and single rhTSH administration has not been assessed. The study sample comprised 147 patients with DTC referred to our center between May 2008 and September 2010. All patients received TE with subsequent RRA. None of these 147 patients had evidence of distant metastasis. 93 patients had endogenous TSH stimulation 4-5 weeks after surgery (group I) and twenty-six received two rhTSH injections (group II). 28 patients were treated with a single rhTSH injection after a brief THW (group III). RRA-Efficacy was assessed three months after therapy by diagnostic whole-body scan and measurement of the tumour marker thyroglobulin (Tg) under TSH stimulation. Three categories of success were defined for remnant ablation. Based on the definition of successful remnant ablation no visible uptake and a Tg ≤ 2.0 ng/ml (category 1) was seen in 62/93 patients in group I, in 17/26 patients in group II (p = n.s.) and in 12/28 patients in group III (p < 0.05). Visible radioiodine uptake and a Tg ≤ 2.0 ng/ml (category 2) was seen in 16/28 patients of group III and thus significantly more frequent than in group I (28/93 patients) (p < 0.01). However, patients in group III (16/28 patients) and group II (8/26 patients) showed no significant difference in this category (p = n.s.). Visible radioiodine uptake and a Tg > 2.0 ng/ml (category 3) was found in 3/93 patients in group I and 1/26 patients in group II but in no patient in group III. The third strategy of remnant ablation using a single injection of rhTSH after a brief THW period resulted in a significant higher rate of patients with residual uptake in the thyroid bed and a Tg level below 2 ng/ml three

TSH Comparison Between Chemiluminescence (Architect) and Electrochemiluminescence (Cobas) Immunoassays: An Indian Population Perspective.

PubMed

Sarkar, Rajarshi

2014-04-01

Although 3rd generation TSH assays are the most widely used immunoassays, credible comparison studies, specially involving Indian sub-populations are practically non-existent. To compare the TSH measurements between chemiluminescence (Architect) and electrochemiluminescence (Cobas) inmmunoassays in an urban ambulatory Indian population. 1,615 subjects were selected randomly from the usual laboratory workflow, their TSH measured in Architect and Cobas and the paired data thus generated were statistically analysed. TSH values of Cobas were observed to be higher than the Architect values by 28.7 %, with a significant proportional difference between the two, but majority of the Cobas values (above 90 %) were within the limits of agreement with Architect values. In situations where both the instruments are in use simultaneously, a standardization of the methods is imperative, in larger interest of the patient populace.

LDSplitDB: a database for studies of meiotic recombination hotspots in MHC using human genomic data.

PubMed

Guo, Jing; Chen, Hao; Yang, Peng; Lee, Yew Ti; Wu, Min; Przytycka, Teresa M; Kwoh, Chee Keong; Zheng, Jie

2018-04-20

Meiotic recombination happens during the process of meiosis when chromosomes inherited from two parents exchange genetic materials to generate chromosomes in the gamete cells. The recombination events tend to occur in narrow genomic regions called recombination hotspots. Its dysregulation could lead to serious human diseases such as birth defects. Although the regulatory mechanism of recombination events is still unclear, DNA sequence polymorphisms have been found to play crucial roles in the regulation of recombination hotspots. To facilitate the studies of the underlying mechanism, we developed a database named LDSplitDB which provides an integrative and interactive data mining and visualization platform for the genome-wide association studies of recombination hotspots. It contains the pre-computed association maps of the major histocompatibility complex (MHC) region in the 1000 Genomes Project and the HapMap Phase III datasets, and a genome-scale study of the European population from the HapMap Phase II dataset. Besides the recombination profiles, related data of genes, SNPs and different types of epigenetic modifications, which could be associated with meiotic recombination, are provided for comprehensive analysis. To meet the computational requirement of the rapidly increasing population genomics data, we prepared a lookup table of 400 haplotypes for recombination rate estimation using the well-known LDhat algorithm which includes all possible two-locus haplotype configurations. To the best of our knowledge, LDSplitDB is the first large-scale database for the association analysis of human recombination hotspots with DNA sequence polymorphisms. It provides valuable resources for the discovery of the mechanism of meiotic recombination hotspots. The information about MHC in this database could help understand the roles of recombination in human immune system. DATABASE URL: http://histone.scse.ntu.edu.sg/LDSplitDB.

The remarkable frequency of human immunodeficiency virus type 1 genetic recombination.

PubMed

Onafuwa-Nuga, Adewunmi; Telesnitsky, Alice

2009-09-01

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination--a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates--occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

Thyrotropin-induced hyperthyroidism caused by selective pituitary resistance to thyroid hormone. A new syndrome of "inappropriate secretion of TSH".

PubMed Central

Gershengorn, M C; Weintraub, B D

1975-01-01

An 18-yr-old woman with clinical and laboratory features of hyperthyroidism had persistently elevated serum levels of immunoreative thyrotropin (TSH). During 11 yr of follow-up there had been no evidence of a pituitary tumor. After thyrotropin-releasing hormone (TRH), there was a marked increase in TSH and secondarily in triiodothyronine (T3), the latter observation confirming the biologic activity of the TSH. Exogenous T3 raised serum T3 and several measurements of peripheral thyroid hormone effect, while decreasing serum TSH, thyroxine (T4), and thyroidal radioiodine uptake. After T3, the TRH-stimulated TSH response was decreased but was still inappropriate for the elevated serum T3 levels. Dexamethasone reduced serum TSH but did not inhibit TRH stimulation of TSH. Propylthiouracil reduced serum T4 and T3 and raised TSH. This patient represents a new syndrome of TSH-induced hyperthyroidism, differing from previous reports in the absence of an obvious pituitary tumor and in the responsiveness of the TSH to TRH stimulation and thyroid hormone suppression. This syndrome appears to be caused by a selective, partial resistance of the pituitary to the action of thyroid hormone. This case is also compared with previous reports in the literature of patients with elevated serum levels of immunoreactive TSH in the presence of elevated total and free thyroid hormones. A classification of these cases, termed "inappropriate secretion of TSH," is proposed. PMID:1159077

The production of recombinant human erythropoietin.

PubMed

Inoue, N; Takeuchi, M; Ohashi, H; Suzuki, T

1995-01-01

Erythropoietin (EPO) is the glycoprotein hormone that promotes differentiation of erythroid progenitor cells in bone marrow. The normal kidney produces EPO to maintain erythrocyte for oxygen supply. This hormone activity was found in the serum of anemic animals in the 1890s. Renal failure results in severe anemia because of reduced EPO production, therefore anemia patients expected EPO treatment for long time. However, this was difficult due to the limited amount of EPO. Many researchers have tried to isolate EPO since the 1950s. Finally Miyake and Goldwasser purified highly active EPO from the urine of aplastic anemia patients. Since then, the characteristics and structural information from the purified material accelerated the cloning of the EPO gene. Mammalian cells were essential to produce EPO, because EPO contains 40% carbohydrate that plays some important roles in its activity, stability and biosynthesis. In 1984, two groups succeeded in cloning the EPO gene and expressing this gene in mammalian cells. Recombinant human EPO is currently available for anemia treatment. In this paper, we review production in mammalian cells, molecular characterization, especially carbohydrate moieties, and clinical applications of recombinant EPO.

Production of biologically active recombinant human factor H in Physcomitrella.

PubMed

Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

2011-04-01

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Short-term effects of recombinant human growth hormone and feeding on gluconeogenesis in humans

USDA-ARS?s Scientific Manuscript database

After a short-term fast, lactating women have increased rates of glucose production but not gluconeogenesis (GNG) despite relative hypoinsulinemia. We explored the effects of non-insulin-dependent increase in glucose utilization and recombinant human growth hormone (rhGH) on glucose production, glyc...

A novel multi-epitope recombined protein for diagnosis of human brucellosis.

PubMed

Yin, Dehui; Li, Li; Song, Xiuling; Li, Han; Wang, Juan; Ju, Wen; Qu, Xiaofeng; Song, Dandan; Liu, Yushen; Meng, Xiangjun; Cao, Hongqian; Song, Weiyi; Meng, Rizeng; Liu, Jinhua; Li, Juan; Xu, Kun

2016-05-21

In epidemic regions of the world, brucellosis is a reemerging zoonosis with minimal mortality but is a serious public hygiene problem. Currently, there are various methods for brucellosis diagnosis, however few of them are available to be used to diagnose, especially for serious cross-reaction with other bacteria. To overcome this disadvantage, we explored a novel multi-epitope recombinant protein as human brucellosis diagnostic antigen. We established an indirect enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein. 248 sera obtained from three different groups including patients with brucellosis (146 samples), non-brucellosis patients (82 samples), and healthy individuals (20 samples) were tested by indirect ELISA. To evaluate the assay, a receiver-operating characteristic (ROC) analysis and immunoblotting were carried out using these characterized serum samples. For this test, the area under the ROC curve was 0.9409 (95 % confidence interval, 0.9108 to 0.9709), and a sensitivity of 88.89 % and a specificity of 85.54 % was given with a cutoff value of 0.3865 from this ROC analysis. The Western blot results indicate that it is feasible to differentiate human brucellosis and non-brucellosis with the newly established method based on this recombinant protein. Our results obtained high diagnostic accuracy of the ELISA assay which encourage the use of this novel recombinant protein as diagnostic antigen to implement serological diagnosis of brucellosis.

25-Hydroxyvitamin D and TSH as Risk Factors or Prognostic Markers in Thyroid Carcinoma

PubMed Central

Danilovic, Debora Lucia Seguro; Ferraz-de-Souza, Bruno; Fabri, Amanda Wictky; Santana, Nathalie Oliveira; Kulcsar, Marco Aurelio; Cernea, Claudio Roberto; Marui, Suemi; Hoff, Ana Oliveira

2016-01-01

Objective The increasing incidence of thyroid nodules demands identification of risk factors for malignant disease. Several studies suggested the association of higher TSH levels with cancer, but influence of 25-hydroxyvitamin D (25OHD) is controversial. This study aimed to identify the relationship of thyroid cancer with higher TSH levels and hypovitaminosis D and to evaluate their influence on prognostic characteristics of papillary thyroid carcinomas (PTC). Methods We retrospectively evaluated 433 patients submitted to thyroidectomy for thyroid nodules. Patients were categorized according to quartiles of TSH and 25OHD levels. Clinicopathological features were analyzed. Results Subjects with thyroid carcinomas were more frequently male and younger compared to those with benign disease. Their median TSH levels were higher and adjusted odds-ratio (OR) for cancer in the highest-quartile of TSH (> 2.4 mUI/mL) was 2.36 (1.36–4.09). Although vitamin D deficiency/insufficiency was prevalent in our cohort (84%), no significant differences in 25OHD levels or quartile distribution were observed between benign and malignant cases. Among 187 patients with PTC, analyses of prognostic features revealed increased risk of lymph nodes metastases for subjects with highest-quartile TSH levels (OR = 3.7, p = 0.029). Decreased 25OHD levels were not overtly associated with poor prognosis in PTC. Conclusions In this cross-sectional cohort, higher TSH levels increased the risk of cancer in thyroid nodules and influenced its prognosis, particularly favoring lymph nodes metastases. On the other hand, no association was found between 25OHD levels and thyroid carcinoma risk or prognosis, suggesting that serum 25OHD determination may not contribute to risk assessment workup of thyroid nodules. PMID:27737011

Recombinant human leptin in women with hypothalamic amenorrhea.

PubMed

Welt, Corrine K; Chan, Jean L; Bullen, John; Murphy, Robyn; Smith, Patricia; DePaoli, Alex M; Karalis, Aspasia; Mantzoros, Christos S

2004-09-02

Disruptions in hypothalamic-gonadal and other endocrine axes due to energy deficits are associated with low levels of the adipocyte-secreted hormone leptin and may result in hypothalamic amenorrhea. We hypothesized that exogenous recombinant leptin replacement would improve reproductive and neuroendocrine function in women with hypothalamic amenorrhea. Eight women with hypothalamic amenorrhea due to strenuous exercise or low weight were studied for one month before receiving recombinant human leptin and then while receiving treatment for up to three months. Six control subjects with hypothalamic amenorrhea received no treatment and were studied for a mean (+/-SD) of 8.5+/-8.1 months. Luteinizing hormone (LH) pulsatility, body weight, ovarian variables, and hormone levels did not change significantly over time in the controls and during a one-month control period before recombinant leptin therapy in the treated subjects. In contrast, recombinant leptin treatment increased mean LH levels and LH pulse frequency after two weeks and increased maximal follicular diameter, the number of dominant follicles, ovarian volume, and estradiol levels over a period of three months. Three patients had an ovulatory menstrual cycle (P<0.05 for the comparison with an expected rate of spontaneous ovulation of 10 percent); two others had preovulatory follicular development and withdrawal bleeding during treatment (P<0.05). Recombinant leptin significantly increased levels of free triiodothyronine, free thyroxine, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, bone alkaline phosphatase, and osteocalcin but not cortisol, corticotropin, or urinary N-telopeptide. Leptin administration for the relative leptin deficiency in women with hypothalamic amenorrhea appears to improve reproductive, thyroid, and growth hormone axes and markers of bone formation, suggesting that leptin, a peripheral signal reflecting the adequacy of energy stores, is required for normal

AIDS and human rights.

PubMed

Tarantola, D; Mann, J

1995-01-01

HIV/AIDS is a health problem that is inseparable from individual and collective behavior and social forces, particularly linked with societal respect for human rights and dignity. In its second decade, the HIV/AIDS pandemic continues to thrive. Where organized communities have access to adequate information, education, and services, the incidence of infection has begun to decline. Elsewhere, HIV continues to reach new populations and new geographic areas. Lessons learned in more than a decade of prevention work point to new directions for expanding national responses, at a time when the UNAIDS program, to be launched in January 1996, offers opportunities for innovative, broad-based, coordinated, and expanded global action. Prevention activities have shown that the spread of HIV can be effectively reduced. Public health interventions, including providing information and applying prevention methods, reduce the probability of infection, the risk of transmission, and the chances of not accessing appropriate care or support once infection has set in. These are proximal interventions that yield the short-term benefits of the decline of incidence and improved quality and duration of life for those infected. Societal vulnerability translates today into the focus the pandemic has on individuals, communities, and nations that are disadvantaged, marginalized, or discriminated against for reasons of gender, age, race, sexual orientation, economic status, or cultural, religious, or political affiliation. A fully expanded response to HIV/AIDS requires a combination of risk-reduction (proximal) and contextual interventions--those directed at reducing vulnerability through social change to enable people to exert control over their own health. Contextual actions can be implemented in the short term (changing laws, policies, practices that discriminate, promoting human rights, developing the most vulnerable communities) and in the long term (cultural changes, gender equality in

Recombinant human prion protein inhibits prion propagation in vitro.

PubMed

Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

2013-10-09

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

PubMed

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Maternal TSH level and TPOAb status in early pregnancy and their relationship to the risk of gestational diabetes mellitus.

PubMed

Ying, Hao; Tang, Yu-Ping; Bao, Yi-Rong; Su, Xiu-Juan; Cai, XueYa; Li, Yu-Hong; Wang, De-Fen

2016-12-01

Subclinical hypothyroidism is common in pregnant women and often related to adverse pregnancy outcomes, but its relationship with gestational diabetes remains controversial. In particular, the impact of thyroperoxidase antibodies status on the relationship between subclinical hypothyroidism and gestational diabetes is not clear. We investigated the association between combined thyroid stimulating hormone (TSH) level and thyroperoxidase antibodies status in early pregnancy (<20 weeks of gestation) and gestational diabetes mellitus. A total of 7084 pregnant women met the inclusion criteria, which included thyroperoxidase antibodies-positive subclinical hypothyroidism [TSH(H)TPOAb(+)] (n = 78), thyroperoxidase antibodies-negative subclinical hypothyroidism [TSH(H)TPOAb(-)] (n = 281), thyroperoxidase antibodies-positive euthyroidism [TSH(N)TPOAb(+)] (n = 648), and thyroperoxidase antibodies-negative euthyroidism [TSH(N)TPOAb(-)] (n = 6077). Of the 7084 cases included in our study, 1141 cases were diagnosed with gestational diabetes mellitus at 24-28 weeks of pregnancy. The prevalence of gestational diabetes mellitus in TSH(N)TPOAb(-), TSH(H)TPOAb(-), TSH(N)TPOAb(+), and TSH(H)TPOAb(+) was 14.65, 19.57, 24.85, and 46.15 %, respectively. Compared with TSH(N)TPOAb(-) women, the risk of gestational diabetes mellitus was increased in all other groups of women in early pregnancy. After dividing early pregnancy into first and second trimesters, we found that TSH(H)TPOAb(-) women in the first trimester do not show this increase. Our study suggests that subclinical hypothyroidism and thyroperoxidase antibodies-positive euthyroidism in early pregnancy are associated with an increased risk of gestational diabetes mellitus.

Production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing 10S-dioxygenase from Nostoc punctiforme PCC 73102 with the aid of a chaperone.

PubMed

Kim, Min-Ji; Seo, Min-Ju; Shin, Kyung-Chul; Oh, Deok-Kun

2017-01-01

To increase the production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing Nostoc punctiforme 10S-dioxygenase with the aid of a chaperone. The optimal conditions for 10S-hydroxy-8(E)-octadecenoic acid production by recombinant cells co-expressing chaperone plasmid were pH 9, 35 °C, 15 % (v/v) dimethyl sulfoxide, 40 g cells l -1 , and 10 g oleic acid l -1 . Under these conditions, recombinant cells co-expressing chaperone plasmid produced 7.2 g 10S-hydroxy-8(E)-octadecenoic acid l -1 within 30 min, with a conversion yield of 72 % (w/w) and a volumetric productivity of 14.4 g l -1 h -1 . The activity of recombinant cells expressing 10S-dioxygenase was increased by 200 % with the aid of a chaperone, demonstrating the first biotechnological production of 10S-hydroxy-8(E)-octadecenoic acid using recombinant cells expressing 10S-dioxygenase.

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

PubMed

Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

2011-05-01

OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

TSH and Thyrotropic Agonists: Key Actors in Thyroid Homeostasis

PubMed Central

Dietrich, Johannes W.; Landgrafe, Gabi; Fotiadou, Elisavet H.

2012-01-01

This paper provides the reader with an overview of our current knowledge of hypothalamic-pituitary-thyroid feedback from a cybernetic standpoint. Over the past decades we have gained a plethora of information from biochemical, clinical, and epidemiological investigation, especially on the role of TSH and other thyrotropic agonists as critical components of this complex relationship. Integrating these data into a systems perspective delivers new insights into static and dynamic behaviour of thyroid homeostasis. Explicit usage of this information with mathematical methods promises to deliver a better understanding of thyrotropic feedback control and new options for personalised diagnosis of thyroid dysfunction and targeted therapy, also by permitting a new perspective on the conundrum of the TSH reference range. PMID:23365787

Thyroid storm induced by TSH-secreting pituitary adenoma: a case report.

PubMed

Fujio, Shingo; Ashari; Habu, Mika; Yamahata, Hitoshi; Moinuddin, F M; Bohara, Manoj; Arimura, Hiroshi; Nishijima, Yui; Arita, Kazunori

2014-01-01

Thyroid stimulating hormone-secreting pituitary adenomas (TSHomas) are uncommon tumors of the anterior pituitary gland. Patients with TSHomas may present with hyperthyroidism, but the incidence of thyroid storm due to TSHomas has yet to be determined. We report a rare case of thyroid storm caused by TSHoma in a 54-year-old woman. Preoperatively she had symptoms of excessive sweating and palpitation. Blood tests showed inappropriate secretion of TSH with blood TSH 6.86 μ U/mL, fT3 19.8 pg/mL, and fT4 5.95 ng/dL. Magnetic resonance imaging (MRI) revealed a pituitary tumor with maximum diameter of 13 mm that was extirpated through transsphenoidal route. After operation the patient was stuporous and thyroid storm occurred presenting with hyperthermia, hypertension, and tachycardia. It was well managed with nicardipine, midazolam, steroids, and potassium iodide. Immunohistochemical staining of tumor specimen was positive for TSH and growth hormone (GH). One year after operation, fT3 and fT4 levels were still high. As her tumor was diagnosed to be GH- and TSH-producing adenoma, octreotide injection therapy was started, which normalized thyroid hormone levels. This is the second reported case with thyroid storm due to TSHoma and emphasizes the importance of strategies with interdisciplinary cooperation for prevention of such emergency conditions.

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

PubMed Central

Foster, Barbara A.; Gangavarapu, Kalyan J.; Mathew, Grinu; Azabdaftari, Gissou; Morrison, Carl D.; Miller, Austin; Huss, Wendy J.

2013-01-01

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population. PMID:23383057

Mortality Benefit of Recombinant Human Interleukin-1 Receptor Antagonist for Sepsis Varies by Initial Interleukin-1 Receptor Antagonist Plasma Concentration.

PubMed

Meyer, Nuala J; Reilly, John P; Anderson, Brian J; Palakshappa, Jessica A; Jones, Tiffanie K; Dunn, Thomas G; Shashaty, Michael G S; Feng, Rui; Christie, Jason D; Opal, Steven M

2018-01-01

Plasma interleukin-1 beta may influence sepsis mortality, yet recombinant human interleukin-1 receptor antagonist did not reduce mortality in randomized trials. We tested for heterogeneity in the treatment effect of recombinant human interleukin-1 receptor antagonist by baseline plasma interleukin-1 beta or interleukin-1 receptor antagonist concentration. Retrospective subgroup analysis of randomized controlled trial. Multicenter North American and European clinical trial. Five hundred twenty-nine subjects with sepsis and hypotension or hypoperfusion, representing 59% of the original trial population. Random assignment of placebo or recombinant human interleukin-1 receptor antagonist × 72 hours. We measured prerandomization plasma interleukin-1 beta and interleukin-1 receptor antagonist and tested for statistical interaction between recombinant human interleukin-1 receptor antagonist treatment and baseline plasma interleukin-1 receptor antagonist or interleukin-1 beta concentration on 28-day mortality. There was significant heterogeneity in the effect of recombinant human interleukin-1 receptor antagonist treatment by plasma interleukin-1 receptor antagonist concentration whether plasma interleukin-1 receptor antagonist was divided into deciles (interaction p = 0.046) or dichotomized (interaction p = 0.028). Interaction remained present across different predicted mortality levels. Among subjects with baseline plasma interleukin-1 receptor antagonist above 2,071 pg/mL (n = 283), recombinant human interleukin-1 receptor antagonist therapy reduced adjusted mortality from 45.4% to 34.3% (adjusted risk difference, -0.12; 95% CI, -0.23 to -0.01), p = 0.044. Mortality in subjects with plasma interleukin-1 receptor antagonist below 2,071 pg/mL was not reduced by recombinant human interleukin-1 receptor antagonist (adjusted risk difference, +0.07; 95% CI, -0.04 to +0.17), p = 0.230. Interaction between plasma interleukin-1 beta concentration and recombinant human

The impact of HIV/AIDS on human development in African countries.

PubMed

Boutayeb, Abdesslam

2009-11-18

In the present paper, we consider the impact of HIV/AIDS on human development in African countries, showing that, beyond health issues, this disease should and must be seen as a global development concern, affecting all components of human development. Consequently, we stress the necessity of multidisciplinary approaches that model, estimate and predict the real impact of HIV/AIDS on human development of African countries in order to optimise the strategies proposed by national countries, international institutions and their partners. In our search strategy, we relied on secondary information, mainly through National Human Development Reports of some African countries and regular publications released by the United Nations (UN), United Nations Development Programme (UNDP), World Health Organization (WHO) and the World Bank. We restricted ourselves to reports dealing explicitly with the impact of HIV/AIDS on human development in African countries. HIV/AIDS is affecting the global human development of African countries through its devastating impact on health and demographic indicators such as life expectancy at birth, healthcare assistance, age and sex distribution, economic indicators like income, work force, and economic growth, education and knowledge acquisition and other indicators like governance, gender inequality and human rights. On the basis of the national reports reviewed, it appears clearly that HIV/AIDS is no longer a crisis only for the healthcare sector, but presents a challenge to all sectors. Consequently, HIV/AIDS is a development question and should be viewed as such. The disease is impeding development by imposing a steady decline in the key indicators of human development and hence reversing the social and economic gains that African countries are striving to attain. Being at the same time a cause and consequence of poverty and underdevelopment, it constitutes a challenge to human security and human development by diminishing the chances of

Image analysis for TSH mRNA in situ hybridization in pituitary glands from rats with thyroid follicular cell hypertrophy after treatment with three different test compounds.

PubMed

Funk, Juergen; Ebeling, Martin; Singer, Thomas; Landes, Christian

2017-10-01

The goal of this in situ hybridization and image analysis technique is to study the effects of new pharmacological/chemical entities on the thyroid and pituitary gland in rats, reveal the pathogenesis of thyroid follicular cell hypertrophy and to retrospectively exclude the risk of thyroid tumor development in humans. In the present study, we describe the increase of thyroid-stimulating hormone- (TSH-) beta subunit mRNA in the pars distalis of the pituitary gland and the quantitative measurement of TSH mRNA positive cells from rats of three 4-week toxicity studies treated with three different test compounds inducing thyroid follicular cell and hepatocellular hypertrophy in rats. Compared to immunohistochemistry (IHC), in situ hybridization (ISH) for TSH was found to be more sensitive. With this technique we are able to exclude a direct effect of the test compound on the thyroid gland by showing the activation of thyrotrope cells from the pituitary gland and therefore this technique retrospectively enables us to exclude a possible risk for humans at an early stage of drug development. Also in case blood serum samples for evaluation of TSH are not available anymore or hepatocellular hypertrophy is not present (close metabolic relationship between thyroid gland and liver in rodents), the described method allows retrospective investigations on thyroid follicular cell hypertrophy or hyperplasia. This can be of high relevance in human safety assessment for certain drugs in order to exclude a primary effect on the thyroid gland especially when it comes to thyroid neoplasia in rodents as previously described. Copyright © 2017 Elsevier Ltd. All rights reserved.

Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology.

PubMed

Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

2017-02-01

Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse -1 . At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 μL) -1 ·mouse -1 ) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

Animal models for HIV/AIDS research

PubMed Central

Hatziioannou, Theodora; Evans, David T.

