This was attributed to decreased Sertoli cell proliferation from E This demonstrates the contribution of activin to establishing the balance between Sertoli and germ cell numbers that is required for male fertility in adulthood. Testis plays an important role in the development of male reproductive system, providing the hormones for sexual maturation as well as the site of spermatogenesis. Spermatogonia B does not appear in testis until onset of puberty in mice and monkeys, although circulating levels of LH, FSH and Testosterone T during infancy are as high as that found during puberty. This implies that mere presence of hormones is not enough, but processes downstream to their action in testicular Sertoli cells Sc are also important in controlling germ-cell Gc division and differentiation.
Lack of noticeable spermatogenesis during infancy in the phase of high hormones is a situation similar to that found in certain categories of male infertility. Therefore, we undertook present study to analyse genes differentially expressed by Sc during infancy and puberty.
To this end, Sc were cultured from infant and pubertal monkeys and maintained in-vitro. Fluorescent differential display was performed using RNA from such treated Sc. Several differentially expressed genes were identified with the help of Bioinformatic tools. Dickoppf homolog 3 Dkk3 was one of the genes which was over expressed by pubertal Sc. Since Dkk3 is a known candidate involved in induction of cell differentiation, a process akin to testis during spermatogenesis, we chose this gene to pursue our study.
In-vivo testicular electroporation technique developed by our laboratory was used to make such transgenic mice.
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Testicular weights of adult transgenic mice were reduced as compared to age matched controls. Histological analysis of atrophic testis revealed a compromised differentiation of germ cells along with multinucleated giant cells and vacuoles. Inhibition of Dkk3 expression had lead to sloughing off of germ cells.
Severe reduction in sperm counts was also observed. Since Dkk3 is a member of Dkk family of Wnt antagonists, interference with Dkk3 expression led to an increase in the expression of beta-catenin. In conformity with this, we found elevated testicular expression of Mullerian inhibiting substance MIS and Glial cell-derived neurotrophic factor GDNF , the known markers of Sc immaturity, in adult Dkk3 knock down mice.
Lack of Sc maturity may underlie defective testicular spermatogenesis and Gc apoptosis in these transgenic mice. Depending on the degree of shRNA mediated inhibition of Dkk3 , the status of spermatogenesis was affected in such transgenic mice. Our findings indicated that Dkk3 is a major regulator of spermatogenesis in mice. This work was supported by the Department of Biotechnology, India. Stem cells have the capacity to self-renew and to generate differentiated cells.
Such cells are found in adult as well as in embryonic tissue. Previous studies have shown that a single injection of adult rats with ethane dimethanesulfonate EDS results in the elimination of the adult Leydig cells. Subsequently, a new generation of Leydig cells is restored to the testes, indicating that the elimination of the adult Leydig cells results in the induction of precursor cells to differentiate into functional Leydig cells. We developed an organ culture system with which to test this hypothesis. Interstitial and seminiferous tubule fractions of rat testes were separated from each other and isolated four days after the rats received an EDS injection.
The two fractions were cultured in vitro with LH for four weeks. Immediately after their isolation, the tubules did not produce testosterone in response to LH. However, after two weeks in culture, testosterone was detected in the medium of the tubule fraction. Testosterone production by the tubules increased at least through four weeks.
Western blots showed that the steroidogenic proteins, Pscc and StAR, which were undetectable immediately after the tubules were isolated, became detectable. In contrast to the tubules, the interstitial fraction failed to produce testosterone. These factors were selected based on previous studies suggesting that they may play roles in Leydig cell differentiation during normal prepubertal development. We suggest that the precursor cells that give rise to the adult cells might be stem Leydig cells. The results support the peritubular localization of these putative stem cells.
The organ culture system used for these studies was particularly useful for identifying the factors hormones and growth factors involved in the regulation of the differentiation of the putative stem cells. Immune-privileged Sertoli cells SC have the unique ability to provide protection to co-transplanted cells and survive long-term when transplanted allogeneically or xenogeneically.
On the other hand, MSC-1 cells a mouse Sertoli cell line lack some of the immunoprotective abilities associated with primary SC, as they are unable to protect co-transplanted cells. The objective of this study was to compare the cell survival rate and gene expression profiles of primary SC and MSC-1 cells, to further understand the mechanism for SC immune-privilege. Additionally, gene expression differences between these cells were investigated by microarray and pathway analyses. Primary SC grafts survived throughout the study and were not rejected, whereas, very few MSC-1 cells were detected by day 11 and MSC-1 cells were completely rejected within 20 days.
Aggregated primary SC, as compared to aggregated MSC-1 cells, expressed immune-related modulators, such as immunosuppressive cytokines and complement inhibitors, regulators of apoptosis and lipid mediators for controlling inflammation. Antibody deposition was not observed until day 14 post-transplantation whereas no complement deposition was observed throughout the study.