2015-01-01

The AIDS pandemic continues to present us with unique scientific and public health challenges. Although the development of effective antiretroviral therapy has been a major triumph, the emergence of drug resistance requires active management of treatment regimens and the continued development of new antiretroviral drugs. Moreover, despite nearly 30 years of intensive investigation, we still lack the basic scientific knowledge necessary to produce a safe and effective vaccine against HIV-1. Animal models offer obvious advantages in the study of HIV/AIDS, allowing for a more invasive investigation of the disease and for preclinical testing of drugs and vaccines. Advances in humanized mouse models, non-human primate immunogenetics and recombinant challenge viruses have greatly increased the number and sophistication of available mouse and simian models. Understanding the advantages and limitations of each of these models is essential for the design of animal studies to guide the development of vaccines and antiretroviral therapies for the prevention and treatment of HIV-1 infection. PMID:23154262

Radioactive body burden measurements in (131)iodine therapy for differentiated thyroid cancer: effect of recombinant thyroid stimulating hormone in whole body (131)iodine clearance.

PubMed

Ravichandran, Ramamoorthy; Al Saadi, Amal; Al Balushi, Naima

2014-01-01

Protocols in the management of differentiated thyroid cancer, recommend adequate thyroid stimulating hormone (TSH) stimulation for radioactive (131)I administrations, both for imaging and subsequent ablations. Commonly followed method is to achieve this by endogenous TSH stimulation by withdrawal of thyroxine. Numerous studies worldwide have reported comparable results with recombinant human thyroid stimulating hormone (rhTSH) intervention as conventional thyroxine hormone withdrawal. Radiation safety applications call for the need to understand radioactive (131)I (RA(131)I) clearance pattern to estimate whole body doses when this new methodology is used in our institution. A study of radiation body burden estimation was undertaken in two groups of patients treated with RA(131)I; (a) one group of patients having thyroxine medication suspended for 5 weeks prior to therapy and (b) in the other group retaining thyroxine support with two rhTSH injections prior to therapy with RA(131)I. Sequential exposure rates at 1 m in the air were measured in these patients using a digital auto-ranging beta gamma survey instrument calibrated for measurement of exposure rates. The mean measured exposure rates at 1 m in μSv/h immediately after administration and at 24 h intervals until 3 days are used for calculating of effective ½ time of clearance of administered activity in both groups of patients, 81 patients in conventionally treated group (stop thyroxine) and 22 patients with rhTSH administration. The (131)I activities ranged from 2.6 to 7.9 GBq. The mean administered (131)I activities were 4.24 ± 0.95 GBq (n = 81) in "stop hormone" group and 5.11 ± 1.40 GBq (n = 22) in rhTSH group. The fall of radioactive body burden showed two clearance patterns within observed 72 h. Calculated T½eff values were 16.45 h (stop hormone group) 12.35 h (rhTSH group) for elapsed period of 48 h. Beyond 48 h post administration, clearance of RA(131)I takes place with T½eff> 20 h in both groups

Recombinant Human Insulin in Global Diabetes Management – Focus on Clinical Efficacy

PubMed Central

Mbanya, Jean Claude; Sandow, Juergen; Landgraf, Wolfgang

2017-01-01

Abstract Biosynthetic human insulin and insulin analogues are the mainstay of insulin therapy for both type 1 and type 2 diabetes although access to human insulin at affordable prices remains a global issue. The world is experiencing an exponential rise in the prevalence of diabetes presenting an urgent need to establish effective diabetes therapy in countries burdened by inadequate health care budgets, malnutrition and infectious diseases. Recombinant human insulin has replaced animal insulins and animal-based semisynthetic human insulin thereby available in sufficient quantities and at affordable prices able to provide global access to insulin therapy. In many patients, analog insulins can offer additional clinical benefit, although at a considerably higher price thus severely restricting availability in low income countries. The approval process for recombinant human insulins (i.e. biosimilars) and analogue insulins is highly variable in the developing countries in contrast to Europe and in North America, where it is well established within a strict regulatory framework. This review aims to discuss the future access to human insulin therapy in a global context with an ever increasing burden of diabetes and significant economic implications. PMID:29632602

Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

PubMed

Grubisha, Olivera; Mogg, Adrian J; Sorge, Jessica L; Ball, Laura-Jayne; Sanger, Helen; Ruble, Cara L A; Folly, Elizabeth A; Ursu, Daniel; Broad, Lisa M

2014-05-01

Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies. © 2013 The British Pharmacological Society.

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

PubMed Central

Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

2016-01-01

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

PubMed

Farahmand, Mahin; Nahrevanian, Hossein

2016-07-01

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

[Low levels of TSH measured by a sensitive assay: do they reflect hyperthyroidism? A critical analysis of 580 cases].

PubMed

Rohmer, V; Ligeard-Ducoroy, A; Perdrisot, R; Beldent, V; Jallet, P; Bigorgne, J C

1990-05-12

Highly sensitive TSH assays make it easier to diagnose thyroid diseases. During one year, we performed 5,300 sensitive TSH assays (normal range: 0.15-4 mU/l) in various patients. The purpose of this work was to test the value of the low TSH plasma concentrations found in 580 patients. In 99.7 percent of the cases, low TSH levels were the consequence of a thyroid disorder or a treatment by thyroid hormones; non thyroidal illnesses were detected in only 0.3 percent. However, not all TSH values below 0.15 mU/l were associated with overt or occult thyrotoxicosis. When TSH was undetectable (less than 0.04 mU/l), and excluding thyroid hormone-treated patients, thyrotoxicosis was present in 97 percent of the cases. On the other hand, when TSH values were between 0.04 and 0.15 mU/l, 41 percent of the patients failed to show any sign or symptom of hyperthyroidism, although they had functioning thyroid nodules, multinodular goitre or iodine overload, or they received thyroid hormones.

Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine.

PubMed

Berg, Michael G; Adams, Robert J; Gambhira, Ratish; Siracusa, Mark C; Scott, Alan L; Roden, Richard B S; Ketner, Gary

2014-09-01

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

TSH Receptor Signaling Abrogation by a Novel Small Molecule

PubMed Central

Latif, Rauf; Realubit, Ronald B.; Karan, Charles; Mezei, Mihaly; Davies, Terry F.

2016-01-01

Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves’ disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3–0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin – a post receptor activator of adenylyl cyclase – confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has

An FSH and TSH pituitary adenoma, presenting with precocious puberty and central hyperthyroidism

PubMed Central

Vargas, Guadalupe; Balcazar-Hernandez, Lourdes-Josefina; Melgar, Virgilio; Magriña-Mercado, Roser-Montserrat; Gonzalez, Baldomero; Baquera, Javier

2017-01-01

A 19-year-old woman with a history of isosexual precocious puberty and bilateral oophorectomy at age 10 years because of giant ovarian cysts, presents with headaches and mild symptoms and signs of hyperthyroidism. Hormonal evaluation revealed elevated FSH and LH levels in the postmenopausal range and free hyperthyroxinemia with an inappropriately normal TSH. Pituitary MRI showed a 2-cm macroadenoma with suprasellar extension. She underwent successful surgical resection of the pituitary tumor, which proved to be composed of two distinct populations of cells, each of them strongly immunoreactive for FSH and TSH, respectively. This mixed adenoma resulted in two different hormonal hypersecretion syndromes: the first one during childhood and consisting of central precocious puberty and ovarian hyperstimulation due to the excessive secretion of biologically active FSH and which was not investigated in detail and 10 years later, central hyperthyroidism due to inappropriate secretion of biologically active TSH. Although infrequent, two cases of isosexual central precocious puberty in girls due to biologically active FSH secreted by a pituitary adenoma have been previously reported in the literature. However, this is the first reported case of a mixed adenoma capable of secreting both, biologically active FSH and TSH. Learning points: Although functioning gonadotrophinomas are infrequent, they should be included in the differential diagnosis of isosexual central precocious puberty. Some functioning gonadotrophinomas are mixed adenomas, secreting other biologically active hormones besides FSH, such as TSH. Early recognition and appropriate treatment of these tumors by transsphenoidal surgery is crucial in order to avoid unnecessary therapeutic interventions that may irreversibly compromise gonadal function. PMID:28721217

An FSH and TSH pituitary adenoma, presenting with precocious puberty and central hyperthyroidism.

PubMed

Vargas, Guadalupe; Balcazar-Hernandez, Lourdes-Josefina; Melgar, Virgilio; Magriña-Mercado, Roser-Montserrat; Gonzalez, Baldomero; Baquera, Javier; Mercado, Moisés

2017-01-01

A 19-year-old woman with a history of isosexual precocious puberty and bilateral oophorectomy at age 10 years because of giant ovarian cysts, presents with headaches and mild symptoms and signs of hyperthyroidism. Hormonal evaluation revealed elevated FSH and LH levels in the postmenopausal range and free hyperthyroxinemia with an inappropriately normal TSH. Pituitary MRI showed a 2-cm macroadenoma with suprasellar extension. She underwent successful surgical resection of the pituitary tumor, which proved to be composed of two distinct populations of cells, each of them strongly immunoreactive for FSH and TSH, respectively. This mixed adenoma resulted in two different hormonal hypersecretion syndromes: the first one during childhood and consisting of central precocious puberty and ovarian hyperstimulation due to the excessive secretion of biologically active FSH and which was not investigated in detail and 10 years later, central hyperthyroidism due to inappropriate secretion of biologically active TSH. Although infrequent, two cases of isosexual central precocious puberty in girls due to biologically active FSH secreted by a pituitary adenoma have been previously reported in the literature. However, this is the first reported case of a mixed adenoma capable of secreting both, biologically active FSH and TSH. Although functioning gonadotrophinomas are infrequent, they should be included in the differential diagnosis of isosexual central precocious puberty.Some functioning gonadotrophinomas are mixed adenomas, secreting other biologically active hormones besides FSH, such as TSH.Early recognition and appropriate treatment of these tumors by transsphenoidal surgery is crucial in order to avoid unnecessary therapeutic interventions that may irreversibly compromise gonadal function.

Human Rights in the Fourth Decade of the HIV/AIDS Response

PubMed Central

Piot, Peter

2017-01-01

Abstract More than 35 years since the HIV/AIDS pandemic began, HIV continues to cause almost two million new infections each year, and the “end of AIDS” by 2030 remains elusive.1 Violations of human rights continue to fuel high rates of new infections among key populations and a generalized epidemic in much of sub-Saharan Africa. Meanwhile, as political shifts worldwide threaten not only HIV funding but also progress toward the globalization of human rights, civil society mobilization and advocacy founded firmly on human rights principles have a more vital role to play than ever. Encouragingly, there are numerous examples of successful integration of human rights-based approaches into HIV prevention and treatment initiatives, and evidence increasingly demonstrates that norms enshrining the respect, protection, and fulfillment of human rights can translate into improved public health.2 This essay will succinctly trace the historic emergence of human rights as an issue at the heart of the HIV/AIDS response; it will then provide examples of progress and setbacks in recent years and consider the potential for rights promotion to address the structural drivers of HIV. Finally, it will consider how the primacy of human rights in HIV/AIDS has affected other fields of global health and will highlight the continuing imperative to work with civil society to protect and promote human rights to reduce the burden of HIV/AIDS. PMID:29302169

Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells.

PubMed

Marshall, G S; Fenger, D P; Stout, G G; Knights, M E; Hunt, L A

1996-07-01

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors.

PubMed

Geithe, Christiane; Andersen, Gaby; Malki, Agne; Krautwurst, Dietmar

2015-11-04

With ∼400 olfactory G protein-coupled receptors (GPCR), humans sensitively perceive ∼230 key aroma compounds as best natural agonists of ∼10000 food volatiles. An understanding of odorant coding, thus, critically depends on the knowledge about interactions of key food aroma chemicals and their mixtures with their cognate receptors. Genetically designed test cell systems enable the screening, deorphaning, and characterization of single odorant receptors (OR). This study shows for the food aroma-specific and quantitative butter aroma recombinate, and its single components, specific in vitro class-I OR activity patterns, as well as the activation of selected OR in a concentration-dependent manner. Recently, chemosensory receptors, especially class-I OR, were demonstrated to be expressed on blood leukocytes, which may encounter foodborne aroma compounds postprandially. This study shows that butter aroma recombinate induced chemotaxis of isolated human neutrophils in a defined gradient, and in a concentration-dependent and pertussis toxin-sensitive manner, suggesting at least a GPCR-mediated activation of blood leukocytes by key food odorants.

Human rights and the requirement for international medical aid.

PubMed

Tolchin, Benjamin

2008-08-01

Every year approximately 18 million people die prematurely from treatable medical conditions including infectious diseases and nutritional deficiencies. The deaths occur primarily amongst the poorest citizens of poor developing nations. Various groups and individuals have advanced plans for major international medical aid to avert many of these unnecessary deaths. For example, the World Health Organization's Commission on Macroeconomics and Health estimated that eight million premature deaths could be prevented annually by interventions costing roughly US$57 bn per year. This essay advances an argument that human rights require high-income nations to provide such aid. The essay briefly examines John Rawls' obligations of justice and the reasons that their applicability to cases of international medical aid remains controversial. Regardless, the essay argues that purely humanitarian obligations bind the governments and citizens of high-income liberal democracies at a minimum to provide major medical aid to avert premature deaths in poor nations. In refusing to undertake such medical relief efforts, developed nations fail to adequately protect a fundamental human right to life.

Effects of recombinant human growth hormone and nandrolone phenylpropionate on the healing of ischemic colon anastomosis in rats.

PubMed

Yarimkaya, Ali; Apaydin, Berat; Unal, Ethem; Karabicak, Ilhan; Aydogan, Fatih; Uslu, Ezel; Erginoz, Ethem; Artis, Tarik; Eyuboglu, Erhun

2003-12-01

Recombinant human growth hormone and nandrolone phenylpropionate are two different anabolic agents. This study was designed to investigate the effects of these anabolic agents on the healing of ischemic colon anastomosis in rats. Seventy adult male Wistar rats were divided into five groups (n = 14). Group I was the sham laparotomy group. In the other groups, surgical procedures consisting of transsection and anastomosis were made at a distance 3 cm from the peritoneal reflection. Group II was the nonischemic control group. Ischemic colon model was produced in the remaining groups. Group III was the untreated control group. Groups IV and V received recombinant human growth hormone and nandrolone phenylpropionate, respectively. Bursting pressure and hydroxyproline levels were measured on the third and seventh postoperative days to evaluate anastomotic healing. Recombinant human growth hormone increased both collagen deposition and bursting pressure significantly at postoperative Days 3 and 7 compared with the sham and untreated control groups (P < 0.005). When compared with the untreated control, nandrolone phenylpropionate significantly increased collagen deposition at postoperative Days 3 and 7 (P < 0.005) and bursting pressure only at postoperative Day 3 (P < 0.005). Recombinant human growth hormone has more favorable therapeutic effects on the healing of ischemic colonic anastomoses than nandrolone phenylpropionate. Recombinant human growth hormone also improves healing of nonischemic colonic anastomosis.

Human factors of intelligent computer aided display design

NASA Technical Reports Server (NTRS)

Hunt, R. M.

1985-01-01

Design concepts for a decision support system being studied at NASA Langley as an aid to visual display unit (VDU) designers are described. Ideally, human factors should be taken into account by VDU designers. In reality, although the human factors database on VDUs is small, such systems must be constantly developed. Human factors are therefore a secondary consideration. An expert system will thus serve mainly in an advisory capacity. Functions can include facilitating the design process by shortening the time to generate and alter drawings, enhancing the capability of breaking design requirements down into simpler functions, and providing visual displays equivalent to the final product. The VDU system could also discriminate, and display the difference, between designer decisions and machine inferences. The system could also aid in analyzing the effects of designer choices on future options and in ennunciating when there are data available on a design selections.

Acute psychological stress increases plasma levels of cortisol, prolactin and TSH.

PubMed

Schedlowski, M; Wiechert, D; Wagner, T O; Tewes, U

1992-01-01

The effects of acute stress during a parachute jump on hormonal responses were studied in 12 experienced and 11 inexperienced military parachutists. Each subject performed two jumps. Prior to and immediately after each jump blood samples were drawn and analysed for plasma levels of cortisol, prolactin, thyrotropin (TSH), somatotropin (STH), and luteinizing hormone (LH). While there was a significant increase in cortisol, prolactin and TSH levels after both jumps, no alterations could be observed in STH and LH levels. Stress-induced hormonal responses were not affected by jump experience. There was also no association between the endocrine variables and anxiety scores.

Convergent Transcription At Intragenic Super-Enhancers Targets AID-initiated Genomic Instability

PubMed Central

Meng, Fei-Long; Du, Zhou; Federation, Alexander; Hu, Jiazhi; Wang, Qiao; Kieffer-Kwon, Kyong-Rim; Meyers, Robin M.; Amor, Corina; Wasserman, Caitlyn R.; Neuberg, Donna; Casellas, Rafael; Nussenzweig, Michel C.; Bradner, James E.; Liu, X. Shirley; Alt, Frederick W.

2015-01-01

Summary Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single stranded DNA targets. While largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-Seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from Super-Enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells. PMID:25483776

Distribution of recombination hotspots in the human genome--a comparison of computer simulations with real data.

PubMed

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.

TSH Compensates Thyroid-Specific IGF-I Receptor Knockout and Causes Papillary Thyroid Hyperplasia

PubMed Central

Müller, Kathrin; Führer, Dagmar; Mittag, Jens; Klöting, Nora; Blüher, Matthias; Weiss, Roy E.; Many, Marie-Christine; Schmid, Kurt Werner

2011-01-01

Although TSH stimulates all aspects of thyroid physiology IGF-I signaling through a tyrosine kinase-containing transmembrane receptor exhibits a permissive impact on TSH action. To better understand the importance of the IGF-I receptor in the thyroid in vivo, we inactivated the Igf1r with a Tg promoter-driven Cre-lox system in mice. We studied male and female mice with thyroidal wild-type, Igf1r+/−, and Igf1r−/− genotypes. Targeted Igf1r inactivation did transiently reduce thyroid hormone levels and significantly increased TSH levels in both heterozygous and homozygous mice without affecting thyroid weight. Histological analysis of thyroid tissue with Igf1r inactivation revealed hyperplasia and heterogeneous follicle structure. From 4 months of age, we detected papillary thyroid architecture in heterozygous and homozygous mice. We also noted increased body weight of male mice with a homozygous thyroidal null mutation in the Igf1r locus, compared with wild-type mice, respectively. A decrease of mRNA and protein for thyroid peroxidase and increased mRNA and protein for IGF-II receptor but no significant mRNA changes for the insulin receptor, the TSH receptor, and the sodium-iodide-symporter in both Igf1r+/− and Igf1r−/− mice were detected. Our results suggest that the strong increase of TSH benefits papillary thyroid hyperplasia and completely compensates the loss of IGF-I receptor signaling at the level of thyroid hormones without significant increase in thyroid weight. This could indicate that the IGF-I receptor signaling is less essential for thyroid hormone synthesis but maintains homeostasis and normal thyroid morphogenesis. PMID:21980075

An intelligent robotic aid system for human services

NASA Technical Reports Server (NTRS)

Kawamura, K.; Bagchi, S.; Iskarous, M.; Pack, R. T.; Saad, A.

1994-01-01

The long term goal of our research at the Intelligent Robotic Laboratory at Vanderbilt University is to develop advanced intelligent robotic aid systems for human services. As a first step toward our goal, the current thrusts of our R&D are centered on the development of an intelligent robotic aid called the ISAC (Intelligent Soft Arm Control). In this paper, we describe the overall system architecture and current activities in intelligent control, adaptive/interactive control and task learning.

Excess Mortality in Treated and Untreated Hyperthyroidism Is Related to Cumulative Periods of Low Serum TSH.

PubMed

Lillevang-Johansen, Mads; Abrahamsen, Bo; Jørgensen, Henrik Løvendahl; Brix, Thomas Heiberg; Hegedüs, Laszlo

2017-07-01

Cumulative time-dependent excess mortality in hyperthyroid patients has been suggested. However, the effect of antithyroid treatment on mortality, especially in subclinical hyperthyroidism, remains unclarified. We investigated the association between hyperthyroidism and mortality in both treated and untreated hyperthyroid individuals. Register-based cohort study of 235,547 individuals who had at least one serum thyroid-stimulating hormone (TSH) measurement in the period 1995 to 2011 (7.3 years median follow-up). Hyperthyroidism was defined as at least two measurements of low serum TSH. Mortality rates for treated and untreated hyperthyroid subjects compared with euthyroid controls were calculated using multivariate Cox regression analyses, controlling for age, sex, and comorbidities. Cumulative periods of decreased serum TSH were analyzed as a time-dependent covariate. Hazard ratio (HR) for mortality was increased in untreated [1.23; 95% confidence interval (CI), 1.12 to 1.37; P < 0.001], but not in treated, hyperthyroid patients. When including cumulative periods of TSH in the Cox regression analyses, HR for mortality per every 6 months of decreased TSH was 1.11 (95% CI, 1.09 to 1.13; P < 0.0001) in untreated hyperthyroid patients (n = 1137) and 1.13 (95% CI, 1.11 to 1.15; P < 0.0001) in treated patients (n = 1656). This corresponds to a 184% and 239% increase in mortality after 5 years of decreased TSH in untreated and treated hyperthyroidism, respectively. Mortality is increased in hyperthyroidism. Cumulative periods of decreased TSH increased mortality in both treated and untreated hyperthyroidism, implying that excess mortality may not be driven by lack of therapy, but rather inability to keep patients euthyroid. Meticulous follow-up during treatment to maintain biochemical euthyroidism may be warranted. Copyright © 2017 by the Endocrine Society

The complete genome sequence of human adenovirus 84, a highly recombinant new Human mastadenovirus D type with a unique fiber gene.

PubMed

Kaján, Győző L; Kajon, Adriana E; Pinto, Alexis Castillo; Bartha, Dániel; Arnberg, Niklas

2017-10-15

A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

A Model Human Sexuality--HIV/AIDS Prevention and Intervention Service-Learning Program

ERIC Educational Resources Information Center

Stewart, Clarence, M., Jr.

2005-01-01

This article deals with a service-learning program focused on human sexuality and HIV/AIDS prevention and intervention at the Howard University Department of Health, Human Performance and Leisure Studies. Topics discussed include how this program was created, an overview of peer education, HIV/AIDS peer education training, and services provided to…

Diagnostic accuracy of basal TSH determinations based on the intravenous TRH stimulation test: an evaluation of 2570 tests and comparison with the literature.

PubMed

Moncayo, Helga; Dapunt, Otto; Moncayo, Roy

2007-08-02

Basal TSH levels reflect the metabolic status of thyroid function, however the definition and interpretation of the basal levels of TSH is a matter of controversial debate. The aim of this study was to evaluate basal TSH levels in relation to the physiological response to i.v. TRH stimulation. A series of 2570 women attending a specialized endocrine unit were evaluated. A standardized i.v. TRH stimulation test was carried out by applying 200 mug of TRH. TSH levels were measured both in the basal and the 30 minute blood sample. The normal response to TRH stimulation had been previously determined to be an absolute value lying between 2.5 and 20 mIU/l. Both TSH values were analyzed by cross tabulation. In addition the results were compared to reference values taken from the literature. Basal TSH values were within the normal range (0.3 to 3.5 mIU/l) in 91,5% of cases, diminished in 3,8% and elevated in 4.7%. Based on the response to TRH, 82.4% were considered euthyroid, 3.3% were latent hyperthyroid, and 14.3% were latent hypothyroid. Combining the data on basal and stimulated TSH levels, latent hypothyroidism was found in the following proportions for different TSH levels: 5.4% for TSH < 2.0 mIU/l, 30.2% for TSH between 2.0 and 3.0 mIU/l, 65,5% for TSH between 3.0 and 3.50 mIU/l, 87.5% for TSH between 3.5 and 4.0 mIU/l, and 88.2% for TSH between 4 and 5 mIU/l. The use of an upper normal range for TSH of 2.5 mIU/l, as recommended in the literature, misclassified 7.7% of euthyroid cases. Our analysis strategy allows us to delineate the predictive value of basal TSH levels in relation to latent hypothyroidism. A grey area can be identified for values between 3.0 and 3.5 mIU/l.

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

USDA-ARS?s Scientific Manuscript database

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal ...

Precise genotyping and recombination detection of Enterovirus

PubMed Central

2015-01-01

Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

Refined genetic maps reveal sexual dimorphism in human meiotic recombination at multiple scales

NASA Astrophysics Data System (ADS)

Bhérer, Claude; Campbell, Christopher L.; Auton, Adam

2017-04-01

In humans, males have lower recombination rates than females over the majority of the genome, but the opposite is usually true near the telomeres. These broad-scale differences have been known for decades, yet little is known about differences at the fine scale. By combining data sets, we have collected recombination events from over 100,000 meioses and have constructed sex-specific genetic maps at a previously unachievable resolution. Here we show that, although a substantial fraction of the genome shows some degree of sexually dimorphic recombination, the vast majority of hotspots are shared between the sexes, with only a small number of putative sex-specific hotspots. Wavelet analysis indicates that most of the differences can be attributed to the fine scale, and that variation in rate between the sexes can mostly be explained by differences in hotspot magnitude, rather than location. Nonetheless, known recombination-associated genomic features, such as THE1B repeat elements, show systematic differences between the sexes.

Doxorubicin-loaded glycyrrhetinic acid modified recombinant human serum albumin nanoparticles for targeting liver tumor chemotherapy.

PubMed

Qi, Wen-Wen; Yu, Hai-Yan; Guo, Hui; Lou, Jun; Wang, Zhi-Ming; Liu, Peng; Sapin-Minet, Anne; Maincent, Philippe; Hong, Xue-Chuan; Hu, Xian-Ming; Xiao, Yu-Ling

2015-03-02

Due to overexpression of glycyrrhetinic acid (GA) receptor in liver cancer cells, glycyrrhetinic acid modified recombinant human serum albumin (rHSA) nanoparticles for targeting liver tumor cells may result in increased therapeutic efficacy and decreased adverse effects of cancer therapy. In this study, doxorubicin (DOX) loaded and glycyrrhetinic acid modified recombinant human serum albumin nanoparticles (DOX/GA-rHSA NPs) were prepared for targeting therapy for liver cancer. GA was covalently coupled to recombinant human serum albumin nanoparticles, which could efficiently deliver DOX into liver cancer cells. The resultant GA-rHSA NPs exhibited uniform spherical shape and high stability in plasma with fixed negative charge (∼-25 mV) and a size about 170 nm. DOX was loaded into GA-rHSA NPs with a maximal encapsulation efficiency of 75.8%. Moreover, the targeted NPs (DOX/GA-rHSA NPs) showed increased cytotoxic activity in liver tumor cells compared to the nontargeted NPs (DOX/rHSA NPs, DOX loaded recombinant human serum albumin nanoparticles without GA conjugating). The targeted NPs exhibited higher cellular uptake in a GA receptor-positive liver cancer cell line than nontargeted NPs as measured by both flow cytometry and confocal laser scanning microscopy. Biodistribution experiments showed that DOX/GA-rHSA NPs exhibited a much higher level of tumor accumulation than nontargeted NPs at 1 h after injection in hepatoma-bearing Balb/c mice. Therefore, the DOX/GA-rHSA NPs could be considered as an efficient nanoplatform for targeting drug delivery system for liver cancer.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep.

PubMed

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura; Gunn, Alistair J

2017-03-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus ( P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes ( P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes ( P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep

PubMed Central

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura

2016-01-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus (P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes (P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes (P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation. PMID:27207167

Prominent mitochondrial DNA recombination intermediates in human heart muscle.

PubMed

Kajander, O A; Karhunen, P J; Holt, I J; Jacobs, H T

2001-11-01

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.

Evaluation of thyroid stimulating hormone (TSH) alone as a first-line thyroid function test (TFT) in Papua New Guinea.

PubMed

Kende, M; Kandapu, S

2002-01-01

In the Port Moresby General Hospital, the Chemical Pathology Department assays both thyroid stimulating hormone (TSH) and free thyroxine (FT4) on all requests for a thyroid function test (TFT). The cost of assaying both tests is obviously higher than either test alone. In order to minimize the cost of a TFT we aimed to determine if TSH or FT4 alone as a first-line test would be adequate in assessing the thyroid hormone status of patients. We analyzed TFT records from January 1996 to May 2000 in the Port Moresby General Hospital. A total of 3089 TSH and 2867 FT4 were assayed at an annual reagent cost of Papua New Guinea kina 14,500. When TSH alone is used as a first-line test at the Port Moresby General Hospital, the biochemical status of 95% of patients will be appropriately categorized as euthyroidism, hypothyroidism or hyperthyroidism with only 5% discrepant (ie, normal TSH with abnormal FT4) results. In contrast, using FT4 alone as a first-line test correctly classifies only 84% of TFTs. Euthyroid status is observed in 50% of patients and FT4 assays on these samples will be excluded appropriately if a TSH-only protocol is adopted. Furthermore, we will save a quarter of the yearly cost of TFTs on reagents alone by performing TSH only. We conclude that TSH alone is an adequate first-line thyroid function test in Papua New Guinea and when it is normal no further FT4 test is necessary unless clinically indicated.

Hypothyroidism in adults. Levothyroxine if warranted by clinical and laboratory findings, not for simple TSH elevation.

PubMed

2015-10-01

Hypothyroidism is a common disorder due to inadequate thyroid hormone secretion. When a patient has signs and symptoms suggestive of hypothyroidism, how is it determined whether thyroid hormone replacement therapy will have a favourable harm-benefit balance? How should treatment be managed? To answer these questions, we conducted a review of the literature using the standard Prescrire methodology. The symptoms of hypothyroidism are due to slow metabolism (constipation, fatigue, sensitivity to cold, weight gain, etc.) and to polysaccharide accumulation in certain tissues, leading to hoarseness, eyelid swelling, etc. A blood TSH concentration of less than 4 or 5 mlU/L rules out peripheral hypothyroidism. TSH levels increase with age. Between 30% and 60% of high TSH levels are not confirmed on a second blood test. In overt hypothyroidism, the TSH level is high and the free T4 (thyroxine) level is low. Most of these patients are symptomatic. So-called subclinical hypothyroidism, which is rarely symptomatic, is characterised by high blood TSH levels and normal free T4 levels. The natural history of hypothyroidism depends on its cause. In chronic autoimmune thyroiditis, the most common form seen in rich countries, hypothyroidism generally worsens over time. However, other situations can lead to transient hypothyroidism that may last several weeks or months. Subclinical hypothyroidism, as the name implies, is usually asymptomatic. The risk of progression to overt hypothyroidism is about 3% to 4% per year overall but increases with the initial TSH level. Treatment guidelines are mainly based on physiological and pharmacological considerations and generally recommend levothyroxine therapy. The adverse effects of levothyroxine are signs of thyrotoxicosis in case of overdose (tachycardia, tremor, sweating, etc.). Even a slight overdose carries a risk of osteoporotic fractures and atrial fibrillation, especially in the elderly. In young adults, levothyroxine is usually started

Influence of thyroid peroxidase antibodies on TSH levels of pregnant women and maternal-fetal complications.

PubMed

Fernández Martínez, Paula; Aguado García, Rocío; Barajas Galindo, David Emilio; Hernández Moreno, Ana; Alejo Ramos, Mirian; García Arias, Sara; Ballesteros Pomar, María D; Cano Rodríguez, Isidoro Manuel

2018-06-14

During pregnancy, thyroid peroxidase (TPO) antibodies may increase the risk of developing subclinical hypothyroidism (SCH). Both conditions appear to be associated to maternal-fetal complications. The objectives of this study were to analyze if a relationship exists between TSH and TPO levels during pregnancy and the potential effects on gestational and perinatal complications, and to assess whether detectable, but not positive, TPO levels have an impact on development of gestational SCH. A prospective study was conducted at the Leon Health Area (CAULE), where universal screening for gestational thyroid dysfunction is performed between weeks 7-13 of pregnancy. Data on TSH and TPO levels and gestational and perinatal complications were collected for all 2016 deliveries. Positive TPO antibodies were defined as values≥35IU/mL. In a previous study, a TSH level>3.72mU/L was established as the cut-off value for gestational SCH. Records corresponding to 1,980 deliveries at CAULE, 21 abortions, and 18 deliveries outside the hospital were analyzed. Of the 1,670 pregnant women screened (84.34%), 142 (8.50%) had positive TPO antibodies and their presence was associated to diagnosis of SCH (P<0.01) and to significantly higher mean TSH levels (3.51mU/L vs. 2.46mU/L, P=0.03). There were no significant differences in gestational or neonatal complications. In the group with undetectable TPO antibodies (<10lU/mL), the mean TSH levels was slightly lower than in the group with TPO values ranging from 10-35 IU/mL, but the difference was not significant (P=0.89). Presence of positive TPO antibodies is associated to higher TSH levels and higher risk of gestational SCH, but does not increase the rate of maternal-fetal complications. Copyright © 2018 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.

The Red Queen Model of Recombination Hotspots Evolution in the Light of Archaic and Modern Human Genomes

PubMed Central

Lesecque, Yann; Glémin, Sylvain; Lartillot, Nicolas; Mouchiroud, Dominique; Duret, Laurent

2014-01-01

Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution. PMID:25393762

The red queen model of recombination hotspots evolution in the light of archaic and modern human genomes.

PubMed

Lesecque, Yann; Glémin, Sylvain; Lartillot, Nicolas; Mouchiroud, Dominique; Duret, Laurent

2014-11-01

Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution.

Gender and human rights dimensions of HIV/AIDS in Nigeria.

PubMed

Aniekwu, Nkoli I

2002-12-01

Until very recently, researchers paid little attention to sex or gender issues in HIV/AIDS. When differences between females and males on health matters were considered at all the focus was clearly on womens' reproductive lives and not on factors affecting the spread of the disease. There was hardly any consideration of the influence of inequalities on the spread of HIV/AIDS and on outcomes of infection between the sexes. Hitherto, health policies and programmes focused on biological aspects of diagnosis, treatment and prevention. In this paper, the author seeks to provide an understanding of the social factors as well as identification of the capacity of human rights to develop an effective response to the disease. It is a gender perspective on human rights with specific implications for women in the context of HIV/AIDS.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

PubMed

Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

2017-12-20

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Thyroid-Stimulating Hormone (TSH) Concentration at Birth in Belgian Neonates and Cognitive Development at Preschool Age

PubMed Central

Trumpff, Caroline; De Schepper, Jean; Vanderfaeillie, Johan; Vercruysse, Nathalie; Van Oyen, Herman; Moreno-Reyes, Rodrigo; Tafforeau, Jean; Vanderpas, Jean; Vandevijvere, Stefanie

2015-01-01

The main objective of the study was to investigate the effect of MID during late pregnancy, assessed by the thyroid-stimulating hormone (TSH) concentration at neonatal screening, on cognitive development of preschool children. A retrospective cohort study including 311 Belgian preschool children of 4–6 years old was conducted. Children were selected at random from the total list of neonates screened in 2008, 2009, and 2010 by the Brussels new-born screening center. Infants with congenital hypothyroidism, low birth weight, and/or prematurity were excluded from the selection. The selected children were stratified by gender and TSH-range (0.45–15 mIU/L). Cognitive abilities were assessed using Wechsler Preschool and Primary Scale of Intelligence—third edition. In addition, several socioeconomic, parental, and child confounding factors were assessed. Neonatal TSH concentration—a surrogate marker for MID—was not associated with Full Scale and Performance IQ scores in children. Lower Verbal IQ scores were found in children with neonatal TSH values comprised between 10–15 mIU/L compared to lower TSH levels in univariate analysis but these results did not hold when adjusting for confounding factors. Current levels of iodine deficiency among pregnant Belgian women may not be severe enough to affect the neurodevelopment of preschool children. PMID:26540070

Thyroid-Stimulating Hormone (TSH) Concentration at Birth in Belgian Neonates and Cognitive Development at Preschool Age.

PubMed

Trumpff, Caroline; De Schepper, Jean; Vanderfaeillie, Johan; Vercruysse, Nathalie; Van Oyen, Herman; Moreno-Reyes, Rodrigo; Tafforeau, Jean; Vanderpas, Jean; Vandevijvere, Stefanie

2015-11-02

The main objective of the study was to investigate the effect of MID during late pregnancy, assessed by the thyroid-stimulating hormone (TSH) concentration at neonatal screening, on cognitive development of preschool children. A retrospective cohort study including 311 Belgian preschool children of 4-6 years old was conducted. Children were selected at random from the total list of neonates screened in 2008, 2009, and 2010 by the Brussels new-born screening center. Infants with congenital hypothyroidism, low birth weight, and/or prematurity were excluded from the selection. The selected children were stratified by gender and TSH-range (0.45-15 mIU/L). Cognitive abilities were assessed using Wechsler Preschool and Primary Scale of Intelligence-third edition. In addition, several socioeconomic, parental, and child confounding factors were assessed. Neonatal TSH concentration-a surrogate marker for MID-was not associated with Full Scale and Performance IQ scores in children. Lower Verbal IQ scores were found in children with neonatal TSH values comprised between 10-15 mIU/L compared to lower TSH levels in univariate analysis but these results did not hold when adjusting for confounding factors. Current levels of iodine deficiency among pregnant Belgian women may not be severe enough to affect the neurodevelopment of preschool children.

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

PubMed Central

Latif, Rauf; Michalek, Krzysztof; Morshed, Syed Ahmed; Davies, Terry F.

2010-01-01

Background The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. Methodology/Principal Findings We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. Conclusions/Significance These data

Effects of recombinant human growth hormone on enterocutaneous fistula patients.

PubMed

Gu, Guo-Sheng; Ren, Jian-An; Li, Ning; Li, Jie-Shou

2008-11-28

To explore the effects of recombinant human growth hormone (rhGH) on intestinal mucosal epithelial cell proliferation and nutritional status in patients with enterocutaneous fistula. Eight patients with enterocutaneous fistulas received recombinant human growth hormone (10 microg/d) for 7 d. Image analysis and immunohistochemical techniques were used to analyse the expression of proliferating cell nuclear antigen (PCNA) in intestinal mucosal epithelial cells in biopsy samples from the patients who had undergone an endoscopic biopsy through the fistula at day 0, 4 and 7. Body weights, nitrogen excretion, serum levels of total proteins, albumin, prealbumin, transferrin and fibronectin were measured at day 0, 4 and 7. Significant improvements occurred in the expression of PCNA in the intestinal mucosal epithelial cells at day 4 and 7 compared to day 0 (24.93 +/- 3.41%, 30.46 +/- 5.24% vs 12.92 +/- 4.20%, P < 0.01). These changes were accompanied by the significant improvement of villus height (500.54 +/- 53.79 microm, 459.03 +/- 88.98 microm vs 210.94 +/- 49.16 microm, P < 0.01), serum levels of total proteins (70.52 +/- 5.13 g/L, 74.89 +/- 5.16 g/L vs 63.51 +/- 2.47 g/L, P < 0.01), albumin (39.44 +/- 1.18 g/L, 42.39 +/- 1.68 g/L vs 35.74 +/- 1.75 g/L, P < 0.01) and fibronectin (236.3 +/- 16.5 mg/L, 275.8 +/- 16.9 mg/L vs 172.5 +/- 21.4 mg/L, P < 0.01) at day 4 and 7, and prealbumin (286.38 +/- 65.61 mg/L vs 180.88 +/- 48.28 mg/L, P < 0.05), transferrin (2.61 +/- 0.12 g/L vs 2.41 +/- 0.14 g/L, P < 0.05) at day 7. Nitrogen excretion was significantly decreased at day 7 (3.40 +/- 1.65 g/d vs 7.25 +/- 3.92 g/d, P < 0.05). No change was observed in the body weight. Recombinant human growth hormone could promote intestinal mucosal epithelial cell proliferation and protein synthesis in patients with enterocutaneous fistula.

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

The intellectual basis for Latino AIDS policy: towards the humanities and health policy.

PubMed

Hayes-Bautista, David E

1992-01-01

The AIDS epidemic touches upon basic humanities themes: sex, death and social worth, to name just three. AIDS policy in general builds upon society's discourse on these topics. The growing Latino population (25% of California and Texas) needs an AIDS policy that builds upon the Latino humanities tradition. The contours of the Latino intellectual tradition, as focused on issues attendant to health, are presented, with examples from Aztec, colonial and modern times.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

[Gender, human rights and socioeconomic impact of AIDS in Brazil].

PubMed

de Oliveira, Rosa Maria Rodrigues

2006-04-01

The paper critically analyzes, from the gender standpoint, official results presented in the Brazilian government report to the Joint United Nations Programme on HIV/AIDS (UNAIDS). Specifically, the fulfillment of 2003 targets set forth in the United Nations Declaration of Commitment on HIV/AIDS, under the category of Human Rights and Reduction of the Economic and Social Impact of AIDS, are evaluated. Key concepts are highlighted, including indicators and strategies that may help civilian society better monitor these targets until 2010.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays

PubMed Central

Grubisha, Olivera; Mogg, Adrian J; Sorge, Jessica L; Ball, Laura-Jayne; Sanger, Helen; Ruble, Cara L A; Folly, Elizabeth A; Ursu, Daniel; Broad, Lisa M

2014-01-01

Background and Purpose Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. Experimental Approach Medium-throughput cellular assays were developed using a Ca2+-sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. Key Results A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. Conclusions and Implications Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies. Linked Articles This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:23848361

Distribution of Recombination Hotspots in the Human Genome – A Comparison of Computer Simulations with Real Data

PubMed Central

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar. PMID:23776462

Interesting coincidence of atypical TSH-secreting pituitary adenoma and chronic lymphocytic leukemia.

PubMed

Bolanowski, Marek; Zieliński, Grzegorz; Jawiarczyk-Przybyłowska, Aleksandra; Maksymowicz, Maria; Potoczek, Stanisław; Syrycka, Joanna; Podgórski, Jan K

2014-01-01

Thyrotropin-secreting adenomas (TSH-oma) are very rare pituitary tumours. They are macroadenomas usually presenting with signs and symptoms of hyperthyroidism, and mass effects. They can co-secrete other hormones such as growth hormone or prolactin. Different malignancies, including haematological ones, are reported in patients with pituitary diseases. Chronic lymphocytic leukemia (CLL) occurs mostly in older patients, more often in males. CLL is associated with increased risk of second malignancies such as other blood neoplasms, skin and solid tumours. We present a successful neurosurgical outcome in a patient with an interesting coincidence of atypical TSH-oma and asymptomatic CLL.

Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein.

PubMed

Okutani, R; Itoh, Y; Yamada, T; Yamaguchi, T; Singh, G; Yagisawa, H; Kawai, T

1996-09-01

Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity. We have expressed this protein in E. coli and characterized its physiochemical and biological properties. Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E. coli culture. The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus. On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1. The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical. Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities. These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1. We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.

Europe's Shifting Response to HIV/AIDS: From Human Rights to Risk Management.

PubMed

Smith, Julia

2016-12-01

Despite a history of championing HIV/AIDS as a human rights issue, and a rhetorical commitment to health as a human right, European states and institutions have shifted from a rights-based response to a risk management approach to HIV/AIDS since the economic recession of 2008. An interdisciplinary perspective is applied to analyze health policy changes at the national, regional, and global levels by drawing on data from key informant interviews, and institutional and civil society documents. It is demonstrated that, in the context of austerity measures, member states such as the UK and Greece reduced commitments to rights associated with HIV/AIDS; at the regional level, the EU failed to develop rights-based approaches to address the vulnerabilities and health care needs of key populations affected by HIV/AIDS, particularly migrants and sex workers; and at the global level, the EU backtracked on commitments to global health and is prioritizing the intellectual property rights of pharmaceutical companies over the human rights of people living with HIV/AIDS. The focus within and from the EU is on containment, efficiency, and cost reduction. The rights of those most affected are no longer prioritized.

Decrease in TSH levels after lactose restriction in Hashimoto's thyroiditis patients with lactose intolerance.

PubMed

Asik, Mehmet; Gunes, Fahri; Binnetoglu, Emine; Eroglu, Mustafa; Bozkurt, Neslihan; Sen, Hacer; Akbal, Erdem; Bakar, Coskum; Beyazit, Yavuz; Ukinc, Kubilay

2014-06-01

We aimed to evaluate the prevalence of lactose intolerance (LI) in patients with Hashimoto's thyroiditis(HT) and the effects of lactose restriction on thyroid function in these patients. Eighty-three HT patients taking L-thyroxine (LT4) were enrolled, and lactose tolerance tests were performed on all patients. Lactose intolerance was diagnosed in 75.9 % of the patients with HT. Thirty-eight patients with LI were started on a lactose-restricted diet for 8 weeks. Thirty-eight patients with LI (30 euthyroid and 8 with subclinical hypothyroidism), and 12 patients without LI were included in the final analysis. The level of TSH significantly decreased in the euthyroid and subclinical hypothyroid patients with LI [from 2.06 ± 1.02 to 1.51 ±1.1 IU/mL and from 5.45 ± 0.74 to 2.25 ± 1.88 IU/mL,respectively (both P<0.05)]. However, the level of TSH in patients without LI did not change significantly over the 8 weeks (P>0.05). Lactose intolerance occurs at a high frequency in HT patients. Lactose restriction leads to decreased levels of TSH, and LI should be considered in hypothyroid patients who require increasing LT4 doses,have irregular TSH levels and are resistant to LT4 treatment.

Higher Incidence Rates of Hypothyroidism and Late TSH Rise in Preterm Very-Low-Birth-Weight Infants at a Tertiary Care Center.

PubMed

Tfayli, Hala; Charafeddine, Lama; Tamim, Hani; Saade, Joanne; Daher, Rose T; Yunis, Khalid

2018-04-11

Preterm newborns with a very low birth weight (VLBW) of < 1,500 g have an atypical form of hypothyroidism with a delayed rise in TSH, necessitating a second newborn screening specimen collection. The aims of this study were to survey the compliance with second newborn screening to detect delayed TSH rise in VLBW preterm infants at a tertiary care center, and to determine the rate of atypical hypothyroidism. Retrospective review of the records of 104 preterm VLBW infants. Late TSH rise was defined as an increase in TSH concentration after 14 days of age in the presence of a normal initial screen. The compliance rate was 92% for the second screening. High rates of hypothyroidism (16.3%) and of late TSH rise (4.8%) were detected. Patients with hypothyroidism had a significantly lower birth weight (p = 0.01) and longer hospital stay (p = 0.004). Patients with late versus those with early TSH rise had a significantly lower mean birth weight (851 ± 302 vs. 1,191 ± 121 g, p = 0.004). The rates of early and late TSH rise in this VLBW population were higher than those in the literature and could be due to the use of povidone-iodine disinfectants. The yield of a second TSH screening in this study was high indicating the need for vigilance in screening VLBW preterm infants. © 2018 S. Karger AG, Basel.

[Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].

PubMed

Shang, Shu-huan; Zhang, Yu-feng; Shi, Bin; Cheng, Xiang-rong

2008-10-01

To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

Protection of Non-Human Primates against Rabies with an Adenovirus Recombinant Vaccine

PubMed Central

Xiang, Z.Q.; Greenberg, L.; Ertl, H. C.; Rupprecht, C.E.

2014-01-01

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. PMID:24503087

Lower-normal TSH is associated with better metabolic risk factors: a cross-sectional study on spanish men

USDA-ARS?s Scientific Manuscript database

Background and aims: Subclinical thyroid conditions, defined by normal thyroxin (T4) but abnormal thyroid-stimulating hormone (TSH) levels, may be associated with cardiovascular and metabolic risk. More recently, TSH levels within the normal range have been suggested to be associated with metabolic ...

Limited infection upon human exposure to a recombinant raccoon pox vaccine vector

USGS Publications Warehouse

Rocke, T.E.; Dein, F.J.; Fuchsberger, M.; Fox, B.C.; Stinchcomb, D.T.; Osorio, J.G.

2004-01-01

A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

The 8p23 inversion polymorphism determines local recombination heterogeneity across human populations.

PubMed

Alves, Joao M; Chikhi, Lounès; Amorim, António; Lopes, Alexandra M

2014-04-01

For decades, chromosomal inversions have been regarded as fascinating evolutionary elements as they are expected to suppress recombination between chromosomes with opposite orientations, leading to the accumulation of genetic differences between the two configurations over time. Here, making use of publicly available population genotype data for the largest polymorphic inversion in the human genome (8p23-inv), we assessed whether this inhibitory effect of inversion rearrangements led to significant differences in the recombination landscape of two homologous DNA segments, with opposite orientation. Our analysis revealed that the accumulation of genetic differentiation is positively correlated with the variation in recombination profiles. The observed recombination dissimilarity between inversion types is consistent across all populations analyzed and surpasses the effects of geographic structure, suggesting that both structures (orientations) have been evolving independently over an extended period of time, despite being subjected to the very same demographic history. Aside this mainly independent evolution, we also identified a short segment (350 kb, <10% of the whole inversion) in the central region of the inversion where the genetic divergence between the two structural haplotypes is diminished. Although it is difficult to demonstrate it, this could be due to gene flow (possibly via double-crossing over events), which is consistent with the higher recombination rates surrounding this segment. This study demonstrates for the first time that chromosomal inversions influence the recombination landscape at a fine-scale and highlights the role of these rearrangements as drivers of genome evolution.

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination.

PubMed

Shao, Renfu; Barker, Stephen C

2011-02-15

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse. Copyright © 2010 Elsevier B.V. All rights reserved.

Mismatch repair proteins and AID deaminase activity are required for the dominant negative function of C terminally-deleted AID in class switching

PubMed Central

Ucher, Anna J.; Ranjit, Sanjay; Kadungure, Tatenda; Linehan, Erin K.; Khair, Lyne; Xie, Elaine; Limauro, Jennifer; Rauch, Katherina S.; Schrader, Carol E.; Stavnezer, Janet

2014-01-01

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR but not for S region DNA DSBs during CSR, and it is not required for SHM. AID lacking the C terminus (ΔAID) is a dominant negative (DN) mutant, as human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that ΔAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. In order to have DN function,ΔAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have previously been shown to contribute to DSB formation in S regions, and here we find that Msh2 is required for the DN activity, as ΔAID is not a DN mutant in msh2−/− cells. Our results suggest that the DNA DSBs induced by ΔAID are unable to participate in CSR, and might interfere with the ability of full-length AID to participate in CSR. We propose thatΔAID is impaired in its ability to recruit non-homologous end joining (NHEJ) repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by ΔAID have longer microhomologies than those induced by full-length AID. In addition, our data suggest that AID binds Sµ regions in vivo as a monomer. PMID:24973444

Structure and receptor binding preferences of recombinant hemagglutinins from avian and human H6 and H10 influenza A virus subtypes.

PubMed

Yang, Hua; Carney, Paul J; Chang, Jessie C; Villanueva, Julie M; Stevens, James

2015-04-01

During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Association between rs12045440 Polymorphism in the CAPZB Intron and Serum TSH Concentrations in Chinese Thyroid Tumor Patients

PubMed Central

Feng, Shouhao; Lin, Shengli; Zou, Jidong; Wang, Yulong; Ji, Qinghai; Lv, Zhenghua

2015-01-01

The aim of this study was to investigate the possible influence of different genotypes of the lead single nucleotide polymorphisms (SNPs) rs10917468 and rs12045440 in the CAPZB gene on the thyroid function in papillary thyroid carcinoma (PTC) and benign thyroid neoplasm (BN) patients. In the study, a significant association was detected between rs12045440 and serum TSH concentrations in thyroid tumor patients (p = 0.001). After the adjustment of relevant covariates, the difference between the mean serum TSH levels in different genotypes of rs12045440 was still significant in the BN group (p = 0.003) but was not significant in the PTC cases (p = 0.115). No significant association of rs10917468 with TSH levels was found. The SNP rs12045440 was associated with the serum TSH concentrations in Chinese thyroid tumor patients, especially in benign thyroid tumor cases. PMID:26273293

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

PubMed

Hanlon, Steven P; Camattari, Andrea; Abad, Sandra; Glieder, Anton; Kittelmann, Matthias; Lütz, Stephan; Wirz, Beat; Winkler, Margit

2012-06-18

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

The effects of HIV/AIDS on economic growth and human capitals: a panel study evidence from Asian countries.