This led to the conclusion that cell-mediated death plays an important role in allograft rejection of MSC-1 cells, and primary SC by inhibiting cell-mediated pathway enjoy long-term survival. Studies are ongoing to analyze MSC-1 and primary SC grafts for cellular infiltrate, cytokines and immune regulating molecules, which may further increase our understanding of the mechanism by which SC establish immune-privilege and thus improve transplantation success.
BDADs Bis- dichloroacetyl -diamines are a group of compounds that inhibit the conversion of vitamin A retinol, ROL to its active metabolite, retinoic acid RA , which is essential for mammalian sperm production. This study investigates the feasibility of using BDADs to inhibit spermatogenesis leading to their potential use as male contraceptives. A novel testis organ culture technique and the expression of a marker of RA activity, Stra8, were utilized to demonstrate that a specific BDAD, WIN 18,, can inhibit the metabolism of ROL to RA in the neonatal and adult murine testis, and the embryonic murine gonad.
The expression of Stra8 was also downregulated in adult mouse testis tubules cultured with BDAD when compared to tubules cultured with the vehicle control. These murine results, combined with the significant reduction in sperm count also observed in rabbits treated with WIN 18,, provide critical insights into how the BDADs can inhibit spermatogenesis through blocking the ability of vitamin A to drive germ cell development. Fertility control is one alternative for managing overabundant wildlife populations; however, current technology is limited by treatment efficacy, duration and unacceptable side-effects.
We previously investigated the effects of a single immunization with gonadotropin-releasing hormone GnRH vaccine on female elk Cervus elaphus nelsoni during mid-gestation. Vaccination did not affect existing pregnancy, calving rates, or neonatal growth rates during the year it was applied; however, it significantly decreased pregnancy rates for three breeding seasons following immunization. Strong immune and inflammatory responses, including robust GnRH antibody concentrations and injection site abscesses, were associated with vaccination. Calves nursing from GnRH vaccinated dams developed high serum GnRH antibody concentrations via colostral antibody transfer.
Antibodies waned by 6 months of age. The effects of exposure to passively acquired GnRH antibodies during the neonatal period, on long-term reproductive development and function, are unknown. This study was designed to test the hypothesis that hypothalmo-pituitary-gonadal HPG axis development and function would be altered due to functional lack of GnRH during the neonatal period. The onset of puberty in male calves was estimated by measuring serum testosterone and secondary sex characteristics such as antler development, neck girth, scrotal size, and semen characteristics.
Additionally, we measured serum luteinizing hormone LH and testosterone concentrations in response to stimulation with a potent GnRH agonist. Similarly, serum progesterone was measured in female calves at 10 day intervals to estimate pubertal onset. They were challenged with GnRH agonist to evaluate pituitary gonadotrope function before the second breeding season and exposed to fertile bulls to determine fertility.
Gross, histologic, and endocrinologic examination of the HPG axis was performed on all calves post-mortem. There were no differences between groups with or without antibodies in pubertal onset for either males or females. Similarly, antibody status did not affect response to GnRH agonist.
All males developed grossly normal antlers and had at least one high quality semen sample during their first breeding season. All females became pregnant during the second breeding season. There were no gross differences in reproductive tracts between groups and pituitary LH and follicle stimulating hormone as well as hypothalamic GnRH concentrations were similar.
We failed to reject the null hypothesis that GnRH antibodies do not affect reproductive development. These results strongly suggest that neonatal passive transfer of maternal GnRH antibodies does not affect long-term reproductive potential of elk and supports the notion that precocial species such as elk do not require functional GnRH stimulation during the neonatal period for complete reproductive development.
A single-dose contraceptive vaccine has several potential applications in felids, most notably for control of feral cat populations. In this study, we evaluated the safety and effectiveness of a single-dose luteinizing hormone-releasing hormone LHRH vaccine for contraception in cats. The single-dose vaccine contained both non-encapsulated and encapsulated forms of ova-LHRH; the latter presented the antigen in agarose microbeads for slow release. Injection sites were monitored for inflammatory responses. Blood samples were collected bi-weekly for one year, and LHRH antibody production assessed using a validated radioimmunoassay.
Progestin metabolite profiles were monitored via a validated enzyme immunoassay in fecal samples collected every 2 to 3 days for one year. Fertility was assessed by housing a proven breeder male with females for four months beginning six months after initial injection. In the saline group, all five females continued to ovulate spontaneously throughout the study until pregnancy.
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In the single-dose group, four of five females were no longer ovulating by the beginning of the fertility trial; however, one of these four resumed ovulatory activity towards the end of the trial and became pregnant. The remaining female in this group ovulated regularly until pregnancy. In the two-dose group, three of five cats were spontaneous ovulators initially; all three ceased ovulating by the time of the fertility trial. One of these cats showed elevated progestin levels towards the end of this time period, but did not become pregnant.