PubMed

Roy, Shongkour

2014-01-01

Human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) affects economic growths by reducing the human capitals are among the most poorly understood aspect of the AIDS epidemic. This article analyzes the effects of the prevalence of HIV and full-blown AIDS on a country's human capitals and economic growths. Using a fixed effect model for panel data 1990-2010 from the Asia, I explored the dynamic relationships among HIV/AIDS, economic growths, and human capitals within countries over time. The econometric effects concerned that HIV/AIDS plays an important role in the field of economic growths and it is measured as a change in real gross domestic product (GDP) per capita and human capitals. The modeling results for the Asian countries indicates HIV/AIDS prevalence that has a hurtful effect on GDP per capita by reducing human capitals within countries over time.

An integrative 'omics' solution to the detection of recombinant human erythropoietin and blood doping.

PubMed

Pitsiladis, Yannis P; Durussel, Jérôme; Rabin, Olivier

2014-05-01

Administration of recombinant human erythropoietin (rHumanEPO) improves sporting performance and hence is frequently subject to abuse by athletes, although rHumanEPO is prohibited by the WADA. Approaches to detect rHumanEPO doping have improved significantly in recent years but remain imperfect. A new transcriptomic-based longitudinal screening approach is being developed that has the potential to improve the analytical performance of current detection methods. In particular, studies are being funded by WADA to identify a 'molecular signature' of rHumanEPO doping and preliminary results are promising. In the first systematic study to be conducted, the expression of hundreds of genes were found to be altered by rHumanEPO with numerous gene transcripts being differentially expressed after the first injection and further transcripts profoundly upregulated during and subsequently downregulated up to 4 weeks postadministration of the drug; with the same transcriptomic pattern observed in all participants. The identification of a blood 'molecular signature' of rHumanEPO administration is the strongest evidence to date that gene biomarkers have the potential to substantially improve the analytical performance of current antidoping methods such as the Athlete Biological Passport for rHumanEPO detection. Given the early promise of transcriptomics, research using an 'omics'-based approach involving genomics, transcriptomics, proteomics and metabolomics should be intensified in order to achieve improved detection of rHumanEPO and other doping substances and methods difficult to detect such a recombinant human growth hormone and blood transfusions.

The rise and fall of a human recombination hot spot.

PubMed

Jeffreys, Alec J; Neumann, Rita

2009-05-01

Human meiotic crossovers mainly cluster into narrow hot spots that profoundly influence patterns of haplotype diversity and that may also affect genome instability and sequence evolution. Hot spots also seem to be ephemeral, but processes of hot-spot activation and their subsequent evolutionary dynamics remain unknown. We now analyze the life cycle of a recombination hot spot. Sperm typing revealed a polymorphic hot spot that was activated in cis by a single base change, providing evidence for a primary sequence determinant necessary, though not sufficient, to activate recombination. This activating mutation occurred roughly 70,000 y ago and has persisted to the present, most likely fortuitously through genetic drift despite its systematic elimination by biased gene conversion. Nonetheless, this self-destructive conversion will eventually lead to hot-spot extinction. These findings define a subclass of highly transient hot spots and highlight the importance of understanding hot-spot turnover and how it influences haplotype diversity.

Human Microbiome and HIV/AIDS

PubMed Central

Li, Yihong; Yang, Liying; Pei, Zhiheng; Poles, Michael; Abrams, William R.; Malamud, Daniel

2013-01-01

Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of “crosstalk” between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against HIV infection. PMID:22193889

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

PubMed

Tanaka, H; Kaneko, T

1992-07-01

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

The TSH levels and risk of hypothyroidism: Results from a population based prospective cohort study in an Iranian adult's population.

PubMed

Aminorroaya, Ashraf; Meamar, Rokhsareh; Amini, Massoud; Feizi, Awat; Nasri, Maryam; Tabatabaei, Azamosadat; Faghihimani, Elham

2017-06-01

The aim of current study was to assess the relationship between serum TSH levels and hypothyroidism risk in the euthyroid population. In a population-based cohort study, a total of 615 individuals with a normal baseline TSH, from of total population (n=2254) in 2006, were followed up for 6years. TSH, total T4, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) were measured. The relative risk (RR) and 95% confidence interval (95%CI) were calculated based on logistic regression. The Receiver Operating Characteristic (ROC) analysis along with area under the curve (AUC) was used to prediction of future hypothyroidism. TSH level in 2006 was a significant predictor for overt hypothyroidism, in the total population (RR=3.5) and female (RR=1.37) (all, P value<0.05). A cutoff value of TSH at 2.05mIU/L [AUC: (CI95 %), 0.68 (0.44-0.92; P=0.05)] was obtained for differentiating the patients with overt hypothyroidism from euthyroid. However, this cut off was not observed when we included only negative TPO and TgAbs people in 2006. The RR of hypothyroidism increased gradually when TSH level increased from 2.06-3.6mIU/L to >3.6mIU/L in the total population and both sexes. In women, the risk of overt hypothyroidism was significantly higher in subjects with TSH above 3.6 than those subject with THS levels≤2.05 [RR: (CI95 %), 20.57(2.-207.04), P value<0.05]. A cutoff value of TSH at 2.05mIU/L could predict the development of overt hypothyroidism in future. However, it was not applicable for people with negative TPOAb and negative TgAb. Copyright © 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis.

PubMed

Mirkalantari, Shiva; Zarnani, Amir-Hassan; Nazari, Mahboobeh; Irajian, Gholam Reza; Amirmozafari, Nour

2017-03-03

The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

PubMed

Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

2011-08-01

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

PubMed Central

Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

2013-01-01

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks. PMID:23881231

Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19

PubMed Central

Weber, James L.; Wang, Zhenyuan; Hansen, Kevin; Stephenson, Matt; Kappel, Clarisse; Salzman, Sherry; Wilkie, Patricia J.; Keats, Bronya; Dracopoli, Nicholas C.; Brandriff, Brigitte F.; Olsen, Anne S.

1993-01-01

An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP “gene” conversion without recombination was calculated as 3 × 10−4/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality. PMID:8213834

Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

PubMed

Leedman, P J; Newman, J D; Harrison, L C

1989-07-01

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Serum TSH within the reference range as a predictor of future hypothyroidism and hyperthyroidism: 11-year follow-up of the HUNT Study in Norway.

PubMed

Åsvold, Bjørn O; Vatten, Lars J; Midthjell, Kristian; Bjøro, Trine

2012-01-01

Serum TSH in the upper part of the reference range may sometimes be a response to autoimmune thyroiditis in early stage and may therefore predict future hypothyroidism. Conversely, relatively low serum TSH could predict future hyperthyroidism. The objective of the study was to assess TSH within the reference range and subsequent risk of hypothyroidism and hyperthyroidism. This was a prospective population-based study with linkage to the Norwegian Prescription Database. A total of 10,083 women and 5,023 men without previous thyroid disease who had a baseline TSH of 0.20-4.5 mU/liter and who participated at a follow-up examination 11 yr later. Predicted probabilities of developing hypothyroidism or hyperthyroidism during follow-up, by categories of baseline TSH, were estimated. During 11 yr of follow-up, 3.5% of women and 1.3% of men developed hypothyroidism, and 1.1% of women and 0.6% of men developed hyperthyroidism. In both sexes, the baseline TSH was positively associated with the risk of subsequent hypothyroidism. The risk increased gradually from TSH of 0.50-1.4 mU/liter [women, 1.1%, 95% confidence interval (CI) 0.8-1.4; men, 0.3%, 95% CI 0.1-0.6] to a TSH of 4.0-4.5 mU/liter (women, 31.5%, 95% CI 24.6-39.3; men, 14.7%, 95% CI 7.7-26.2). The risk of hyperthyroidism was higher in women with a baseline TSH of 0.20-0.49 mU/liter (3.9%, 95% CI 1.8-8.4) than in women with a TSH of 0.50-0.99 mU/liter (1.4%, 95% CI 0.9-2.1) or higher (∼1.0%). TSH within the reference range is positively and strongly associated with the risk of future hypothyroidism. TSH at the lower limit of the reference range may be associated with an increased risk of hyperthyroidism.

Vitamin D status in Egyptian euthyroid multinodular non-toxic goiter patients and its correlation with TSH levels.

PubMed

Aboelnaga, Mohamed M; Elshafei, Maha M; Elsayed, Eman

2016-10-01

Although the prevalence of MNG is widespread throughout the world, its pathogenesis is poorly understood, and the complex interactions of both genetic predisposition and the individuals' environment are likely. However, to the best of our knowledge, it remains unknown whether there is a relationship between vitamin D status and prevalence or pathogenesis of euthyroid MNG. Therefore, the goal of the present study was determination of vitamin D status in euthyroid MNG as well as exploration of the correlation between vitamin D status & TSH levels. A total of 77 patients diagnosed with euthyroid MNG and 50 subjects without goiter were matched according to age, weight and BMI as control group in this case control study. We found that patients with euthyroid MNG had statistically significant lower mean of [25(OH)D] (24.21±8.68ng/mL) in comparison with its mean in control subjects (28.37±10.91ng/mL, P value=0.019). The 28 sufficient vitamin D MNG patients had statistically significant lower level of TSH than 49 insufficient vitamin D MNG patients. Vitamin D and TSH levels correlate with vitamin D levels in MNG patients in Pearson correlation. Also 25 OH vitamin D was a significant independent predictor for TSH levels among euthyroid MNG patients in regression analysis. Patients with euthyroid MNG have lower levels of vitamin D and TSH levels correlate with vitamin D levels in euthyroid MNG patients. In addition, 25 OH vitamin D was a significant independent predictor for TSH levels among euthyroid MNG patients. We recommend hypovitaminosis D evaluation and correction in patients with MNG. Copyright © 2016 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

Drug eruption caused by recombinant human G-CSF.

PubMed

Sasaki, O; Yokoyama, A; Uemura, S; Fujino, S; Inoue, Y; Kohno, N; Hiwada, K

1994-10-01

Two types of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are available, and equally used for mitigation of neutropenia. One is a glycosylated natural product from mammalian cells, and the other a non-glycosylated form from Escherichia coli. Though only minimal adverse effects have been reported for both, we treated two patients with rhG-CSF-induced systemic eruption. Based on these patients, the following should be noted: 1) drug eruption may occur in both types of rhG-CSF without detectable antibodies, 2) intradermal test is useful for determination of the causal drug, and 3) if one rhG-CSF product causes eruption, the alternative one may possibly be safe and effective.

Conceptualizing a Human Right to Prevention in Global HIV/AIDS Policy

PubMed Central

Meier, Benjamin Mason; Brugh, Kristen Nichole; Halima, Yasmin

2012-01-01

Given current constraints on universal treatment campaigns, recent advances in public health prevention initiatives have revitalized efforts to stem the tide of HIV transmission. Yet, despite a growing imperative for prevention—supported by the promise of behavioral, structural and biomedical approaches to lower the incidence of HIV—human rights frameworks remain limited in addressing collective prevention policy through global health governance. Assessing the evolution of rights-based approaches to global HIV/AIDS policy, this review finds that human rights have shifted from collective public health to individual treatment access. While the advent of the HIV/AIDS pandemic gave meaning to rights in framing global health policy, the application of rights in treatment access litigation came at the expense of public health prevention efforts. Where the human rights framework remains limited to individual rights enforced against a state duty bearer, such rights have faced constrained application in framing population-level policy to realize the public good of HIV prevention. Concluding that human rights frameworks must be developed to reflect the complementarity of individual treatment and collective prevention, this article conceptualizes collective rights to public health, structuring collective combination prevention to alleviate limitations on individual rights frameworks and frame rights-based global HIV/AIDS policy to assure research expansion, prevention access and health system integration. PMID:23226723

Thyroid function. Pathogenesis of Graves ophthalmopathy--a role for TSH-R?

PubMed

Wall, Jack R

2014-05-01

A new study highlights the complexities of anti-TSH-receptor antibody function and the differences between adult and paediatric patients with Graves disease, adding to the controversy regarding the possible role of these antibodies in the development of ophthalmopathy.

Modeling strategic behavior in human-automation interaction - Why an 'aid' can (and should) go unused

NASA Technical Reports Server (NTRS)

Kirlik, Alex

1993-01-01

Task-offload aids (e.g., an autopilot, an 'intelligent' assistant) can be selectively engaged by the human operator to dynamically delegate tasks to automation. Introducing such aids eliminates some task demands but creates new ones associated with programming, engaging, and disengaging the aiding device via an interface. The burdens associated with managing automation can sometimes outweigh the potential benefits of automation to improved system performance. Aid design parameters and features of the overall multitask context combine to determine whether or not a task-offload aid will effectively support the operator. A modeling and sensitivity analysis approach is presented that identifies effective strategies for human-automation interaction as a function of three task-context parameters and three aid design parameters. The analysis and modeling approaches provide resources for predicting how a well-adapted operator will use a given task-offload aid, and for specifying aid design features that ensure that automation will provide effective operator support in a multitask environment.

National HIV/AIDS mortality, prevalence, and incidence rates are associated with the Human Development Index.

PubMed

Lou, Li-Xia; Chen, Yi; Yu, Chao-Hui; Li, You-Ming; Ye, Juan

2014-10-01

HIV/AIDS is a worldwide threat to human health with mortality, prevalence, and incidence rates varying widely. We evaluated the association between the global HIV/AIDS epidemic and national socioeconomic development. We obtained global age-standardized HIV/AIDS mortality, prevalence, and incidence rates from World Health Statistics Report of the World Health Organization. The human development indexes (HDIs) of 141 countries were obtained from a Human Development Report. Countries were divided into 4 groups according to the HDI distribution. We explored the association between HIV/AIDS epidemic and HDI information using Spearman correlation analysis, regression analysis, and the Kruskal-Wallis test. HIV/AIDS mortality, prevalence, and incidence rates were inversely correlated with national HDI (r = -0.675, -0.519, and -0.398, respectively; P < .001), as well as the 4 indicators of HDI (ie, life expectancy at birth, mean years of schooling, expected years of schooling, and gross national income per capita). Low HDI countries had higher HIV/AIDS mortality, prevalence, and incidence rates than that of medium, high, and very high HDI countries. Quantile regression results indicated that HDI had a greater negative effect on the HIV/AIDS epidemic in countries with more severe HIV/AIDS epidemic. Less-developed countries are likely to have more severe HIV/AIDS epidemic. There is a need to pay more attention to HIV/AIDS control in less-developed countries, where lower socioeconomic status might have accelerated the HIV/AIDS epidemic more rapidly. Copyright © 2014 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Immunoglobulin class switch DNA recombination: induction, targeting and beyond

PubMed Central

Xu, Zhenming; Zan, Hong; Pone, Egest J.; Mai, Thach; Casali, Paolo

2012-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus is central to the maturation of the antibody response and critically requires the AID cytidine deaminase. CSR entails changes of the chromatin state and transcriptional activation of the IgH locus upstream and downstream switch (S) regions that are to undergo S-S DNA recombination, induction of AID, and targeting of CSR factors to S regions by 14-3-3 adaptors and as enabled by the transcription machinery and histone modifications. In this Review, we focus on recent advances in CSR induction and targeting. We also outline an integrated model of the assembly of macromolecular complexes that transduce critical epigenetic information to enzymatic effectors of the CSR machinery. PMID:22728528

Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

PubMed

Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

2014-01-01

No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

A case of methimazole-induced hypothyroidism in a patient with endemic goiter: effects of endogenous TSH hyperstimulation after discontinuation of the drug.

PubMed

Messina, M; Manieri, C; Spagnuolo, F; Sardi, E; Allegramente, L; Monaco, A; Ciccarelli, E

1989-04-01

Serum thyroid hormone and TSH concentrations were monitored in a patient with multinodular endemic goiter and severe methimazole (MMI) induced hypothyrodism up to 190 days after drug withdrawal. Serum concentrations of TT3, TT4 and TSH returned to normal values at the 6th., the 140th, and the 120th. day respectively. Within the first 20 days after MMI withdrawal the increase of serum T3 levels was correlated with the observed decrease of serum TSH concentrations. Successively T3 values decreased and T4 levels progressively increased. Six months after MMI withdrawal basal serum TSH concentration was normal while an exaggerated response to TRH was observed. We think that this peculiar hormone pattern is due to iodine depletion. In this case TSH hyperstimulation increases predominantly T3 secretion demonstrating the reduced thyroidal ability to produce T4 when hyperstimulated.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

PubMed

Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

1996-07-15

Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S

EPA Science Inventory

ABSTRACT
We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

PubMed

Levin, W; Daniel, R F; Stoner, C R; Stoller, T J; Wardwell-Swanson, J A; Angelillo, Y M; Familletti, P C; Crowl, R M

1992-02-01

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.

Production of recombinant human lysozyme in the milk of transgenic pigs.

PubMed

Tong, Jia; Wei, HengXi; Liu, XiaoFang; Hu, WenPing; Bi, MingJun; Wang, YuanYuan; Li, QiuYan; Li, Ning

2011-04-01

In the swine industry pathogenic infections have a significant negative impact on neonatal survival. Piglets fed with human lysozyme, a natural antibiotic, might be more resistant to gastrointestinal infections. Here we describe the generation of transgenic swine expressing recombinant human lysozyme by somatic cell nuclear transfer. Three cloned female pigs were born, one of which expressed rhLZ at 0.32 ± 0.01 μg/ml in milk, 50-fold higher than that of the pig native lysozyme. Both the transgenic gilts and their progeny appear healthy. Introducing human lysozyme into pigs' milk has a potential to benefit the piglets by enhancing immune function and defending against pathogenic bacteria, thereby increasing the new born survival rate. This advance could be of great value to commercial swine producers.

[Use of recombinant Human Growth Hormone (rHGH)].

PubMed

Calzada-León, Raúl

2017-01-01

Recombinant human growth hormone, synthesized in E.coli or mammalian cells cultures, is since 1985, a useful therapeutic resource to increase growth velocity and final height. In this paper are discussed the four phases (aims, security and efficacy, utility and efficiency) indispensables to define the start of treatment, as well as the absolute, relative and metabolic indications and the transitory and permanent conditions that contraindicate its use. It is commented the way to optimize the results (simple but indispensables indications for the physician, the patients and their family). Finally it is analyzed the results of treatment in patients with growth hormone deficiency, Turner syndrome, chronic renal failure, Prader-Willi syndrome, Noonan syndrome, SHOX deficiency, intrauterine growth retardation and idiopathic short stature.

International Validation of Two Human Recombinant Estrogen ...

EPA Pesticide Factsheets

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay using a ligand-binding domain of the human ER. Twenty three compounds were tested in 6 laboratories for the FW assay and 5 for the CERJ assay, which included three controls (used with every run), 9 uncoded, and 14 coded chemicals across 3 subtasks. The overall goal of this validation study was to demonstrate the ability of each of the two assays to reliably classify the test chemicals as binders or non-binders. Laboratories had little trouble with the ER binders that produced a full binding curve when using either the CERI or FW assays. As is typical with all ER competitive binding assays, the weak binders proved to be more challenging. However, overall results from both the FW and CERI assays were consistent and in agreement with expected classifications regardless of the form of the hrER (i.e., full length ER versus an ER ligand binding domain) or the subtle differences in the protocols for conducting each assay. The reproducibility and accuracy for classification of chemicals as potential ER binders and non- binders using the FW and CERI hrER binding assays were comparable to that of the U.S.EPA’s existing ER binding test guideline OPPTS 890.1250, while providing an improved, highe

Human-aided and natural dispersal drive gene flow across the range of an invasive mosquito.

PubMed

Medley, Kim A; Jenkins, David G; Hoffman, Eric A

2015-01-01

Human-aided transport is responsible for many contemporary species introductions, yet the contribution of human-aided transport to dispersal within non-native regions is less clear. Understanding dispersal dynamics for invasive species can streamline mitigation efforts by targeting routes that contribute disproportionally to spread. Because of its limited natural dispersal ability, rapid spread of the Asian tiger mosquito (Aedes albopictus) has been attributed to human-aided transport, but until now, the relative roles of human-aided and natural movement have not been rigorously evaluated. Here, we use landscape genetics and information-theoretic model selection to evaluate 52 models representing 9240 pairwise dispersal paths among sites across the US range for Ae. albopictus and show that recent gene flow reflects a combination of natural and human-aided dispersal. Highways and water availability facilitate dispersal at a broad spatial scale, but gene flow is hindered by forests at the current distributional limit (range edge) and by agriculture among sites within the mosquito's native climatic niche (range core). Our results show that highways are important to genetic structure between range-edge and range-core pairs, suggesting a role for human-aided mosquito transport to the range edge. In contrast, natural dispersal is dominant at smaller spatial scales, reflecting a shifting dominance to natural movement two decades after introduction. These conclusions highlight the importance of (i) early intervention for species introductions, particularly those with readily dispersed dormant stages and short generation times, and (ii) strict monitoring of commercial shipments for transported immature stages of Ae. albopictus, particularly towards the northern edge of the US range. © 2014 John Wiley & Sons Ltd.

Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

PubMed Central

Ponnazhagan, Selvarangan; Weigel, Kirsten A.; Raikwar, Sudhanshu P.; Mukherjee, Pinku; Yoder, Mervin C.; Srivastava, Arun

1998-01-01

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited

Impact of light exposure on thyroid-stimulating hormone results using the Siemens Advia Centaur TSH-3Ultra assay.

PubMed

Armer, Jane; Giles, Diane; Lancaster, Ian; Brownbill, Kathryn

2017-09-01

Background Thyroid-stimulating hormone (TSH) is used as the first-line test of thyroid function. Siemens Healthcare Diagnostics recommend that Siemens Centaur reagents must be protected from light in the assay information and on reagent packaging. We have compared the effect of light exposure on results using Siemens TSH-3Ultra and follicle-stimulating hormone reagents. The thyroid-stimulating hormone reagent includes fluoroscein thiocyanate whereas the follicle-stimulating hormone reagent does not. Methods Three levels of quality controls were analysed using SiemensTSH-3Ultra and follicle-stimulating hormone reagent packs that had been kept protected from light or exposed to light at 6-h intervals for 48 h and then at 96 h. Results Thyroid-stimulating hormone results were significantly lower after exposure of TSH-3Ultra reagent packs to light. Results were >15% lower at all three levels of quality control following 18 h of light exposure and continued to decrease until 96 h. There was no significant difference in follicle-stimulating hormone results whether reagents had been exposed to or protected from light. Conclusions Thyroid-stimulating hormone results but not follicle-stimulating hormone results are lowered after exposure of reagent packs to light. Laboratories must ensure that TSH-3Ultra reagents are not exposed to light and analyse quality control samples on every reagent pack to check that there has not been light exposure prior to delivery. The labelling on TSH-3Ultra reagent packs should reflect the significant effect of light exposure compared with the follicle-stimulating hormone reagent. We propose that the effect of light exposure on binding of fluoroscein thiocyanate to the solid phase antibody causes the falsely low results.

TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

PubMed Central

Opitz, Robert; Maquet, Emilie; Zoenen, Maxime; Dadhich, Rajesh

2011-01-01

TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid. PMID:21737742

Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

DTIC Science & Technology

2009-01-01

Literature 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Dramatic Differences in Organophosphorus Hydrolase Activity between Human and 5a... activity , V-agents, VX, bioscavenger, medical countermeasures 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes† Tamara C. Otto,‡ Christina K. Harsch,§ David T

Identification of Recombinant Human Rhinovirus A and C in Circulating Strains from Upper and Lower Respiratory Infections

PubMed Central

Kim, Hak; Kim, Kisoon; Kim, Dae-Won; Jung, Hee-Dong; Min Cheong, Hyang; Kim, Ki Hwan; Soo Kim, Dong; Kim, You-Jin

2013-01-01

Human rhinoviruses (HRVs), in the Enterovirus genus within the family Picornaviridae, are a highly prevalent cause of acute respiratory infection (ARI). Enteroviruses are genetically highly variable, and recombination between serotypes is known to be a major contribution to their diversity. Recently it was reported that recombination events in HRVs cause the diversity of HRV-C. This study analyzed parts of the viral genes spanning the 5′ non- coding region (NCR) through to the viral protein (VP) encoding sequences of 105 HRV field isolates from 51 outpatient cases of Acute Respiratory Infectious Network (ARINET) and 54 inpatient cases of severe lower respiratory infection (SLRI) surveillance, in order to identify recombination in field samples. When analyzing parts of the 5′NCR and VP4/VP2 encoding sequences, we found intra- and interspecies recombinants in field strains of HRV-A and -C. Nineteen cases of recombination events (18.1%) were found among 105 field strains. For HRV-A, there were five cases (4.8%) of intraspecies recombination events and three cases (2.8%) of interspecies recombination events. For HRV-C, there were four cases (3.8%) of intraspecies recombination events and seven cases (6.7%) of interspecies recombination events. Recombination events were significantly more frequently observed in the ARINET samples (18 cases) than in the SLRI samples (1 case; P< 0.0001). The recombination breakpoints were located in nucleotides (nt) 472–554, which comprise stem-loop 5 in the internal ribosomal entry site (IRES), based on the HRV-B 35 sequence (accession no. FJ445187). Our findings regarding genomic recombination in circulating HRV-A and -C strains suggest that recombination might play a role in HRV fitness and could be a possible determinant of disease severity caused by various HRV infections in patients with ARI. PMID:23826363

Mathematical Modeling of the Pituitary–Thyroid Feedback Loop: Role of a TSH-T3-Shunt and Sensitivity Analysis

PubMed Central

Berberich, Julian; Dietrich, Johannes W.; Hoermann, Rudolf; Müller, Matthias A.