No pregnancy occurred in the two-dose group. In the single-dose group, two of five females became pregnant litter size, Subcutaneous granulomas were observed at the injection sites in all immunized females, with the most severe granulomas occurring in the two-dose group including two with ulceration.
Granulomas reduced in size spontaneously but were still palpable one year after injection. We previously reported that both follicle stimulating hormone FSH and luteinizing hormone LH may play an essential role in dog follicle growth in vitro. In the present study, we determined the influence of 1 follicle size and 2 the gonadotropin microenvironment on in vitro growth of dog preantral follicles.
In Study 1, preantral follicles were enzymatically-isolated by incubating ovarian cortices at All follicles were individually encapsulated in 0. In both studies, follicle diameter and survival based on morphology were determined every 48 h with growth rate assessed by ANOVA followed by the Duncan's Multiple Comparison Method and survival by Chi-square testing.
In Study 1, follicle size influenced in vitro follicle growth, but had no effect on survival. In Study 2, FSH and LH concentration and the interaction of these gonadotropins influenced in vitro follicle growth and survival. Our findings indicate that the stage of preantral development influences the culture environment required to promote dog follicle development in vitro. It is possible to demonstrate a dose-dependency for FSH in culture, illustrating the critical importance of this gonadotropin in dog folliculogenesis.
Furthermore, results confirm a role for LH in dog follicle growth, especially in a microenvironment where FSH is available only at a modest concentration. These findings are relevant for two opposing challenges in canids, exploring: Captive breeding programs have been developed for sand cats in the U. Since in vitro fertilization IVF and embryo transfer ET could reduce or eliminate the need to transport living animals between regions, these technologies could facilitate the integration of disjunct populations and allow for the development of global, rather than regional, management programs.
Similarly, IVF and ET would allow offspring to be produced in zoos using spermatozoa collected from wild males, effectively integrating captive and wild populations without removing animals from the wild. However, production of viable offspring in sand cats has never been demonstrated using these technologies. Our objectives were to: Cumulus-oocyte complexes were collected from gonadotropin-treated I. A total of high-quality oocytes Although pregnancy and embryo survival percentages were relatively low in this initial embryo transfer study, the successful production of kittens using IVF and ET demonstrates the value of these reproductive technologies for global management of sand cat populations.
Interspecies somatic cell nucleus transfer could not only be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos but can also be used as a possible approach to conserve endangered species. It could provide a useful technique to preserve the wild Bactrian camels, which are threatened with extinction. In the present study embryos were reconstructed by using skin fibroblast cells from a Bactrian camel Camelus bactrianus as donor karyoplasts and dromedary camel Camelus dromedarius oocytes as recipient cytoplasts for investigating the developmental potential of the these embryos.
Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up, 26 h after GnRH administration. Serum-starved adult Bactrian camel skin fibroblast cells were injected in to the perivitalline space of enucleated dromedary oocytes. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. The embryos reconstructed from dromedary camel Camelus dromedarius fibroblasts and oocytes were treated in similar manner and worked as control.
Out of 58 reconstructed embryos, in 3 replicates, The embryos that cleaved This study demonstrated, for the first time, that somatic cells from Bactrian camel can be reprogrammed in the dromedary camel ooplasm and such interspecies nuclear transfer embryos can develop to the blastocyst stage. Further studies are needed to see the feasibility of transferring these embryos into dromedary camel recipients and the possibility of producing Bactrian camel calves from dromedary recipient camels. The most recent report from the CDC shows that more than one third of American adult women are obese.
The Developmental Origins Hypothesis postulates that potentially adverse conditions in utero, such as exposure to a maternal high fat diet and maternal obesity, are associated with fetal alterations, rendering susceptibility to adult metabolic disorders. We have previously reported in a non-human primate model that exposure to a maternal high fat diet results in both pathological and epigenetic changes to the fetal liver. We have shown that high fat diet exposure results in non-alcoholic fatty liver disease, concomitant with covalent modifications to fetal hepatic chromatin structure, specifically increasing histone H3 lysine 14 acetylation H3K14ac levels.
We also show that this increased acetylation correlates with the reprogramming of several metabolic and circadian regulatory genes. The expression of Npas2 , a peripheral circadian transcription factor, is disrupted in fetal hepatic tissue from animals exposed to a maternal high fat diet, returning to those of control diet upon high fat diet cessation. We also report the disruption of other circadian genes regulated by Npas2 , such as Per1 and Reverb-alpha in the high fat diet exposed animals. Extensive analysis of the Npas2 promoter region shows that DNA methylation levels do not differ due to maternal diet.
However, using chromatin immunoprecipitation of an important regulatory region of the Npas2 promoter, we find enrichment of H3K14ac in animals exposed to a high fat diet compared with those on a control diet. This enrichment is lost from the diet reverted group. We conclude that H3K14ac levels are perturbed by maternal high fat diet exposure, which correlates with altered gene expression.