2018-01-01

Despite significant progress in assay technology, diagnosis of functional thyroid disorders may still be a challenge, as illustrated by the vague upper limit of the reference range for serum thyrotropin (TSH). Diagnostical problems also apply to subjects affected by syndrome T, i.e., those 10% of hypothyroid patients who continue to suffer from poor quality of life despite normal TSH concentrations under substitution therapy with levothyroxine (L-T4). In this paper, we extend a mathematical model of the pituitary–thyroid feedback loop in order to improve the understanding of thyroid hormone homeostasis. In particular, we incorporate a TSH-T3-shunt inside the thyroid, whose existence has recently been demonstrated in several clinical studies. The resulting extended model shows good accordance with various clinical observations, such as a circadian rhythm in free peripheral triiodothyronine (FT3). Furthermore, we perform a sensitivity analysis of the derived model, revealing the dependence of TSH and hormone concentrations on different system parameters. The results have implications for clinical interpretation of thyroid tests, e.g., in the differential diagnosis of subclinical hypothyroidism. PMID:29619006

Mathematical Modeling of the Pituitary-Thyroid Feedback Loop: Role of a TSH-T3-Shunt and Sensitivity Analysis.

PubMed

Berberich, Julian; Dietrich, Johannes W; Hoermann, Rudolf; Müller, Matthias A

2018-01-01

Despite significant progress in assay technology, diagnosis of functional thyroid disorders may still be a challenge, as illustrated by the vague upper limit of the reference range for serum thyrotropin ( TSH ). Diagnostical problems also apply to subjects affected by syndrome T, i.e., those 10% of hypothyroid patients who continue to suffer from poor quality of life despite normal TSH concentrations under substitution therapy with levothyroxine ( L - T 4 ). In this paper, we extend a mathematical model of the pituitary-thyroid feedback loop in order to improve the understanding of thyroid hormone homeostasis. In particular, we incorporate a TSH - T 3 -shunt inside the thyroid, whose existence has recently been demonstrated in several clinical studies. The resulting extended model shows good accordance with various clinical observations, such as a circadian rhythm in free peripheral triiodothyronine ( FT 3 ). Furthermore, we perform a sensitivity analysis of the derived model, revealing the dependence of TSH and hormone concentrations on different system parameters. The results have implications for clinical interpretation of thyroid tests, e.g., in the differential diagnosis of subclinical hypothyroidism.

AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells

PubMed Central

Huemer, Michael; Rebhandl, Stefan; Zaborsky, Nadja; Gassner, Franz J; Hainzl, Stefan; Weiss, Lukas; Hebenstreit, Daniel; Greil, Richard; Geisberger, Roland

2014-01-01

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sμ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL. PMID:25179679

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

PubMed

Coppen, S R; Newsam, R; Bull, A T; Baines, A J

1995-04-20

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

Functional and morphological changes in the adenohypophysis of dogs with induced primary hypothyroidism: loss of TSH hypersecretion, hypersomatotropism, hypoprolactinemia, and pituitary enlargement with transdifferentiation.

PubMed

Diaz-Espiñeira, M M; Mol, J A; van den Ingh, T S G A M; van der Vlugt-Meijer, R H; Rijnberk, A; Kooistra, H S

2008-07-01

From case studies in humans it is known that primary hypothyroidism (PH) may be associated with morphological and functional changes of the pituitary. There is no insight into the time scale of these changes. In this study, seven beagle dogs were followed up for 3 years after the induction of primary hypothyroidism. Three of these dogs were followed up for another 1.5 years while receiving l-thyroxine. Adenohypophyseal function was investigated at 2-month intervals with the combined intravenous injection of CRH, GHRH, GnRH, and TRH, and measurement of the plasma concentrations of ACTH, GH, LH, PRL, and TSH. In addition, after 2 years of hypothyroidism a single TRH-stimulation test and a somatostatin test were performed, with measurements of the same pituitary hormones. Every 6 months the pituitary gland was visualized by computed tomography (CT). Induction of PH led to high plasma TSH concentrations for a few months, where after concentrations gradually declined to values no longer significantly different from pre-PH values. A blunted response to stimulation of TSH release preceded this decline. Basal plasma GH concentrations increased during PH and there was a paradoxical hyperresponsiveness to TRH stimulation. Basal GH concentrations remained elevated and returned only to low values during l-thyroxine treatment. Basal PRL concentrations decreased significantly during PH and normalized after several months of l-thyroxine treatment. The pituitary gland became enlarged in all dogs. Histomorphology and immunohistochemical studies in 4 dogs, after 3 years of PH, revealed thyrotroph hyperplasia, large vacuolated thyroid deficiency cells, and decreased numbers of mammotrophs. Several cells stained for both GH and TSH. In conclusion, with time PH led to a loss of the TSH response to low T4 concentrations, hypersecretion of GH, and hyposecretion of PRL. The enlarged pituitaries were characterized by thyrotroph hyperplasia, large vacuolated thyroid deficiency cells, and

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

Comparative effect of Citrus sinensis and carbimazole on serum T4, T3 and TSH levels.

PubMed

Uduak, Okon Akpan; Ani, Elemi John; Etoh, Emmauel Columba Inyang; Macstephen, Adienbo Ologbagno

2014-05-01

There are previous independent reports on the anti-thyroid property of Citrus sinensis. This isoflavones and phenolic acid-rich natural agent is widely consumed as dietary supplement, thus the need to investigate its comparative effect with a standard anti-thyroid drug on T4, T3 and thyroid stimulating hormone (TSH) levels. To compare the effect of Citrus sinensis and carbimazole (CARB) on blood levels of thyroid hormones (T4 and T3) and TSH. Male wistar albino rats weighing 100-150 g were employed in this research. The rats were randomly assigned to four groups of seven rats per group. Group I served as control and were administered distilled water while groups II-IV were administered with 1500 mg/kg of Citrus sinensis (fresh orange juice; FOJ), 0.1 μg/g of levothyroxine (LVT) and 0.01 mg/g of CARB, respectively, per oral once daily for 28 days. The animals were sacrificed under chloroform anaesthesia and blood sample collected by cardiac puncture and processed by standard method to obtain serum. TSH, T4 and T3 were assayed with the serum using ARIA II automated radioimmunoassay instrument. The results showed that TSH level was significantly (P < 0.05) decreased in LVT treated group compared with the FOJ group. T4 was significantly (P < 0.05) decreased in the FOJ and CARB groups compared with the control and LVT groups. LVT significantly increased T4 when compared with FOJ group. T3 was significantly (P < 0.05) decreased in the CARB group compared with the control. These findings suggest that FOJ alters thyroid hormones metabolism to reduce their serum levels with a compensatory elevations of TSH level in a direction similar to CARB.

Comparative effect of Citrus sinensis and carbimazole on serum T4, T3 and TSH levels

PubMed Central

Uduak, Okon Akpan; Ani, Elemi John; Etoh, Emmauel Columba Inyang; Macstephen, Adienbo Ologbagno

2014-01-01

Background: There are previous independent reports on the anti-thyroid property of Citrus sinensis. This isoflavones and phenolic acid-rich natural agent is widely consumed as dietary supplement, thus the need to investigate its comparative effect with a standard anti-thyroid drug on T4, T3 and thyroid stimulating hormone (TSH) levels. Objective: To compare the effect of Citrus sinensis and carbimazole (CARB) on blood levels of thyroid hormones (T4 and T3) and TSH. Materials and Methods: Male wistar albino rats weighing 100-150 g were employed in this research. The rats were randomly assigned to four groups of seven rats per group. Group I served as control and were administered distilled water while groups II-IV were administered with 1500 mg/kg of Citrus sinensis (fresh orange juice; FOJ), 0.1 μg/g of levothyroxine (LVT) and 0.01 mg/g of CARB, respectively, per oral once daily for 28 days. The animals were sacrificed under chloroform anaesthesia and blood sample collected by cardiac puncture and processed by standard method to obtain serum. TSH, T4 and T3 were assayed with the serum using ARIA II automated radioimmunoassay instrument. Results: The results showed that TSH level was significantly (P < 0.05) decreased in LVT treated group compared with the FOJ group. T4 was significantly (P < 0.05) decreased in the FOJ and CARB groups compared with the control and LVT groups. LVT significantly increased T4 when compared with FOJ group. T3 was significantly (P < 0.05) decreased in the CARB group compared with the control. Conclusion: These findings suggest that FOJ alters thyroid hormones metabolism to reduce their serum levels with a compensatory elevations of TSH level in a direction similar to CARB. PMID:25013255

Reference values for TSH may be inadequate to define hypothyroidism in persons with morbid obesity: Di@bet.es study.

PubMed

Valdés, Sergio; Maldonado-Araque, Cristina; Lago-Sampedro, Ana; Lillo-Muñoz, Juan Antonio; Garcia-Fuentes, Eduardo; Perez-Valero, Vidal; Gutiérrez-Repiso, Carolina; Garcia-Escobar, Eva; Goday, Albert; Urrutia, Inés; Peláez, Laura; Calle-Pascual, Alfonso; Bordiú, Elena; Castaño, Luis; Castell, Conxa; Delgado, Elias; Menéndez, Edelmiro; Franch-Nadal, Josep; Gaztambide, Sonia; Girbés, Joan; Ortega, Emilio; Vendrell, Joan; Chacón, Matilde R; Javier Chaves, F; Soriguer, Federico; Rojo-Martínez, Gemma

2017-04-01

To analyze the reference range of thyroid-stimulating hormone (TSH) in different BMI categories and its impact on the classification of hypothyroidism. The study included 3,928 individuals free of thyroid disease (without previous thyroid disease, no interfering medications, TSH <10 µUI/mL and thyroid peroxidase antibodies [TPO Abs] <50 IU/mL) who participated in a national, cross-sectional, population-based study and were representative of the adult population of Spain. Data gathered included clinical and demographic characteristics, physical examination, and blood and urine sampling. TSH, free thyroxine, free triiodothyronine, and TPO Ab were analyzed by electrochemiluminescence (E170, Roche Diagnostics, Basel, Switzerland). The reference range (p2.5-97.5) for TSH was estimated as 0.6 to 4.8 µUI/mL in the underweight category (BMI<20 kg/m 2 ), 0.6 to 5.5 µUI/mL in the normal-weight category (BMI 20-24.9 kg/m 2 ), 0.6 to 5.5 µUI/mL in the overweight category (BMI 25-29.9 kg/m 2 ), 0.5 to 5.9 µUI/mL in the obesity category (BMI 30-39.9 kg/m 2 ), and 0.7 to 7.5 µUI/mL in the morbid obesity category (BMI ≥40). By using the reference criteria for the normal-weight population, the prevalence of high TSH levels increased threefold in the morbid obesity category (P < 0.01). Persons with morbid obesity might be inappropriately classified if the standard ranges of normality of TSH for the normal-weight population are applied to them. © 2017 The Obesity Society.

Preparation of Tc-99m-macroaggregated albumin from recombinant human albumin for lung perfusion imaging.

PubMed

Hunt, A P; Frier, M; Johnson, R A; Berezenko, S; Perkins, A C

2006-01-01

Human serum albumin (HSA) extracted from pooled blood taken from human donors is used in the production of (99m)Tc-labelled macroaggregated albumin (MAA) for lung perfusion imaging. However, concerns for the safety of blood-derived products due to potential contamination by infective agents (e.g. new variant CJD), make alternative production methods necessary. Recombinant DNA technology is a promising method of albumin production avoiding problems associated with human-derived HSA. This paper presents results comparing MAA prepared from recombinant human albumin (rHA, Recombumin) (rMAA) with in-house produced HSA MAA (hMAA) and commercially available MAA (cMAA). (99m)Tc-MAA was prepared using previously published production methods by heating a mixture of albumin and stannous chloride in acetate buffer (pH 5.4) at 70 degrees C for 20 min. Parameters investigated include aggregate size, radiolabelling efficiency, radiochemical and aggregate stability at 4 degrees C and in vitro (in whole human blood) at 37 degrees C and biodistribution studies. Results showed that rMAA could be produced with similar morphology, labelling efficiency and stability to hMAA and cMAA. Our findings confirm that rHA shows significant potential as a direct replacement for HSA in commercially available MAA.

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

PubMed

Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

2016-05-01

Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1

PubMed Central

Varghese, Vici; Mitsuya, Yumi; Fessel, W. Jeffrey; Liu, Tommy F.; Melikian, George L.; Katzenstein, David A.; Schiffer, Celia A.; Holmes, Susan P.

2013-01-01

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses. PMID:23796938

Thyrotropin (TSH) regulates triiodothyronine (T3) production in the unicellular Tetrahymena.

PubMed

Csaba, G; Pállinger, Eva

2011-09-01

The aim of the experiments was to study the regulation of triiodothyronine (T3) production in the unicellular Tetrahymena. Untreated and troph-hormone treated specimen were prepared and in different timepoints T3 content was measured and compared by immunocytochemical flow cytometry. 0.1 or 0.001 IU TSH in tryptone-yeast medium stimulated T3 synthesis at 10, 20, 30 min, but does not stimulate after 1 h. The overlapping gonadotropic hormone (GTH) also did it, however only at 10 min. In Losina salt solution (physiological for Tetrahymena) the effect was weaker, however outer amino acid source was not absolutely needed for the production of the hormone. The results show that the TSH regulation of thyroid hormone synthesis (storage, secretion) and troph-hormone overlap can be deduced to a unicellular level. This may allow the hypothesis that the endocrine mechanisms proved at a low level of phylogeny are preserved for the higher ranked organisms.

How sensitive (second-generation) thyroglobulin measurement is changing paradigms for monitoring patients with differentiated thyroid cancer, in the absence or presence of thyroglobulin autoantibodies

PubMed Central

Spencer, Carole; LoPresti, Jonathan; Fatemi, Shireen

2014-01-01

Purpose of review To discuss new insights regarding how sensitive (second-generation) thyroglobulin immunometric assays (Tg2GIMAs), (functional sensitivities ≤0.10 μg/L) necessitate different approaches for postoperative thyroglobulin monitoring of patients with differentiated thyroid cancer (DTC), depending on the presence of thyroglobulin autoantibodies (TgAbs). Recent findings Reliable low-range serum thyroglobulin measurement has both enhanced clinical utility and economic advantages, provided TgAb is absent (∼75% DTC patients). Basal [nonthyroid-stimulating hormone (TSH) stimulated] Tg2GIMA measurement obviates the need for recombinant human TSH stimulation because basal Tg2GIMA below 0.20 μg/L has comparable negative predictive value (>95%) to recombinant human TSH-stimulated thyroglobulin values below the cutoff of 2 μg/L. Now that radioiodine remnant ablation is no longer considered necessary to treat low-risk DTC, the trend and doubling time of low basal thyroglobulin values arising from postsurgical thyroid remnants have recognized prognostic significance. The major limitation of Tg2GIMA testing is interference by TgAb (∼25% DTC patients), causing Tg2GIMA underestimation that can mask disease. When TgAb is present, the trend in TgAb concentrations (measured by the same method) can serve as the primary (surrogate) tumor-marker and be augmented by thyroglobulin measured by a TgAb-resistant class of method (radioimmunoassay or liquid chromatography-tandem mass spectrometry). Summary The growing use of Tg2GIMA measurement is changing paradigms for postoperative DTC monitoring. When TgAb is absent, it is optimal to monitor the basal Tg2GIMA trend and doubling time (using the same method) in preference to recombinant human TSH-stimulated thyroglobulin testing. When TgAb is present, interference renders Tg2GIMA testing unreliable and the trend in serum TgAb concentrations per se (same method) can serve as a (surrogate) tumor-marker. PMID:25122493

AID Mediates Hypermutation by Deaminating Single Stranded DNA

PubMed Central

Dickerson, Sarah K.; Market, Eleonora; Besmer, Eva; Papavasiliou, F. Nina

2003-01-01

Activation-induced deaminase (AID) is a protein indispensable for the diversification of immunoglobulin (Ig) genes by somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion. To date, the precise role of AID in these processes has not been determined. Here we demonstrate that purified, tetrameric AID can deaminate cytidine residues in DNA, but not in RNA. Furthermore, we show that AID will bind and deaminate only single-stranded DNA, which implies a direct, functional link between hypermutation and transcription. Finally, AID does not target mutational hotspots, thus mutational targeting to specific residues must be attributed to different factors. PMID:12756266

Human Rights in the Fourth Decade of the HIV/AIDS Response: An Inspiring Legacy and Urgent Imperative.

PubMed

Enoch, Jamie; Piot, Peter

2017-12-01

More than 35 years since the HIV/AIDS pandemic began, HIV continues to cause almost two million new infections each year, and the "end of AIDS" by 2030 remains elusive. 1 Violations of human rights continue to fuel high rates of new infections among key populations and a generalized epidemic in much of sub-Saharan Africa. Meanwhile, as political shifts worldwide threaten not only HIV funding but also progress toward the globalization of human rights, civil society mobilization and advocacy founded firmly on human rights principles have a more vital role to play than ever. Encouragingly, there are numerous examples of successful integration of human rights-based approaches into HIV prevention and treatment initiatives, and evidence increasingly demonstrates that norms enshrining the respect, protection, and fulfillment of human rights can translate into improved public health. 2 This essay will succinctly trace the historic emergence of human rights as an issue at the heart of the HIV/AIDS response; it will then provide examples of progress and setbacks in recent years and consider the potential for rights promotion to address the structural drivers of HIV. Finally, it will consider how the primacy of human rights in HIV/AIDS has affected other fields of global health and will highlight the continuing imperative to work with civil society to protect and promote human rights to reduce the burden of HIV/AIDS.

Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History

PubMed Central

Tao, Ying; Shi, Mang; Chommanard, Christina; Queen, Krista; Zhang, Jing; Markotter, Wanda; Kuzmin, Ivan V.; Holmes, Edward C.

2017-01-01

ABSTRACT Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. PMID:28077633

Severe Toxoplasma gondii I/III Recombinant-Genotype Encephalitis in a Human Immunodeficiency Virus Patient▿

PubMed Central

Genot, Séverine; Franck, Jacqueline; Forel, Jean-Marie; Rebaudet, Stanislas; Ajzenberg, Daniel; de Paula, Andre Maues; Dardé, Marie-Laure; Stein, Andreas; Ranque, Stéphane

2007-01-01

The reactivation of an uncommon type I/III recombinant-genotype Toxoplasma gondii strain resulted in unusually severe encephalitis and chorioretinitis associated with a cerebral salt wasting syndrome in an African human immunodeficiency virus patient. This observation suggests an influence of the parasite genotype on disease expression in immunocompromised patients. PMID:17634310

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

PubMed Central

White, Clayton A.; Li, Guideng; Pone, Egest J.; Xu, Zhenming; Casali, Paolo

2013-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5′-AGCT-3′ repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S–S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180–198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR. PMID:24282540

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells.

PubMed Central

Takeuchi, M; Inoue, N; Strickland, T W; Kubota, M; Wada, M; Shimizu, R; Hoshi, S; Kozutsumi, H; Takasaki, S; Kobata, A

1989-01-01

Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO. PMID:2813359

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

PubMed

Palacios, Florencia; Moreno, Pilar; Morande, Pablo; Abreu, Cecilia; Correa, Agustín; Porro, Valentina; Landoni, Ana Ines; Gabus, Raul; Giordano, Mirta; Dighiero, Guillermo; Pritsch, Otto; Oppezzo, Pablo

2010-06-03

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Flavonoids of cocoa inhibit recombinant human 5-lipoxygenase.

PubMed

Schewe, Tankred; Kühn, Hartmut; Sies, Helmut

2002-07-01

(-)-Epicatechin and its related oligomers, the procyanidins, are present in sizable amounts in some cocoas and chocolates. Intake of flavonoid-rich chocolate in humans has been reported to increase the plasma level of (-)-epicatechin and concomitantly to significantly decrease the plasma level of proinflammatory cysteinyl leukotrienes. Because leukotrienes are formed via the 5-lipoxygenase pathway of arachidonic acid metabolism, we examined whether 5-lipoxygenase is a possible target for the flavonoids of cocoa. Recombinant human 5-lipoxygenase was reacted with arachidonic acid and yielded a mixture of mainly 5-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-HpETE) and hydrolysis products of 5,6-leukotriene A(4) (LTA(4)). The formation of these products was significantly inhibited by (-)-epicatechin in a dose-dependent manner with 50% inhibitory concentrations (IC(50)) of 22 and 50 micromol/L, respectively. Among the procyanidin fractions isolated from the seeds of Theobroma cacao, only the dimer fraction and, to a lesser extent, the trimer through pentamer fractions exhibited comparable effects, whereas the larger procyanidins (hexamer through nonamer) were almost inactive. We conclude that (-)-epicatechin and its low-molecular procyanidins inhibit both dioxygenase and LTA(4) synthase activities of human 5-lipoxygenase and that this action may contribute to a putative anti-inflammatory effect of cocoa products.

Characteristics of recombinantly expressed rat and human histamine H3 receptors.

PubMed

Wulff, Birgitte S; Hastrup, Sven; Rimvall, Karin

2002-10-18

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor. Copyright 2002 Elsevier Science B.V.

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

2008-05-01

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Treatment of dwarfism with recombinant human insulin-like growth factor-1.

PubMed

Ranke, Michael B; Wölfle, Joachim; Schnabel, Dirk; Bettendorf, Markus

2009-10-01

The growth hormone-IGF (insulin-like growth factor) system plays a central role in hormonal growth regulation. Recombinant human (rh) growth hormone (GH) has been available since the late 1980s for replacement therapy in GH-deficient patients and for the stimulation of growth in patients with short stature of various causes. Growth promotion by GH occurs in part indirectly through the induction of IGF-1 synthesis. In primary disturbances of IGF-1 production, short stature can only be treated with recombinant human IGF-1 (rhIGF-1). rhIGF-1 was recently approved for this indication but can also be used to treat other conditions. Selective review of the literature on IGF-1 therapy, based on a PubMed search. In children with severe primary IGF-1 deficiency (a rare condition whose prevalence is less than 1:10,000), the prognosis for final height is very poor (ca. 130 cm), and IGF-1 therapy is the appropriate form of pathophysiologically based treatment. There is no alternative treatment at present. The subcutaneous administration of IGF-1 twice daily in doses of 80 to 120 microg/kg accelerates growth and increases final height by 12 to 15 cm, according to current data. There is, however, a risk of hypoglycemia, as IGF-1 has an insulin-like effect. As treatment with IGF-1 is complex, this new medication should only be prescribed, for the time being, by experienced pediatric endocrinologists and diabetologists.

Protection of non-human primates against rabies with an adenovirus recombinant vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Z.Q.; Greenberg, L.; Ertl, H.C., E-mail: ertl@wistar.upenn.edu

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protectsmore » against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.« less

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

PubMed

Geng, Zhirong; Song, Xiaoli; Xing, Zhi; Geng, Jinlong; Zhang, Sichun; Zhang, Xinrong; Wang, Zhilin

2009-05-01

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

PubMed

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

Site-specific genetic recombination: hops, flips, and flops.

PubMed

Sadowski, P D

1993-06-01

Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Impact of TSH during the first trimester of pregnancy on obstetric and foetal complications: Usefulness of 2.5 mIU/L cut-off value.

PubMed

Hernández, Marta; López, Carolina; Soldevila, Berta; Cecenarro, Laura; Martínez-Barahona, María; Palomera, Elisabet; Rius, Ferran; Lecube, Albert; Pelegay, Maria José; García, Jordi; Mauricio, Dídac; Puig Domingo, Manel

2018-05-01

An association of pregnancy outcomes with subclinical hypothyroidism has been reported; however, there still exists a strong controversy regarding whether subclinical hypothyroidism ought to be dealt with or not. The objective of the study was to evaluate the association of foetal-maternal complications with first trimester maternal Thyrotropin (TSH) values. A retrospective study in a single tertiary care hospital was performed. A total of 1981 pregnant women were studied during 2012. Thyrotropin (TSH) universal screening was performed between 9 and 12 weeks of gestation. Outcomes included foetal-maternal complications and newborn health parameters. Median TSH was 1.72 (0.99-2.61) mIU/L. The incidence of perinatal loss, miscarriage and stillbirth was 7.2%, 5.9% and 1.1%, respectively. Median TSH of women with and without miscarriage was 1.97 (1.29-3.28) vs 1.71 (0.96-2.58) mIU/L (P = .009). Incidence of pre-eclampsia was 3.2%; TSH in these women was 2.10 (1.40-2.74) vs 1.71 (0.98-2.59) mIU/L in those without (P = .027). TSH in women with dystocia in labour was 1.76 (1.00-2.53) vs 1.68 (0.94-2.59) mIU/L in those who gave birth with normal progression (P = .044). Women with TSH 2.5-5.1 mIU/L had a higher risk of perinatal loss [OR 1.589 (1.085-2.329)], miscarriage [OR 1.702 (1.126-2.572)] and premature birth [OR 1.39 (1.013-1.876)], adjusted by mother's age. There was no association with the other outcomes analysed. There is a positive association between maternal TSH in the first trimester of pregnancy and the incidence of perinatal loss and miscarriage. The TSH cut-off value of 2.5 mIU/L identified women with higher adverse pregnancy outcomes. © 2018 John Wiley & Sons Ltd.

Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

PubMed Central

2013-01-01

Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis. PMID:23784855

A novel platform for biologically active recombinant human interleukin-13 production.

PubMed

Wang, David J; Brandsma, Martin; Yin, Ziqin; Wang, Aiming; Jevnikar, Anthony M; Ma, Shengwu

2008-06-01

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Integrated Summary Report: Validation of Two Binding Assays Using Human Recombinant Estrogen Receptor Alpha (hrERa)

EPA Science Inventory

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (h...

Human-centered automation and AI - Ideas, insights, and issues from the Intelligent Cockpit Aids research effort

NASA Technical Reports Server (NTRS)

Abbott, Kathy H.; Schutte, Paul C.

1989-01-01

A development status evaluation is presented for the NASA-Langley Intelligent Cockpit Aids research program, which encompasses AI, human/machine interfaces, and conventional automation. Attention is being given to decision-aiding concepts for human-centered automation, with emphasis on inflight subsystem fault management, inflight mission replanning, and communications management. The cockpit envisioned is for advanced commercial transport aircraft.

78 FR 78838 - Grant of Interim Extension of the Term of U.S. Patent No. 5,496,801; Recombinant Human...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-27

... Hormone AGENCY: United States Patent and Trademark Office, Commerce. ACTION: Notice of interim patent term... No. 5,496,801. The patent claims the human biological product recombinant human parathyroid hormone... human parathyroid hormone, was filed on October 24, 2013, and is currently undergoing regulatory review...

Non-hyperfunctioning nodules from multinodular goiters: a minor role in pathogenesis for somatic activating mutations in the TSH-receptor and Gsalpha subunit genes.

PubMed

Derrien, C; Sonnet, E; Gicquel, I; Le Gall, J Y; Poirier, J Y; David, V; Maugendre, D

2001-05-01

Constitutive activation of the cAMP pathway stimulates thyrocyte proliferation. Gain-of-function mutations in Gsalpha protein have already been identified in thyroid nodules which have lost the ability to trap iodine. In contrast, most of the studies failed to detect somatic activating mutations in the thyrotropin receptor (TSH-R) in non-hyperfunctioning thyroid tumors. The aim of this study was to screen for mutations TSH-R exon 10, encoding the whole intracytoplasmic area involved in signal transduction, and Gsalpha exons 8 and 9, containing the two hot-spot codons 201 and 227, in a subset of non-hyperfunctioning nodules from multinodular goiter. Identified by matching ultrasonography and scintiscan, 22 eufunctioning (normal 99Tc uptake) and 15 nonfunctioning (decreased 99Tc uptake) nodules from 27 non-toxic multinodular goiters were isolated. After DNA extraction, TSH-R exon 10 was analyzed by direct sequencing of the PCR products and Gsalpha exons 8 and 9 by Denaturing Gradient Gel Electrophoresis. No mutation of TSH-R or Gsalpha was detected in the 37 nodules analyzed. This absence of mutation, despite the use of two sensitive screening methods associated with the analysis of the TSH-R whole intracytoplasmic area and Gsalpha two hot-spot codons, suggests that TSH-R and Gsalpha play a minor role in the pathogenesis of non-toxic nodules from multinodular goiters.

Genetic Variants Associated with Serum Thyroid Stimulating Hormone (TSH) Levels in European Americans and African Americans from the eMERGE Network

PubMed Central

Malinowski, Jennifer R.; Denny, Joshua C.; Bielinski, Suzette J.; Basford, Melissa A.; Bradford, Yuki; Peissig, Peggy L.; Carrell, David; Crosslin, David R.; Pathak, Jyotishman; Rasmussen, Luke; Pacheco, Jennifer; Kho, Abel; Newton, Katherine M.; Li, Rongling; Kullo, Iftikhar J.; Chute, Christopher G.; Chisholm, Rex L.; Jarvik, Gail P.; Larson, Eric B.; McCarty, Catherine A.; Masys, Daniel R.; Roden, Dan M.; de Andrade, Mariza; Ritchie, Marylyn D.; Crawford, Dana C.

2014-01-01

Thyroid stimulating hormone (TSH) hormone levels are normally tightly regulated within an individual; thus, relatively small variations may indicate thyroid disease. Genome-wide association studies (GWAS) have identified variants in PDE8B and FOXE1 that are associated with TSH levels. However, prior studies lacked racial/ethnic diversity, limiting the generalization of these findings to individuals of non-European ethnicities. The Electronic Medical Records and Genomics (eMERGE) Network is a collaboration across institutions with biobanks linked to electronic medical records (EMRs). The eMERGE Network uses EMR-derived phenotypes to perform GWAS in diverse populations for a variety of phenotypes. In this report, we identified serum TSH levels from 4,501 European American and 351 African American euthyroid individuals in the eMERGE Network with existing GWAS data. Tests of association were performed using linear regression and adjusted for age, sex, body mass index (BMI), and principal components, assuming an additive genetic model. Our results replicate the known association of PDE8B with serum TSH levels in European Americans (rs2046045 p = 1.85×10−17, β = 0.09). FOXE1 variants, associated with hypothyroidism, were not genome-wide significant (rs10759944: p = 1.08×10−6, β = −0.05). No SNPs reached genome-wide significance in African Americans. However, multiple known associations with TSH levels in European ancestry were nominally significant in African Americans, including PDE8B (rs2046045 p = 0.03, β = −0.09), VEGFA (rs11755845 p = 0.01, β = −0.13), and NFIA (rs334699 p = 1.50×10−3, β = −0.17). We found little evidence that SNPs previously associated with other thyroid-related disorders were associated with serum TSH levels in this study. These results support the previously reported association between PDE8B and serum TSH levels in European Americans and emphasize the need for additional genetic

Genetic variants associated with serum thyroid stimulating hormone (TSH) levels in European Americans and African Americans from the eMERGE Network.

PubMed

Malinowski, Jennifer R; Denny, Joshua C; Bielinski, Suzette J; Basford, Melissa A; Bradford, Yuki; Peissig, Peggy L; Carrell, David; Crosslin, David R; Pathak, Jyotishman; Rasmussen, Luke; Pacheco, Jennifer; Kho, Abel; Newton, Katherine M; Li, Rongling; Kullo, Iftikhar J; Chute, Christopher G; Chisholm, Rex L; Jarvik, Gail P; Larson, Eric B; McCarty, Catherine A; Masys, Daniel R; Roden, Dan M; de Andrade, Mariza; Ritchie, Marylyn D; Crawford, Dana C

2014-01-01

Thyroid stimulating hormone (TSH) hormone levels are normally tightly regulated within an individual; thus, relatively small variations may indicate thyroid disease. Genome-wide association studies (GWAS) have identified variants in PDE8B and FOXE1 that are associated with TSH levels. However, prior studies lacked racial/ethnic diversity, limiting the generalization of these findings to individuals of non-European ethnicities. The Electronic Medical Records and Genomics (eMERGE) Network is a collaboration across institutions with biobanks linked to electronic medical records (EMRs). The eMERGE Network uses EMR-derived phenotypes to perform GWAS in diverse populations for a variety of phenotypes. In this report, we identified serum TSH levels from 4,501 European American and 351 African American euthyroid individuals in the eMERGE Network with existing GWAS data. Tests of association were performed using linear regression and adjusted for age, sex, body mass index (BMI), and principal components, assuming an additive genetic model. Our results replicate the known association of PDE8B with serum TSH levels in European Americans (rs2046045 p = 1.85×10-17, β = 0.09). FOXE1 variants, associated with hypothyroidism, were not genome-wide significant (rs10759944: p = 1.08×10-6, β = -0.05). No SNPs reached genome-wide significance in African Americans. However, multiple known associations with TSH levels in European ancestry were nominally significant in African Americans, including PDE8B (rs2046045 p = 0.03, β = -0.09), VEGFA (rs11755845 p = 0.01, β = -0.13), and NFIA (rs334699 p = 1.50×10-3, β = -0.17). We found little evidence that SNPs previously associated with other thyroid-related disorders were associated with serum TSH levels in this study. These results support the previously reported association between PDE8B and serum TSH levels in European Americans and emphasize the need for additional genetic studies in more

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

PubMed

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens

PubMed Central

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies. PMID:26713728

Update in TSH Receptor Agonists and Antagonists

PubMed Central

Neumann, Susanne

2012-01-01

The physiological role of the TSH receptor (TSHR) as a major regulator of thyroid function is well understood, but TSHRs are also expressed in multiple normal extrathyroidal tissues, and the physiological roles of TSHRs in these tissues are unclear. Moreover, TSHRs play a major role in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Small molecule, “drug-like” TSHR agonists, neutral antagonists, and inverse agonists may be useful as probes of TSHR function in extrathyroidal tissues and as leads to develop drugs for several diseases of the thyroid. In this Update, we review the most recent findings regarding the development and use of these small molecule TSHR ligands. PMID:23019348

Computer aided systems human engineering: A hypermedia tool

NASA Technical Reports Server (NTRS)

Boff, Kenneth R.; Monk, Donald L.; Cody, William J.

1992-01-01

The Computer Aided Systems Human Engineering (CASHE) system, Version 1.0, is a multimedia ergonomics database on CD-ROM for the Apple Macintosh II computer, being developed for use by human system designers, educators, and researchers. It will initially be available on CD-ROM and will allow users to access ergonomics data and models stored electronically as text, graphics, and audio. The CASHE CD-ROM, Version 1.0 will contain the Boff and Lincoln (1988) Engineering Data Compendium, MIL-STD-1472D and a unique, interactive simulation capability, the Perception and Performance Prototyper. Its features also include a specialized data retrieval, scaling, and analysis capability and the state of the art in information retrieval, browsing, and navigation.

Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis.

PubMed

Pathak, Prachi; Kumar, Ashu; Thavaselvam, Duraipandian

2017-07-11

Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). The rOmp2a was found to be

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

PubMed Central

Le, Quy; Maizels, Nancy

2015-01-01

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome. PMID:26355458

Recombination gives a new insight in the effective population size and the history of the old world human populations.

PubMed

Melé, Marta; Javed, Asif; Pybus, Marc; Zalloua, Pierre; Haber, Marc; Comas, David; Netea, Mihai G; Balanovsky, Oleg; Balanovska, Elena; Jin, Li; Yang, Yajun; Pitchappan, R M; Arunkumar, G; Parida, Laxmi; Calafell, Francesc; Bertranpetit, Jaume

2012-01-01

The information left by recombination in our genomes can be used to make inferences on our recent evolutionary history. Specifically, the number of past recombination events in a population sample is a function of its effective population size (Ne). We have applied a method, Identifying Recombination in Sequences (IRiS), to detect specific past recombination events in 30 Old World populations to infer their Ne. We have found that sub-Saharan African populations have an Ne that is approximately four times greater than those of non-African populations and that outside of Africa, South Asian populations had the largest Ne. We also observe that the patterns of recombinational diversity of these populations correlate with distance out of Africa if that distance is measured along a path crossing South Arabia. No such correlation is found through a Sinai route, suggesting that anatomically modern humans first left Africa through the Bab-el-Mandeb strait rather than through present Egypt.

Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering.

PubMed

Bozzini, Sabrina; Giuliano, Liliana; Altomare, Lina; Petrini, Paola; Bandiera, Antonella; Conconi, Maria Teresa; Farè, Silvia; Tanzi, Maria Cristina

2011-12-01

The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.

Models of Human Information Requirements: "When Reasonable Aiding Systems Disagree"

NASA Technical Reports Server (NTRS)

Corker, Kevin; Pisanich, Gregory; Shafto, Michael (Technical Monitor)

1994-01-01

Aircraft flight management and Air Traffic Control (ATC) automation are under development to maximize the economy of flight and to increase the capacity of the terminal area airspace while maintaining levels of flight safety equal to or better than current system performance. These goals are being realized by the introduction of flight management automation aiding and operations support systems on the flight deck and by new developments of ATC aiding systems that seek to optimize scheduling of aircraft while potentially reducing required separation and accounting for weather and wake vortex turbulence. Aiding systems on both the flight deck and the ground operate through algorithmic functions on models of the aircraft and of the airspace. These models may differ from each other as a result of variations in their models of the immediate environment. The resultant flight operations or ATC commands may differ in their response requirements (e.g. different preferred descent speeds or descent initiation points). The human operators in the system must then interact with the automation to reconcile differences and resolve conflicts. We have developed a model of human performance including cognitive functions (decision-making, rule-based reasoning, procedural interruption recovery and forgetting) that supports analysis of the information requirements for resolution of flight aiding and ATC conflicts. The model represents multiple individuals in the flight crew and in ATC. The model is supported in simulation on a Silicon Graphics' workstation using Allegro Lisp. Design guidelines for aviation automation aiding systems have been developed using the model's specification of information and team procedural requirements. Empirical data on flight deck operations from full-mission flight simulation are provided to support the model's predictions. The paper describes the model, its development and implementation, the simulation test of the model predictions, and the empirical

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase.

PubMed

Zhou, Min; Kitagawa, Yoshinori; Yamaguchi, Mayu; Uchiyama, Chika; Itoh, Masae; Gotoh, Bin

2013-01-01

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

PubMed

Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

2016-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Reference Values for TSH and Free Thyroid Hormones in Healthy Pregnant Women in Poland: A Prospective, Multicenter Study.

PubMed

Kostecka-Matyja, Marta; Fedorowicz, Anna; Bar-Andziak, Ewa; Bednarczuk, Tomasz; Buziak-Bereza, Monika; Dumnicka, Paulina; Górska, Maria; Krasnodębska, Małgorzata; Niedźwiedzka, Beata; Pach, Dorota; Ruchała, Marek; Siewko, Katarzyna; Solnica, Bogdan; Sowiński, Jerzy; Szelachowska, Małgorzata; Trofimiuk-Müldner, Małgorzata; Wachowiak-Ochmańska, Katarzyna; Hubalewska-Dydejczyk, Alicja

2017-04-01

The diagnosis and treatment of thyroid diseases in pregnant women remains a challenge. Various medical associations recommend establishing the reference intervals for thyroid hormones by a local laboratory. Considering differences within geophysical, socioeconomic conditions, and iodine prophylaxis in various populations, it is advisable to assess reference intervals for thyroid hormones specific to a region of residence. The objective was to assess trimester-specific reference intervals for TSH, fT 3 , and fT 4 for pregnant women in the Polish population. We conducted a prospective study in 4 centers representing different regions of Poland (Krakow, Warsaw, Poznan, and Bialystok). Our study included consecutive, healthy pregnant women (172 patients), with an age range of 27-47 years. All women had a negative history for thyroid diseases, normal thyroid peroxidase antibody levels, and proper iodine prophylaxis. All newborns had TSH levels in the appropriate reference range. Serum TSH, fT 3 , fT 4 , and thyroid-peroxidase antibodies were measured in each trimester. The reference intervals were calculated using the percentile method, as recommended by the International Federation of Clinical Chemistry. The reference values calculated were 0.009-3.177, 0.05-3.442, and 0.11-3.53 mIU/L for TSH; 3.63-6.55, 3.29-5.45, and 3.1-5.37 pmol/L for fT 3 ; and 11.99-21.89, 10.46-16.67, and 8.96-17.23 pmol/L for fT 4 in consecutive trimesters of pregnancy. Reference intervals for pregnant women when compared to the general population showed a lower concentration of TSH in every trimester of pregnancy and lower fT 4 in the 2nd and 3rd trimesters. Using appropriate trimester-specific reference intervals may improve care of pregnant women by preventing misdiagnosis and inadequate treatment.

A large-scale association analysis of 68 thyroid hormone pathway genes with serum TSH and FT4 levels.

PubMed

Medici, Marco; van der Deure, Wendy M; Verbiest, Michael; Vermeulen, Sita H; Hansen, Pia S; Kiemeney, Lambertus A; Hermus, Ad R M M; Breteler, Monique M; Hofman, Albert; Hegedüs, Laszlo; Kyvik, Kirsten Ohm; den Heijer, Martin; Uitterlinden, André G; Visser, Theo J; Peeters, Robin P

2011-05-01

Minor variation in serum thyroid hormone (TH) levels can have important effects on various clinical endpoints. Although 45-65% of the inter-individual variation in serum TH levels is due to genetic factors, the causative genes are not well established. We therefore studied the effects of genetic variation in 68 TH pathway genes on serum TSH and free thyroxine (FT(4)) levels. Sixty-eight genes (1512 polymorphisms) were studied in relation to serum TSH and FT(4) levels in 1121 Caucasian subjects. Promising hits (P<0.01) were studied in three independent Caucasian populations (2656 subjects) for confirmation. A meta-analysis of all four studies was performed. For TSH, eight PDE8B polymorphisms (P=4×10(-17)) remained significant in the meta-analysis. For FT(4), two DIO1 (P=8×10(-12)) and one FOXE1 (P=0.0003) polymorphisms remained significant in the meta-analysis. Suggestive associations were detected for one FOXE1 (P=0.0028) and three THRB (P=0.0045) polymorphisms with TSH, and one SLC16A10 polymorphism (P=0.0110) with FT(4), but failed to reach the significant multiple-testing corrected P value (P<0.0022 and P<0.0033 respectively). Using a large-scale association analysis, we replicated previously reported associations with genetic variation in PDE8B, THRB, and DIO1. We demonstrate effects of genetic variation in FOXE1 on serum FT(4) levels, and borderline significant effects on serum TSH levels. A suggestive association of genetic variation in SLC16A10 with serum FT(4) levels was found. These data provide insight into the molecular basis of inter-individual variation in TH serum levels.

Corticotropin-releasing factor accelerates metamorphosis in Bufo arenarum: effect on pituitary ACTH and TSH cells.

PubMed

Miranda, L A; Affanni, J M; Paz, D A

2000-04-01

The actions of several neuropeptides as hypothalamic mediators in the regulation of Bufo arenarum metamorphosis were investigated. Prometamorphic larvae were injected with 1.5 microg thyrotropin-releasing hormone (TRH), 2 microg ovine corticotropin-releasing factor (oCRF), 2 microg mammalian gonadotropin-releasing hormone (mGnRH), 2 microg human growth hormone-releasing hormone (hGHRH), or Holtfreter solution (control group). Larvae received two injections with the same dose: one at the beginning of the experiment and the other 7 days later. Several morphologic parameters (total length, tail length, wet weight, hind limb length, and metamorphic stages) were measured as indicators of growth and metamorphic development. These measurements were taken in 20 larvae per treatment or control group at the beginning of the experiment, at day 7 and at day 14 when the experiment ended. We observed that only the administration of exogenous CRF stimulated resorption of the tail and accelerated the rate of metamorphosis. In the pituitary of CRF-treated larvae we observed that thyrotropin (TSH) and adrenocorticotropic hormone (ACTH) producing cells showed a weaker immunoreactivity, a decrease in cell number and a reduction of volume density when compared with normal larvae. In conclusion, the results obtained indicate a possible role for CRF in Bufo arenarum metamorphosis. CRF may regulate interrenal and thyroid activity by acting directly upon TSH and ACTH cells. On the other hand, TRH, GnRH and GHRH were inactive in stimulating growth or metamorphosis of Bufo arenarum. J. Exp. Zool. 286:473-480, 2000. Copyright 2000 Wiley-Liss, Inc.

Guidelines to reach high-quality purified recombinant proteins.

PubMed

Oliveira, Carla; Domingues, Lucília

2018-01-01

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain.

PubMed

Nandi, Somen; Yalda, Dorice; Lu, Stephen; Nikolov, Zivko; Misaki, Ryo; Fujiyama, Kazuhito; Huang, Ning

2005-06-01

In this paper, we show that recombinant human lactoferrin (rhLF) has been stably expressed at 0.5% brown rice flour weight for nine generations. Process development indicates that rhLF can be efficiently extracted from rice flour in 20 mM phosphate buffer (pH 7.0) containing up to 0.5 M NaCl and at a ratio of 1 kg flour to 10 L buffer. After solid/liquid separation, the extract can then be loaded directly onto an ion-exchange column and rhLF can be eluted using 0.8 M NaCl. The resulting rhLF is about 95% pure. A range of biochemical and biophysical analyses were carried out and results indicated that the purified rhLF was identical to its native human counterpart other than its glycosylation. Economic analysis shows that at 600 kg/year scale, the cash cost to produce 1 g of rhLF of pharmaceutical grade is US$ 5.90. Analysis also indicates that the expression level has profound impact on costs related to planting, milling, extraction and purification, thus high level expression of recombinant protein in plants is one of the key parameters for the success of plant made pharmaceuticals.

[Genetic evidence for recombination and mutation in the emergence of human enterovirus 71].

PubMed

Liu, Ai-Ping; Tan, Hui; Xie, Qun; Chen, Bai-Tang; Liu, Xiao-Feng; Zhang, Yong

2014-09-01

We wished to understand the genetic recombination and phylogenetic characteristics of human en- terovirus A71 (EV-A71) and to explore its potential virulence-related sites. Full-length genomes of three EV-A71 strains isolated from patients in Chenzhou City (China) were sequenced and analyzed. Possible re- combination events and crossover sites were analyzed with Recombination Detection Program v4. 1. 6 by comparison with the complete genome sequences of 231 strains of EV-A71. Similarly, plot and bootscanning analyses were undertaken with SimPlot v3. 5. 1. Phylogenetic trees based on the sequences of VP1 regions were constructed with MEGA v5. 2 using the Kimura two-parameter model and neighbor-joining method. Results suggested that recombination events were detected among the three EV-A71 isolates from Chenzhou City. The common main parent sequence was from JF799986 isolated from samples in Guang- zhou City (China) in 2009, and the minor parent sequence was TW/70516/08. Intertypic recombination e- vents were found in the C4b strain (strain SHZH98 isolated in 1998) and C4a strain (Fuyang strain isola- ted in 2008) with the prototype strains of CVA4 and CVA14 in the 3D region. The chi-square test was used to screen-out potential virulence-related sites with nucleotide substitutions of different types of hand, foot, and mouth disease (HFMD) cases using SPSS v19.0. Results suggested that there were no significant nucleotide substitutions between death cases and severe-HFMD cases. Eighteen significant nucleotide substitutions were found between death/severe-HFMD cases and mild-HFMD cases, and all these 18 substitutions were distributed only in P2 and P3 regions. Intertypic recombination among the predominant circulating EV-A71 strains in the Chinese mainland and other EV-A strains probably dates before 1998, and intratypic recombination might have occurred frequently in the HFMD outbreak from 2008 to 2012. Substitutions in the non-capsid region may be correlated with the

Recombinant human erythropoietin therapy in low-birthweight preterm infants: a prospective controlled study.

PubMed

Arif, Bahar; Ferhan, Karademir

2005-02-01

This study aimed to detect the effectiveness of recombinant human erythropoietin therapy in preventing premature anemia in low-birthweight preterm infants. A total of 292 premature infants who were born earlier than 33 gestational weeks and smaller than 1500 g birthweight were enrolled into the study. In addition to their conventional supportive therapy (medications), recombinant human erythropoietin 200 U/kg twice a week, subcutaneously, was given to randomly selected 142 premature infants for 6 weeks. The blood count variables and need for transfusions were compared with the remaining 150 premature infants during 6 months follow up. Serum erythropoietin levels were 11.3 +/- 6.1 mU/mL and 38.3 +/- 19.1 mU/mL in the erythropoietin group before and at the fourth week of the study, respectively (P < 0.001). Reticulocyte counts of the group treated with erythropoietin were 146 x 10(6) +/- 28 x 10(6)/mL and 122 x 10(6) +/- 27 x 10(6)/mL at the fourth and seventh week of the study, respectively, and these values were significantly higher when compared with the control group (P < 0.001 and P < 0.001). At the same period, hematocrit values were also found to be higher in the treatment group than the control group (P < 0.001). Serum ferritin levels were lower in the treatment group compared with the control group at the fourth week of the study. No side-effects related to erythropoietin usage were encountered. The need for packed cell transfusions were 47% in the group treated with erythropoietin and 62.6% in the control group. A statistically significant difference was found for transfusion needs between the control and treatment groups (P < 0.001). Recombinant erythropoietin is effective therapy for maintaining stable hematocrit levels in low-birthweight preterm infants and prevents the need for blood transfusions.

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atsmon, Jacob; Sackler Faculty of Medicine, Tel Aviv University; Brill-Almon, Einat

PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2more » min before being exposed to 1.33 × LD{sub 50} and 1.5 × LD{sub 50} of toxin and 10 min after exposure to 1.5 × LD{sub 50} survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t{sub ½}) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t{sub ½} in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human

Assessment of thyroid function during first-trimester pregnancy: what is the rational upper limit of serum TSH during the first trimester in Chinese pregnant women?

PubMed

Li, Chenyan; Shan, Zhongyan; Mao, Jinyuan; Wang, Weiwei; Xie, Xiaochen; Zhou, Weiwei; Li, Chenyang; Xu, Bin; Bi, Lihua; Meng, Tao; Du, Jianling; Zhang, Shaowei; Gao, Zhengnan; Zhang, Xiaomei; Yang, Liu; Fan, Chenling; Teng, Weiping

2014-01-01

Guidelines of the American Thyroid Association (ATA) proposed that the upper limit of the TSH reference range should be 2.5 mIU/L in first trimester, but the reported ranges in China are significantly higher. Our objective was to establish a rational reference range of serum TSH for diagnosis of subclinical hypothyroidism in the first trimester of pregnant women in China. We screened 4800 pregnant women in the first trimester and 2000 women who planned to become pregnant and evaluated 535 pregnant women in follow-up visits during the second and third trimester. Median concentrations of serum TSH decreased significantly from the seventh week of gestation. The median of TSH from 4 to 6 weeks was significantly higher than from 7 to 12 weeks (2.15 [0.56-5.31] mIU/L vs 1.47 [0.10-4.34] mIU/L, P<.001); however, there was no significant difference compared with nonpregnant women (2.07 [0.69-5.64] mIU/L; P=.784). The median of free T4 was not significantly altered in the first trimester. The prevalence of subclinical hypothyroidism in the 4800 pregnant women was 27.8% on the diagnostic criteria of TSH>2.5 mIU/L and 4.0% using the reference interval derived by our laboratory (0.14-4.87 mIU/L).Additionally, of 118 pregnant women who had serum TSH>2.5 mIU/L in the first trimester, only 30.0% and 20.3% of them at the 20th and 30th week of gestation had TSH>3.0 mIU/L. The reference range for nonpregnant women can be used for the assessment of pregnant women at 4 to 6 weeks of gestation. The upper limit of serum TSH in the first trimester was much higher than 2.5 mIU/L in Chinese pregnant women.

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

PubMed

Bian, Liujiao; Ji, Xu; Hu, Wei

2014-07-01

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.

TSH evaluation in hypothyroid patients assuming liquid levothyroxine at breakfast or 30 min before breakfast.

PubMed

Pirola, I; Gandossi, E; Brancato, D; Marini, F; Cristiano, A; Delbarba, A; Agosti, B; Castellano, M; Cappelli, C

2018-03-26

To compare TSH levels of hypothyroid patients treated with liquid LT4 at breakfast or 30 min before breakfast. Subjects, aged 18-75 years old, were eligible if they presented hypothyroidism, due to Hashimoto's thyroiditis or after thyroidectomy for proven benign goiter. Seven hundred ninety-eight patients were recruited and enrolled in the study. Thirty-seven subjects withdrew from the trial. A total of 761 patients (mean age 46.2 ± 10.8 years) completed the study. The starting dose of LT4 was determined through clinical judgment, taking into account TSH levels, estimated residual thyroid function, age, body weight and comorbidities. All patients underwent TSH, fT4, and fT3 evaluation to verify achievement of euthyroidism with their initial fasting state assumption of LT4 after 8 weeks of therapy. If euthyroidism was not achieved, an appropriately adjusted LT4 dose was administered for 8 weeks, after which thyroid function parameters were checked again. If euthyroidism was achieved, the patients were asked to take LT4 at breakfast and hormone levels were checked again after 6 months. At the end of the study period, no significant differences in serum TSH level were observed whether LT4 was ingested at breakfast or 30 min prior in a fasting state: 2.61 ± 1.79 vs. 2.54 ± 1.86 mIU/L, respectively (p = 0.455). This study confirms in a large set of patients that a liquid LT4 formulation can be taken directly at breakfast and potentially improve therapeutic compliance.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

PubMed Central

Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

1993-01-01

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii.

PubMed

Xu, Jianping; Yan, Zhun; Guo, Hong

2009-06-01

The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii. The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii, but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.

Loss of Tumor Suppressor STAG2 Promotes Telomere Recombination and Extends the Replicative Lifespan of Normal Human Cells.

PubMed

Daniloski, Zharko; Smith, Susan

2017-10-15

Sister chromatids are held together by cohesin, a tripartite ring with a peripheral SA1/2 subunit, where SA1 is required for telomere cohesion and SA2 for centromere cohesion. The STAG2 gene encoding SA2 is often inactivated in human cancer, but not in in a manner associated with aneuploidy. Thus, how these tumors maintain chromosomal cohesion and how STAG2 loss contributes to tumorigenesis remain open questions. Here we show that, despite a loss in centromere cohesion, sister chromatids in STAG2 mutant tumor cells maintain cohesion in mitosis at chromosome arms and telomeres. Telomere maintenance in STAG2 mutant tumor cells occurred by either telomere recombination or telomerase activation mechanisms. Notably, these cells were refractory to telomerase inhibitors, indicating recombination can provide an alternative means of telomere maintenance. STAG2 silencing in normal human cells that lack telomerase led to increased recombination at telomeres, delayed telomere shortening, and postponed senescence onset. Insofar as telomere shortening and replicative senescence prevent genomic instability and cancer by limiting the number of cell divisions, our findings suggest that extending the lifespan of normal human cells due to inactivation of STAG2 could promote tumorigenesis by extending the period during which tumor-driving mutations occur. Cancer Res; 77(20); 5530-42. ©2017 AACR . ©2017 American Association for Cancer Research.

Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

PubMed Central

Dinarello, C A; O'Connor, J V; LoPreste, G; Swift, R L

1984-01-01

Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot

Motion sickness susceptibility related to ACTH, ADH and TSH

NASA Technical Reports Server (NTRS)

Kohl, R. L.; Leach, C.; Homick, J. L.; Larochelle, F. T.

1983-01-01

The hypothesis that endogenous levels of certain hormones might be indicative of an individual's susceptibility to stressful motion is tested in a comparison of subjects classified as less prone to motion sickness with those of higher susceptibility. The levels of ACTH and vasopressin measured before exposure to stressful motion were twice as high in the less-suceptible group. No significant differences were noted in the levels of angiotensin, aldosterone, or TSH. The differences between the two groups were greater for a given hormone than for any of the changes induced by exposure to stressful motion.

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

PubMed Central

Crow, J. Allen; Herring, Katye L.; Xie, Shuqi; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2009-01-01

Summary Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC50=8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (Kiapp=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (Kiapp=1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which

Circulating IGF-1, IGFB-3, GH and TSH levels in multiple sclerosis and their relationship with treatment.

PubMed

Akcali, Aylin; Bal, Berrin; Erbagci, Binnur

2017-07-01

Improving the proficiency of oligodendrocytes in their ability to repair myelin damage is one of the major goals of multiple sclerosis treatment. Insulin-like growth factor-1 (IGF-1) is one of several polypeptides that are considered to have potential benefits in that sense. In the present study, we aimed to determine serum levels of IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3), thyroid stimulating hormone (TSH) and growth hormone (GH) among treated and non-treated patients with Relapsing-Remitting Multiple Sclerosis (RRMS) and a healthy control group. The study enrolled 100 RRMS patients and 100 age- and sex-matched control subjects diagnosed with definite multiple sclerosis (MS). Serum GH, IGF-1, IGFBP-3, and TSH levels were studied. The number of relapses and Expanded Disability Status Scale were negatively correlated and IGFBP-3 and GH were positively correlated with IGF-1. A statistically significant difference was not observed when patients were divided into two subgroups as patients treated with a MS-specific therapy (n = 54) and non-treated patients (n = 46). TSH and IGFBP-3 values were significantly lower in patient group vs. While no difference was determined with in IGF-1 levels, low levels of IGF-1 was in correlation with the least levels of IGFBP-3. To understand the relation between IGF-1 and IGFBP-3, the role of low levels of IGFBP-3 and TSH may be studied with clinic isolated syndrome patients and the evolution of these patients to definite MS.

Brazil and Access to HIV/AIDS Drugs: A Question of Human Rights and Public Health

PubMed Central

Galvão, Jane

2005-01-01

I explore the relationship between public health and human rights by examining the Brazilian government’s policy of free and universal access to anti-retroviral medicines for people with HIV/AIDS. The Brazilian government’s management of the HIV/AIDS epidemic arose from initiatives in both civil society and the governmental sector following the democratization of the country. The dismantling of authoritarian rule in Brazil was accompanied by a strong orientation toward human rights, which formed the sociopolitical framework of Brazil’s response to the HIV/AIDS epidemic. Even if the Brazilian experience cannot be easily transferred to other countries, the model of the Brazilian government’s response may nonetheless serve as inspiration for finding appropriate and lifesaving solutions in other national contexts. PMID:15933238

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

PubMed Central

Tscheppe, Angelika; Breiteneder, Heimo

2017-01-01

The years 1988–1995 witnessed the beginning of allergen cloning and the generation of recombinant allergens, which opened up new avenues for the diagnosis and research of human allergic diseases. Most crystal and solution structures of allergens have been obtained using recombinant allergens. Structural information on allergens allows insights into their evolutionary biology, illustrates clinically observed cross-reactivities, and makes the design of hypoallergenic derivatives for allergy vaccines possible. Recombinant allergens are widely used in molecule-based allergy diagnosis such as protein microarrays or suspension arrays. Recombinant technologies have been used to produce well-characterized, noncontaminated vaccine components with known biological activities including a variety of allergen derivatives with reduced IgE reactivity. Such recombinant hypoallergens as well as wild-type recombinant allergens have been used successfully in several immunotherapy trials for more than a decade to treat birch and grass pollen allergy. As a more recent application, the development of antibody repertoires directed against conformational epitopes during immunotherapy has been monitored by recombinant allergen chimeras. Although much progress has been made, the number and quality of recombinant allergens will undoubtedly increase and keep improving our knowledge in basic scientific investigations, diagnosis, and therapy of human allergic diseases. PMID:28467993

Population Demographic History Can Cause the Appearance of Recombination Hotspots

PubMed Central

Johnston, Henry R.; Cutler, David J.

2012-01-01

Although the prevailing view among geneticists suggests that recombination hotspots exist ubiquitously across the human genome, there is only limited experimental evidence from a few genomic regions to support the generality of this claim. A small number of true recombination hotspots are well supported experimentally, but the vast majority of hotspots have been identified on the basis of population genetic inferences from the patterns of linkage disequilibrium (LD) seen in the human population. These inferences are made assuming a particular model of human history, and one of the assumptions of that model is that the effective population size of humans has remained constant throughout our history. Our results show that relaxation of the constant population size assumption can create LD and variation patterns that are qualitatively and quantitatively similar to human populations without any need to invoke localized hotspots of recombination. In other words, apparent recombination hotspots could be an artifact of variable population size over time. Several lines of evidence suggest that the vast majority of hotspots identified on the basis of LD information are unlikely to have elevated recombination rates. PMID:22560089

Snakebites: First Aid

MedlinePlus

First aid Snakebites: First aid Snakebites: First aid By Mayo Clinic Staff Most North American snakes aren't dangerous to humans. Some exceptions include the rattlesnake, coral snake, water moccasin ...

Congenital Hypothyroidism with Neurological and Respiratory Alterations: A Case Detected Using a Variable Diagnostic Threshold for TSH

PubMed Central

Barreiro, Jesús; Castro-Feijoo, Lidia; Colón, Cristóbal; Cabanas, Paloma; Heredia, Claudia; Castaño, Luis Antonio; Gómez-Lado, Carmen; Couce, M.Luz; Pombo, Manuel

2011-01-01

We report a case of congenital hypothyroidism (CH) with neurological and respiratory alterations due to a heterozygotic c.374-1G > A mutation of TITF1/NKX2-1. The hypothyroidism was detected using a neonatal screening protocol in which the thyroid stimulating hormone (TSH) threshold is re-set each day on the basis of within-day variability and between-day variation. In this case, the threshold on the day of the initial analysis was 8.2 mIU/L, and the measured TSH level in heel-prick blood was 8.3 mIU/L. Conflict of interest:None declared. PMID:22155464

Recombination of Globally Circulating Varicella-Zoster Virus

PubMed Central

Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas

2015-01-01

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the

Beyond erythropoiesis: novel applications for recombinant human erythropoietin.

PubMed

Cerami, A

2001-07-01

Erythropoietin (EPO) primarily is produced in the kidney and acts as a principal mediator of the physiologic response to hypoxia by increasing red blood cell production. Astrocytes and neurons in the central nervous system (CNS) also are known to produce EPO in response to hypoxia/ischemia. EPO appears to play a neuroprotective role based on preclinical data demonstrating the ability of recombinant human erythropoietin (r-HuEPO) to shield neurons from hypoxic/ischemic stress when administered intracerebraventricularly. In CNS models, systemically administered r-HuEPO has not been intensely investigated because large glycosylated molecules generally were deemed incapable of crossing the blood-brain barrier (BBB). A collaborative research effort identified expression of EPO receptors on human brain capillaries and a specific receptor-mediated transport of r-HuEPO across the BBB after a single intraperitoneal (IP) injection in rodents, with subsequent protection against various types of neuronal damage. For example, administration of r-HuEPO 24 hours before or up to 6 hours after focal ischemic stroke significantly reduced the extent of infarction. r-HuEPO also attenuated concussive brain injury, kainate-induced seizure activity, and autoimmune encephalomyelitis. These preclinical findings suggest that r-HuEPO may have therapeutic potential for stroke, head trauma, and epilepsy; additional studies are needed to confirm and extend these encouraging observations in animal models. Copyright 2001 by W.B. Saunders Company.

MosaicSolver: a tool for determining recombinants of viral genomes from pileup data

PubMed Central

Wood, Graham R.; Ryabov, Eugene V.; Fannon, Jessica M.; Moore, Jonathan D.; Evans, David J.; Burroughs, Nigel

2014-01-01

Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, contributing to changes in tropism and possibly assisting transmission across species barriers. The ability to determine whether recombination has occurred and to locate associated specific recombination junctions is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method from both siRNAs and reads sampling the viral genome population (cDNA library); our results are confirmed experimentally. Matlab software (MosaicSolver) is available. PMID:25120266

One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

PubMed

Caldinelli, Laura; Albani, Diego; Pollegioni, Loredano

2013-04-04

Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vita, N.; Magazin, M.; Marchese, E.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with (35S)-methionine, or with (3H)-glucosamine and (3H)-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the (35S)-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and themore » structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.« less

Purification and Characterization of Tagless Recombinant Human Elongation Factor 2 Kinase (eEF-2K) Expressed in Escherichia coli

PubMed Central

Abramczyk, Olga; Tavares, Clint D. J.; Devkota, Ashwini K.; Ryazanov, Alexey G.; Turk, Benjamin E.; Riggs, Austen F.; Ozpolat, Bulent; Dalby, Kevin N.

2012-01-01

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ~ 85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme. PMID:21605678

Short-term preoperative octreotide treatment for TSH-secreting pituitary adenoma.

PubMed

Fukuhara, Noriaki; Horiguchi, Kentaro; Nishioka, Hiroshi; Suzuki, Hisanori; Takeshita, Akira; Takeuchi, Yasuhiro; Inoshita, Naoko; Yamada, Shozo

2015-01-01

Preoperative control of hyperthyroidism in patients with TSH-secreting pituitary adenomas (TSHoma) may avoid perioperative thyroid storm. Perioperative administration of octreotide may control hyperthyroidism, as well as shrink tumor size. The effects of preoperative octreotide treatment were assessed in a large number of patients with TSHomas. Of 81 patients who underwent surgery for TSHoma at Toranomon Hospital between January 2001 and May 2013, 44 received preoperative short-term octreotide. After excluding one patient because of side effects, 19 received octreotide as a subcutaneous injection, and 24 as a long-acting release (LAR) injection. Median duration between initiation of octreotide treatment and surgery was 33.5 days. Octreotide normalized free T4 in 36 of 43 patients (84%) and shrank tumors in 23 of 38 (61%). Length of octreotide treatment did not differ significantly in patients with and without hormonal normalization (p=0.09) and with and without tumor shrinkage (p=0.84). Serum TSH and free T4 concentrations, duration of treatment, incidence of growth hormone (GH) co-secretion, results of octreotide loading tests, form of administration (subcutaneous injection or LAR), tumor volume, and tumor consistency did not differ significantly in patients with and without hormonal normalization and with and without tumor shrinkage. Short-term preoperative octreotide administration was highly effective for TSHoma shrinkage and normalization of excess hormone concentrations, with tolerable side effects.

AID-initiated purposeful mutations in immunoglobulin genes.

PubMed

Goodman, Myron F; Scharff, Matthew D; Romesberg, Floyd E

2007-01-01

Exposure brings risk to all living organisms. Using a remarkably effective strategy, higher vertebrates mitigate risk by mounting a complex and sophisticated immune response to counter the potentially toxic invasion by a virtually limitless army of chemical and biological antagonists. Mutations are almost always deleterious, but in the case of antibody diversification there are mutations occurring at hugely elevated rates within the variable (V) and switch regions (SR) of the immunoglobulin (Ig) genes that are responsible for binding to and neutralizing foreign antigens throughout the body. These mutations are truly purposeful. This chapter is centered on activation-induced cytidine deaminase (AID). AID is required for initiating somatic hypermutation (SHM) in the V regions and class switch recombination (CSR) in the SR portions of Ig genes. By converting C --> U, while transcription takes place, AID instigates a cascade of mutational events involving error-prone DNA polymerases, base excision and mismatch repair enzymes, and recombination pathways. Together, these processes culminate in highly mutated antibody genes and the B cells expressing antibodies that have achieved optimal antigenic binding undergo positive selection in germinal centers. We will discuss the biological role of AID in this complex process, primarily in terms of its biochemical properties in relation to SHM in vivo. The chapter also discusses recent advances in experimental methods to characterize antibody dynamics as a function of SHM to help elucidate the role that the AID-induced mutations play in tailoring molecular recognition. The emerging experimental techniques help to address long-standing conundrums concerning evolution-imposed constraints on antibody structure and function.

Regulation of AID, the B-cell genome mutator

PubMed Central

Keim, Celia; Kazadi, David; Rothschild, Gerson; Basu, Uttiya

2013-01-01

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis. PMID:23307864

Definition of reference ranges for free T4, TSH, and thyroglobulin levels in healthy subjects of the Jaén Health District.

PubMed

Olmedo Carrillo, Pablo; Santiago Fernández, Piedad; García Fuentes, Eduardo; Ureña Fernández, Tomás; Gutiérrez Alcántara, Carmen; Sánchez-Malo, Carolina; Gassó Campos, Manuela; Martínez Ramírez, María José

2017-10-01

The treatment guidelines for thyroid dysfunction recommend defining reference ranges for thyroid hormones in each area through assessment of local population data considering the iodine nutritional status. The aim of this study was to define the reference ranges of free thyroxine (FT4), TSH, and thyroglobulin levels in a general population from Jaen, an area of southern Spain with an adequate iodine nutritional status, and whether they were associated with urinary iodine levels. A cross-sectional study was conducted in 1,003 subjects of the general population of the Jaen Health District. Levels of urinary iodine, FT4, TSH, thyroglobulin, and thyroid peroxidase (TPO) antibodies were measured according to age and sex. Median and mean urinary iodine levels were 110.59μg/L and 130.11μg/L respectively. Median TSH level was 1.83μIU/mL (p2.5=0.56μIU/mL, p97.5=4.66μIU/mL). Median FT4 level was 0.84ng/dL (p2.5=0.62ng/dL, p97.5=1.18ng/dL). TPO antibodies were detected in 5.7% of subjects. There was no correlation between urinary iodine levels and FT4, TSH or TPO antibodies. Subjects with positive TPO antibodies had higher TSH levels (3.34μIU/L versus 2.14μIU/mL, P=.001; odds ratio=2.42). Urinary iodine levels in Jaen are optimal according to World Health Organization standards. Reference ranges of FT4, TSH, and thyroglobulin do not differ from those reported in the literature and are no associated to urinary iodine levels. The prevalence of positive TPO antibodies was similar to that reported in other Spanish areas. Copyright © 2017 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.

The etiologies and incidences of congenital hypothyroidism before and after neonatal TSH screening program implementation: a study in southern Thailand.

PubMed

Jaruratanasirikul, Somchit; Piriyaphan, Jutarat; Saengkaew, Tansit; Janjindamai, Waricha; Sriplung, Hutcha

2018-06-27

Congenital hypothyroidism (CH) is one of the common causes of intellectual disability which can be prevented by early detection of an elevated thyroid stimulating hormone (TSH) level in the newborn and by treatment with thyroxine. In Thailand, neonatal TSH screening was implemented nationwide in 2005. The objective of the study was to determine the etiologies and the estimated incidences of CH in southern Thailand before and after the implementation of a neonatal TSH screening program in 2005. The medical records of pediatric patients who were diagnosed with primary CH at Songklanagarind Hospital during 1995-2013 were retrospectively reviewed. The study was divided into two time periods: study period 1 (SP1) (1995-2004) and study period 2 (SP2) (2005-2013), the time before and after TSH program implementation. The most common form of CH during SP1 was overt permanent CH (66%), mostly caused by athyreosis or ectopic thyroid. In SP2, the most common form of CH was mild permanent CH (39%) (mostly due to dyshormonogenesis), followed by overt CH (32%) and transient CH (29%). The overall annual estimated incidence of CH per 10,000 live births in Songkhla Province was 1.69 (1:5021) in SP1, increasing to 4.77 (1:2238) in SP2; in all 14 provinces in southern Thailand, the estimated incidence was 1.24 (1:8094) in SP1 and 2.33 (1:4274) in SP2. Neonatal TSH screening has a significant impact on the increased detection of the mild form of permanent and transient CH cases, which may be important for the prevention of brain damage from less severe CH although this remains to be documented.

Association between the clinical classification of hypothyroidism and reduced TSH in LT4 supplemental replacement treatment for pregnancy in China.

PubMed

Zhang, Lyu; Zhang, Zhaoyun; Ye, Hongying; Zhu, Xiaoming; Li, Yiming

2016-01-01

The study was aimed to evaluate the effects of levothyroxine (LT4) supplemental replacement treatment for pregnancy and analyze the associations between the clinical classification of hypothyroidism and reduced thyroid-stimulating hormone (TSH) in LT4 therapy. Totally, 195 pregnant women with hypothyroidism receiving routine prenatal care were enrolled. They were categorized into three groups: overt hypothyroidism (OH), subclinical hypothyroidism (SCH) with negative thyroperoxidase antibody (TPOAb), and SCH with positive TPOAb. The association between the clinical classification and reduced TSH in LT4 supplemental replacement treatment was assessed. The results indicated that reduced TSH was significantly different among the groups according to the clinical classifications (p = 0.043). The result was also significantly different between patients with OH and patients with SCH and negative TPOAb (p = 0.036). Similar result was reported for the comparison between patients with OH and patients with SCH and positive TPOAb (p = 0.016). Multiple variable analyses showed that LT4 supplementation, gestational age and the variable of clinical classifications were associated with reduced TSH independently. Our data suggested that the therapeutic effect of substitutive treatment with LT4 was significantly associated with different clinical classifications of hypothyroidism in pregnancy and the treatment should begin as soon as possible after diagnosis.

Production and purification of recombinant human glucagon overexpressed as intein fusion protein in Escherichia coli.

PubMed

Esipov, Roman S; Stepanenko, Vasily N; Gurevich, Alexandr I; Chupova, Larisa A; Miroshnikov, Anatoly I

2006-01-01

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.

[Effect of selenium on serum TGAb, TMAb, FT3, FT4 and TSH of rats with excessive intake of iodine].

PubMed

Chi, Haiyan; Zhou, Yuping; Li, Li

2012-07-01

To investigate the effect of selenium on the TGAb, TMAb, FT3, FT4 and TSH level of rats with excessive intake of iodine. Wistar rats were divided into three groups by random:normal control, high iodine group and high iodine plus selenium group. Rats in the high iodine plus selenium group were lavaged with sodium selenite for 10 weeks. The levels of serum TGAb, TMAb, FT3, FT4 and TSH were tested at different time of the experiment. There were no significant change on levels of FT3, FT4 and TSH (P > 0.05). The levels of TGAb and TMAb in the high iodine group were increased slowly (P < 0.05), but no significant change was observed in the high iodine plus selenium group. Excessive intake of iodine might induce goiter, and selenium might have antagonistic effect on it.

Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4.

PubMed

Khamrin, Pattara; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

2013-07-01

Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1-4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011-11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2. Copyright © 2013 Elsevier B.V. All rights reserved.

Effective visualization of suppressed thyroid tissue by means of baseline 99mTc-methoxy isobutyl isonitrile in comparison with 99mTc-pertechnetate scintigraphy after TSH stimulation.

PubMed

Vattimo, A; Bertelli, P; Burroni, L

1992-01-01

Baseline 99mTc-MIBI thyroid scintigraphy was compared with 99mTc-pertechnetate scintigraphy after TSH stimulation in seven patients with suppressed thyroid tissue due to an autonomously functioning thyroid nodule (AFTN). In all patients the suppressed thyroid tissue was visualized by means of both baseline 99mTc-MIBI and post-TSH 99mTc-pertechnetate scintigraphy, and in some cases the former technique provided better visualization. In one patient presenting a "warm" nodule T3-suppression did not affect the nodular/extranodular uptake ratio of 99mTc-MIBI, whereas the 99mTc-pertechnetate uptake ratio increased significantly. This leads us to hypothesize that the thyroid uptake of 99mTc-MIBI is not related to TSH control, but rather to other mechanisms such as the blood flow. Since exogenous TSH is no longer available, 99mTc-MIBI scintigraphy can be successfully used in the place of repeated 99mTc-pertechnetate scintigraphy after TSH stimulation in the assessment of AFTN.

AID targeting: old mysteries and new challenges.

PubMed

Chandra, Vivek; Bortnick, Alexandra; Murre, Cornelis

2015-09-01

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for noncoding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multilevel regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research. Copyright © 2015 Elsevier Ltd. All rights reserved.

AID Targeting: Old Mysteries and New Challenges

PubMed Central

Chandra, Vivek; Bortnick, Alexandra; Murre, Cornelis

2015-01-01

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus, stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for non-coding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multi-level regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research. PMID:26254147

Major psychological factors affecting acceptance of gene-recombination technology.

PubMed

Tanaka, Yutaka

2004-12-01

The purpose of this study was to verify the validity of a causal model that was made to predict the acceptance of gene-recombination technology. A structural equation model was used as a causal model. First of all, based on preceding studies, the factors of perceived risk, perceived benefit, and trust were set up as important psychological factors determining acceptance of gene-recombination technology in the structural equation model. An additional factor, "sense of bioethics," which I consider to be important for acceptance of biotechnology, was added to the model. Based on previous studies, trust was set up to have an indirect influence on the acceptance of gene-recombination technology through perceived risk and perceived benefit in the model. Participants were 231 undergraduate students in Japan who answered a questionnaire with a 5-point bipolar scale. The results indicated that the proposed model fits the data well, and showed that acceptance of gene-recombination technology is explained largely by four factors, that is, perceived risk, perceived benefit, trust, and sense of bioethics, whether the technology is applied to plants, animals, or human beings. However, the relative importance of the four factors was found to vary depending on whether the gene-recombination technology was applied to plants, animals, or human beings. Specifically, the factor of sense of bioethics is the most important factor in acceptance of plant gene-recombination technology and animal gene-recombination technology, and the factors of trust and perceived risk are the most important factors in acceptance of human being gene-recombination technology.

Production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk.

PubMed

Lu, Dan; Liu, Shen; Shang, Shengzhe; Wu, Fangfang; Wen, Xiao; Li, Zhiyuan; Li, Yan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Qiuyan; Li, Ning

2015-01-01

Human lysozyme is a natural non-specific immune factor in human milk that plays an important role in the defense of breastfed infants against pathogen infection. Although lysozyme is abundant in human milk, there is only trace quantities in pig milk. Here, we successfully generated transgenic cloned pigs with the expression vector pBAC-hLF-hLZ-Neo and their first generation hybrids (F1). The highest concentration of recombinant human lysozyme (rhLZ) with in vitro bioactivity was 2759.6 ± 265.0 mg/L in the milk of F0 sows. Compared with wild-type milk, rhLZ milk inhibited growth of Escherichia coli K88 during the exponential growth phase. Moreover, rhLZ in milk from transgenic sows was directly absorbed by the intestine of piglets with no observable anaphylactic reaction. Our strategy may provide a powerful tool for large-scale production of this important human protein in pigs to improve resistance to pathogen infection.

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

PubMed

Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

2015-10-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Generation and Repair of AID-initiated DNA Lesions in B Lymphocytes

PubMed Central

Chen, Zhangguo; Wang, Jing H.

2014-01-01

Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions. PMID:24748462

HIV/AIDS and Pediatric AIDS. The Arc Q & A.

ERIC Educational Resources Information Center

Kowalski, Jo Anne T.

This fact sheet uses a question-and-answer format to summarize what is known about HIV/AIDS (Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome) and pediatric AIDS and applies this information to children in school settings. Questions and answers address the following topics: what the AIDS disease and HIV infection are; estimates…

Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli.

PubMed

Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

2015-11-01

Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate. Copyright © 2015 Elsevier Inc. All rights reserved.

[The use of growth hormone to treat endocrine-metabolic disturbances in acquired immunodeficiency syndrome (AIDS) patients].

PubMed

Spinola-Castro, Angela Maria; Siviero-Miachon, Adriana A; da Silva, Marcos Tadeu Nolasco; Guerra-Junior, Gil

2008-07-01

Acquired Immunodeficiency Syndrome (Aids) was initially related to HIV-associated wasting syndrome, and its metabolic disturbances to altered body composition. After Highly Active Antiretroviral Therapy (HAART) was started, malnutrition has declined and HIV-associated lipodystrophy syndrome has emerged as an important metabolic disorder. Aids is also characterized by hormonal disturbances, principally in growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis. The use of recombinant human GH (hrGH) was formerly indicated to treat wasting syndrome, in order to increase lean body mass. Even though the use of hrGH in lipodystrophy syndrome has been considered, the decrease in insulin sensitivity is a limitation for its use, which has not been officially approved yet. Diversity in therapeutic regimen is another limitation to its use in Aids patients. The present study has reviewed the main HIV-related endocrine-metabolic disorders as well as the use of hrGH in such conditions.

AIDS (image)

MedlinePlus

AIDS (acquired immune deficiency syndrome) is caused by HIV (human immunodeficiency virus), and is a syndrome that ... life-threatening illnesses. There is no cure for AIDS, but treatment with antiviral medicine can suppress symptoms. ...

Development of a Highly Specific Recombinant Toxocara canis Second-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis

PubMed Central

Yamasaki, Hiroshi; Araki, Kunioki; Lim, Patricia Kim Chooi; Zasmy, Ngah; Mak, Joon Wah; Taib, Radzan; Aoki, Takashi

2000-01-01

The specificity of the recombinant Toxocara canis antigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen from T. canis second-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 μg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis with Paragonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected with Ascaris and hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected with Ascaris and hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods. PMID:10747116

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

PubMed Central

Zahn, Astrid; Eranki, Anil K.; Patenaude, Anne-Marie; Methot, Stephen P.; Fifield, Heather; Cortizas, Elena M.; Foster, Paul; Imai, Kohsuke; Durandy, Anne; Larijani, Mani; Verdun, Ramiro E.; Di Noia, Javier M.

2014-01-01

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR. PMID:24591601

Bacteriophage recombination systems and biotechnical applications.

PubMed

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

Paediatric reference interval and biological variation trends of thyrotropin (TSH) and free thyroxine (T4) in an Asian population.

PubMed

Loh, Tze Ping; Sethi, Sunil Kumar; Metz, Michael Patrick

2015-08-01

To describe the reference intervals and biological variation data for thyrotropin (TSH) and free thyroxine (FT4) in a mixed Asian population using an indirect sampling approach and to compare them with published reports. TSH and FT4 of children measured once or twice over a 7-year period (2008-2014) at primary-care and tertiary-care settings were extracted from the laboratory information system. After excluding outliers, age-related reference intervals were derived using the Lambda-Mu-Sigma (LMS) approach, while age-partitioned biological variation data were obtained according to recommendations by Fraser and Harris. Both TSH and FT4 were very high at birth and declined with age. Similarly within-individual and between-individual biological variations were higher for both TSH and FT4 at birth and also declined with age. Our data were broadly similar to previous studies. Significant heterogeneity in study population and methods prohibited direct numerical comparison between this and previously published studies. This study fills two important gaps in our knowledge of paediatric thyroid function by reporting the centile trends (and reference values) in a mixed Asian population, as well as providing age-partitioned biological variation data. The variation in published reference intervals highlights the difficulty in harmonising paediatric thyroid reference intervals or recommending universal clinical cut-offs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Studies and Analyses of Aided Adversarial Decision Making. Phase 2: Research on Human Trust in Automation

DTIC Science & Technology

1998-04-01

34AFRL-HE-WP-TR-1999-0216 UNITED STATES AIR FORCE RESEARCH LABORATORY STUDIES AND ANALYSES OF AIDED ADVERSARIAL DECISION MAKING PHASE 2: RESEARCH ON...Analyses of Aided Adversarial Decision Making . C: F41624-94-D-6000 Phase 2: Research on Human Trust in Automation PE: 62202F PR: 7184 6. AUTHOR(S) TA...Buffalo. This work focused on Aided Adversarial Decision Making (AADM) in Information Warfare (1W) environments. Previous work examined informational

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

[Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

PubMed

Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

2008-06-01

This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

Effects of potassium iodide in concentrations of TSH, tT3 and tT4 in serum of subjects with sporotrichosis.

PubMed

Ramírez Soto, Max Carlos

2014-08-01

The saturated potassium iodide solution (SSKI) as treatment for sporotrichosis may cause hypothyroidism by suppressing the synthesis of thyroid hormones (tT3 and tT4 ) and the iodine excess could lead to thyrotoxicosis. Evaluating the changes in serum levels of TSH, tT3 and tT4 in euthyroid patients with sporotrichosis treated with SSKI. For the selection of euthyroid patients, TSH, tT3 and tT4 concentrations were measured for those adults and children diagnosed with sporotrichosis. Each paediatric patient was administered SSKI orally in increasing doses of 2-20 drops/3 times/day and 4-40 drops/3 times/day in adults. Serum concentrations of TSH, tT3 and tT4 were measured 20 days after started the treatment and 15 days posttreatment. Eight euthyroid patients aged between 2 to 65 years old were included. After 20 days of treatment, two suffered subclinical hypothyroidism, one developed subclinical hyperthyroidism, and one hyperthyroxinaemia euthyroid. At 15 days posttreatment only four patients were evaluated and all serum levels of TSH, tT3 and tT4 were normal. Some euthyroid patients with sporotrichosis can develop hyperthyroidism or subclinical iodine-induced hypothyroidism, during the administration of 3 or 6 g SSKI/day. © 2014 Blackwell Verlag GmbH.

Bidirectional TSH and IGF-1 receptor cross talk mediates stimulation of hyaluronan secretion by Graves' disease immunoglobins.

PubMed

Krieger, Christine C; Neumann, Susanne; Place, Robert F; Marcus-Samuels, Bernice; Gershengorn, Marvin C

2015-03-01

There is no pathogenetically linked medical therapy for Graves' ophthalmopathy (GO). Lack of animal models and conflicting in vitro studies have hindered the development of such therapy. Recent reports propose that Graves' Igs bind to and activate thyrotropin receptors (TSHRs) and IGF-1 receptors (IGF-1Rs) on cells in orbital fat, stimulating hyaluronan (HA) secretion, a component of GO. The objective of the study was to investigate potential cross talk between TSHRs and IGF-1Rs in the pathogenesis of GO using a sensitive HA assay. Orbital fibroblasts from GO patients were collected in an academic clinical practice and cultured in a research laboratory. Cells were treated with TSH, IGF-1, and a monoclonal Graves' Ig M22. HA was measured by a modified ELISA. Simultaneous activation by TSH and IGF-1 synergistically increased HA secretion from 320 ± 52 for TSH and 430 ± 65 μg/mL for IGF-1 alone, to 1300 ± 95 μg/mL. IGF-1 shifted the TSH EC50 19-fold to higher potency. The dose response to M22 was biphasic. An IGF-1R antagonist inhibited the higher potency phase but had no effect on the lower potency phase. M22 did not cause IGF-1R autophosphorylation. A TSHR antagonist abolished both phases of M22-stimulated HA secretion. M22 stimulation of HA secretion by GO fibroblasts/preadipocytes involves cross talk between TSHR and IGF-1R. This cross talk relies on TSHR activation rather than direct activation of IGF-1R and leads to synergistic stimulation of HA secretion. These data propose a model for GO pathogenesis that explains previous contradictory results and argues for TSHR as the primary therapeutic target for GO.

GH safety workshop position paper: A critical appraisal of recombinant human GH therapy in children and adults

USDA-ARS?s Scientific Manuscript database

Recombinant human Growth Hormone (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, t...

Power spool test, TSH-002, SPTF No. 19

DOE Office of Scientific and Technical Information (OSTI.GOV)

McInturff, A.D.

1982-05-28

The data presented in this Technical Memo will pertain to the operating characteristics of Power Spool TSH-002. This spool had a large number of thermometers built into it. These thermometers monitored most of the thermal characteristics of the 5000 A American Magnetics, Inc. vapor-cooled leads used in this power spool. Operating conditions, such as peak temperatures, ramp and dc lead cooling gas flow requirements, voltage as an indicator of stable conditions (ac and dc) and general voltage characteristics (i.e., amount of ice formed outside of leads vs high-pot voltage) were measured and observed. It was found that previous operating conditionsmore » of the power leads influenced the temperature gradients of the leads in certain cases.« less

The use of recombinant human erythropoietin in lung transplantation.

PubMed

End, A; Stift, A; Ringl, H; Inhauser, T; Grimm, M; Geissler, K; Stockenhuber, F; Klepetko, W

1995-01-01

Recombinant human erythropoietin (r-HuEPO) was administered in 4 lung transplant recipients to treat chronic anemia beyond the immediate postoperative period. There were 2 males and 2 females with a mean age of 38 years (range 22-49). None of the patients had major infection or rejection problems, and no blood products were used. Because of different individual responses duration of therapy was 1 to 17 weeks (median 5) with a total dosage ranging from 8 to 36 x 10(3) IU (mean 21 x 10(3)). The median single dose was 58 IU/kg (range 36-100). Hemoglobin levels increased significantly from 9.1 +/- 0.2 to 12.7 +/- 0.3 g/dl (mean +/- SE; p < 0.01). There were no side effects. r-HuEPO is recommended in treatment of chronic anemia in lung transplant patients to save blood products and to exclude the potential risk of transfusion-transmitted viral infections.

A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

PubMed

Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

2015-11-10

Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

Human retroviruses in leukaemia and AIDS: reflections on their discovery, biology and epidemiology.

PubMed

Karpas, Abraham

2004-11-01

The study of retroviruses has had a profound impact by unveiling an unusual form of viral replication: the multiplication of RNA viruses via a proviral DNA, for which Jan Svoboda provided the experimental model over forty years ago. In 1970 Temin, Mizutani and Baltimore discovered that this group of viruses contains a unique enzyme catalysing the synthesis of a DNA copy of the viral RNA: reverse transcriptase (RT). The discovery of RT has itself had an enormous impact on molecular biology in general, but also stimulated many premature claims of its detection in human disease. Claims by Gallo's laboratory that the cytoplasm of human leukaemia cells contained RT proved to be unfounded, as did his report in collaboration with Weiss that myeloid leukaemia contained HL23 virus, this organism proving not to be human but a laboratory contaminant of three monkey viruses. Conclusive demonstration of a retroviral involvement in human leukaemia was first provided in 1981 by Hinuma and his associates, showing that adult T-cell leukaemia (ATL), a rare form of leukaemia endemic to south-west Japan, is caused by a new retrovirus (ATLV). Other publications in December 1980 and through 1981 claimed the discovery of a new human T-cell leukaemia virus involved in mycosis fungoides (MF) and Sézary's syndrome (SS). This virus was termed HTLV by Gallo. The nucleotide sequence of ATLV is strongly conserved, that of my 1983 isolate from a black British ATL patient being practically identical with the Japanese virus isolates. After AIDS was recognised in 1981 by Gottlieb and coworkers as a new human disease, several papers were published by Gallo and his associates during 1983-4, invoking the oncovirus responsible for adult T-cell leukaemia as the cause of AIDS. In 1983 the French scientist Barré-Sinoussi and her colleagues succeeded in isolating a new agent in the disease, a lentivirus, which they named LAV. The French immunologist Klatzmann and his colleagues discovered that LAV killed

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Human Immunodeficiency Virus Vaccine Trials

PubMed Central

O’Connell, Robert J.; Kim, Jerome H.; Corey, Lawrence; Michael, Nelson L.

2012-01-01

More than 2 million AIDS-related deaths occurred globally in 2008, and more than 33 million people are living with HIV/AIDS. Despite promising advances in prevention, an estimated 2.7 million new HIV infections occurred in that year, so that for every two patients placed on combination antiretroviral treatment, five people became infected. The pandemic poses a formidable challenge to the development, progress, and stability of global society 30 years after it was recognized. Experimental preventive HIV-1 vaccines have been administered to more than 44,000 human volunteers in more than 187 separate trials since 1987. Only five candidate vaccine strategies have been advanced to efficacy testing. The recombinant glycoprotein (rgp)120 subunit vaccines, AIDSVAX B/B and AIDSVAX B/E, and the Merck Adenovirus serotype (Ad)5 viral-vector expressing HIV-1 Gag, Pol, and Nef failed to show a reduction in infection rate or lowering of postinfection viral set point. Most recently, a phase III trial that tested a heterologous prime-boost vaccine combination of ALVAC-HIV vCP1521 and bivalent rgp120 (AIDSVAX B/E) showed 31% efficacy in protection from infection among community-risk Thai participants. A fifth efficacy trial testing a DNA/recombinant(r) Ad5 prime-boost combination is currently under way. We review the clinical trials of HIV vaccines that have provided insight into human immunogenicity or efficacy in preventing HIV-1 infection. PMID:23209178

Effect of Hemodilution on Coagulation and Recombinant Factor VIIa Efficacy in Human Blood In Vitro

DTIC Science & Technology

2011-11-01

thrombasthenia.12 In trauma, when a blood vessel is injured, tissue factor on subendothelial pericytes is exposed and binds to endogenous FVII ...a more complex effect on coagulation than simply dilution of any single coagulation factor like FVII or fibrinogen (Fig. 1). It is interesting to note...ORIGINAL ARTICLE Effect of Hemodilution on Coagulation and Recombinant Factor VIIa Efficacy in Human Blood In Vitro Daniel N. Darlington, PhD, Angel

A facile method for isolation of recombinant human apolipoprotein A-I from E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikon, Nikita; Shearer, Jennifer; Liu, Jianfang

Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets “Good Manufacturing Practice” standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scalemore » are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. In conclusion, purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.« less

A facile method for isolation of recombinant human apolipoprotein A-I from E. coli

DOE PAGES

Ikon, Nikita; Shearer, Jennifer; Liu, Jianfang; ...

2017-03-20

Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets “Good Manufacturing Practice” standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scalemore » are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. In conclusion, purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.« less

Analysis of European mtDNAs for recombination.

PubMed

Elson, J L; Andrews, R M; Chinnery, P F; Lightowlers, R N; Turnbull, D M; Howell, N

2001-01-01

The standard paradigm postulates that the human mitochondrial genome (mtDNA) is strictly maternally inherited and that, consequently, mtDNA lineages are clonal. As a result of mtDNA clonality, phylogenetic and population genetic analyses should therefore be free of the complexities imposed by biparental recombination. The use of mtDNA in analyses of human molecular evolution is contingent, in fact, on clonality, which is also a condition that is critical both for forensic studies and for understanding the transmission of pathogenic mtDNA mutations within families. This paradigm, however, has been challenged recently by Eyre-Walker and colleagues. Using two different tests, they have concluded that recombination has contributed to the distribution of mtDNA polymorphisms within the human population. We have assembled a database that comprises the complete sequences of 64 European and 2 African mtDNAs. When this set of sequences was analyzed using any of three measures of linkage disequilibrium, one of the tests of Eyre-Walker and colleagues, there was no evidence for mtDNA recombination. When their test for excess homoplasies was applied to our set of sequences, only a slight excess of homoplasies was observed. We discuss possible reasons that our results differ from those of Eyre-Walker and colleagues. When we take the various results together, our conclusion is that mtDNA recombination has not been sufficiently frequent during human evolution to overturn the standard paradigm.

Impairment and restoration of response to TSH in dog thyroid slices after treatment with phospholipase A and lubrol PX.

PubMed

Yamashita, K; Oka, H; Kaneko, T; Ogata, E

1976-01-01

Incubation of dog thyroid slices with phospholipase A (10-40 U/Ml) or Lubrol PX (0.08-0.4%) caused a diminution in the subsequent TSH effect on the tissue cyclic AMP level and glucose oxidation. The same treatment had no effect on the basal level of these parameters. When the phospholipase A or Lubrol PX-treated slices were rinsed intensively with a Krebs-Ringer bicarbonate buffer and then incubated at 37degreesC in the same buffer for a further 1 to 3 hours, responsiveness to TSH recovered progressively reaching almost completely that of the control slices. Again, these procedures were without any significant effect on the responsiveness of the control slices. The above results together with those reported previously suggest strongly that phospholipids are an essential component of the plasma membrane system by which TSH stimulates adenylate cyclase activity. In addition, these essential lipids in the membrane appear to be renewed rather efficiently in this tissue, thus securing the functional integrity of the thyroid in the face of various deleterious situations.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

PubMed

Berg, Ingrid L; Neumann, Rita; Lam, Kwan-Wood G; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A; Jeffreys, Alec J

2010-10-01

PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans

PubMed Central

Berg, Ingrid L.; Neumann, Rita; Lam, Kwan-Wood G.; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A.; Jeffreys, Alec J.

2011-01-01

PRDM9 has recently been identified as a likely trans-regulator of meiotic recombination hot spots in humans and mice1-3. The protein contains a zinc finger array that in humans can recognise a short sequence motif associated with hot spots4, with binding to this motif possibly triggering hot-spot activity via chromatin remodelling5. We now show that variation in the zinc finger array in humans has a profound effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Very subtle changes within the array can create hot-spot non-activating and enhancing alleles, and even trigger the appearance of a new hot spot. PRDM9 thus appears to be the preeminent global regulator of hot spots in humans. Variation at this locus also influences aspects of genome instability, specifically a megabase-scale rearrangement underlying two genomic disorders6 as well as minisatellite instability7, implicating PRDM9 as a risk factor for some pathological genome rearrangements. PMID:20818382

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

PubMed

Szpakowski, Piotr; Biet, Franck; Locht, Camille; Paszkiewicz, Małgorzata; Rudnicka, Wiesława; Druszczyńska, Magdalena; Allain, Fabrice; Fol, Marek; Pestel, Joël; Kowalewicz-Kulbat, Magdalena

2015-01-01

Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults

PubMed Central

Biet, Franck; Rudnicka, Wiesława; Druszczyńska, Magdalena; Fol, Marek; Pestel, Joël

2015-01-01

Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4+ T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs. PMID:26339658

AID Biology: A pathological and clinical perspective.

PubMed

Choudhary, Meenal; Tamrakar, Anubhav; Singh, Amit Kumar; Jain, Monika; Jaiswal, Ankit; Kodgire, Prashant

2018-01-02

Activation-induced cytidine deaminase (AID), primarily expressed in activated mature B lymphocytes in germinal centers, is the key factor in adaptive immune response against foreign antigens. AID is responsible for producing high-affinity and high-specificity antibodies against an infectious agent, through the physiological DNA alteration processes of antibody genes by somatic hypermutation (SHM) and class-switch recombination (CSR) and functions by deaminating deoxycytidines (dC) to deoxyuridines (dU), thereby introducing point mutations and double-stranded chromosomal breaks (DSBs). The beneficial physiological role of AID in antibody diversification is outweighed by its detrimental role in the genesis of several chronic immune diseases, under non-physiological conditions. This review offers a comprehensive and better understanding of AID biology and its pathological aspects, as well as addresses the challenges involved in AID-related cancer therapeutics, based on various recent advances and evidence available in the literature till date. In this article, we discuss ways through which our interpretation of AID biology may reflect upon novel clinical insights, which could be successfully translated into designing clinical trials and improving patient prognosis and disease management.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Non-transference of biological reference interval of TSH by electrochemiluminescence immunoassay: an Indian population perspective.

PubMed

Sarkar, Rajarshi

2013-08-23

Although TSH measurement by electrochemiluminescence immunoassay has become commonplace in India, significant discrepancy has been observed on interpretation of the test results when the manufacturer supplied biological reference interval (BRI) criteria were applied. This report determined whether the manufacturer's BRI (Roche Cobas) is transferable to the Indian population. Three hundred seventy-eight age- and sex-matched healthy subjects were selected from an urban Indian population. TSH reference measurements were acquired, and the reference data were statistically analysed. BRI of the Indian urban reference population was determined by non-parametric means. BRI was found to be 1.134 to 7.280μIU/ml. BRI thus calculated was found to be significantly different from that mentioned by the manufacturer (0.27 to 4.20μIU/ml), which, needless to mention, has profound clinical implications in this part of the globe. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the

Recombinant human bone morphogenetic protein 2 in augmentation procedures: case reports.

PubMed

Luiz, Jaques; Padovan, Luis Eduardo Marques; Claudino, Marcela

2014-01-01

To successfully rehabilitate edentulous patients using endosseous implants, there must be enough available bone. Several techniques have been proposed for augmentation of sites with insufficient bone volume. Although autogenous bone has long been considered the gold standard for such procedures, the limited availability of graft material and a high morbidity rate are potential disadvantages of this type of graft. An alternative is to use recombinant human bone morphogenetic protein 2 (rhBMP-2), which is able to support bone regeneration in the oral environment. These cases demonstrate the applicability of rhBMP-2 in maxillary sinus elevation and augmentation procedures in the maxilla to enable dental implant placement. The use of rhBMP-2 in alveolar augmentation procedures had several clinical benefits for these patients.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

PubMed Central

Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

2012-01-01

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

Negative correlation between bone mineral density and TSH receptor antibodies in long-term euthyroid postmenopausal women with treated Graves’ disease

PubMed Central

2013-01-01

Background Thyrotoxicosis is a cause of secondary osteoporosis. High concentrations of triiodotironine (T3) in Graves’ disease stimulate bone turnover, but it is unclear if euthyroidism will always normalize bone metabolism. Thyrotropin (TSH) is known to affect directly the bone metabolism through the TSH receptor and TSH receptor antibodies (TRAb) may have an important role in bone turn-over. The aim of our study was to determine, in pre and postmenopausal euthyroidism patients with previous overt hyperthyroidism due to Graves’ disease the bone mineral density (BMD) as well as factors that could affect BMD in each group, including TRAb. Methods Cross-sectional, non-interventional study. Fifty-seven patients with previous hyperthyroidism due to Graves’ disease (premenopausal: 30, postmenopausal: 27) that remained euthyroid for at least 6 months prior to study were included and compared with fifty- two matched respective controls. Thyrotoxine (T4), TSH, TRAb and BMD were measured. Results Only euthyroid postmenopausal patients with a history of hyperthyroidism due to Graves’ disease showed lower whole body BMD than matched controls. The BMD expressed as Z-score was less in whole body and lumbar spine in postmenopausal in relation to premenopausal women with previous overt hyperthyroidism due to Graves’ disease. In the postmenopausal patients, the Z-score of lumbar spine BMD correlated negatively with TRAb (r = −0,53, p < 0.008), positively with the time of evolution of the disease (r = +0.42, p < 0.032) and positively with the time of euthyroidism (r = + 0.50, p < 0.008), but neither with serum T4 nor TSH. In a multiple regression analysis TRAb was the only significant independent variable in relation to lumbar spine BMD (F = 3. 90, p < 0.01). Conclusions In euthyroid women with a history of Graves’ hyperthyroidism, BMD was only affected in the postmenopausal group. The negative correlation of Z-score of lumbar spine BMD with TRAb suggests that this

Ikaros controls isotype selection during immunoglobulin class switch recombination.

PubMed

Sellars, MacLean; Reina-San-Martin, Bernardo; Kastner, Philippe; Chan, Susan

2009-05-11

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG(2b) and IgG(2a), and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the gamma2b and gamma2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of gamma2b and gamma2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

PubMed

Rozov, S M; Permyakova, N V; Deineko, E V

2018-03-01

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

Large-scale purification and characterization of recombinant human stem cell factor in Escherichia coli.

PubMed

Chen, Liang-Hua; Cai, Feng; Zhang, Dan-Ju; Zhang, Li; Zhu, Peng; Gao, Shun

2017-07-01

The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 10 6  IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.

PubMed

Ng, Elizabeth S; Davis, Richard; Stanley, Edouard G; Elefanty, Andrew G

2008-01-01

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

Legal rights, human rights and AIDS: the first decade. Report from South Africa 2.

PubMed

Cameron, E

1993-01-01

A broad range of coercive measures has been considered internationally and applied in some countries in the interest of controlling the spread of HIV. Although a couple such measures are on the books in South Africa, they have never been invoked and will soon be officially repealed. There is, however, a problem in South Africa with the violation by health care workers, employers, and others of individuals' rights to dignity, privacy, and autonomy. The exaggerated and undue fear that doctors and other health workers have of being infected by patients with HIV has led to widespread and gross human rights abuses in clinical management and treatment. Abuses include the refusal of treatment, testing patients for HIV without their informed or any consent, insisting upon HIV testing devoid of diagnostic or therapeutic justification, and widespread breaches of confidentiality. Persons with AIDS and HIV are also denied access to their fair share of national resources. This latter phenomenon is likely to become the principal form of human rights abuse, with racism and class differences exacerbating the problem. The practice is proliferating and takes many forms including pre-employment HIV testing; exclusionary discrimination in insurance; discrimination between HIV and other life-threatening conditions in corporate medical, pension, and provident funds; and the discriminatory denial of fair and adequate health care to people with HIV or AIDS. Discrimination of all kinds, however, retards preventive efforts. Public health therefore demands the recognition and enforcement of individual human rights and that structures of discrimination be eliminated. Human rights protection may, by limiting the effect of discrimination, play a significant part in fighting the epidemic. Protective measures could include enacting legislation to prohibit pre-employment testing, legislation to regulate the provision of insurance and to prohibit or regulate pre-insurance HIV testing and the

SequenceLDhot: detecting recombination hotspots.

PubMed

Fearnhead, Paul

2006-12-15

There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (< 1/kb). Program, data sets, and full details of results are available at: http://www.maths.lancs.ac.uk/~fearnhea/Hotspot.

Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins.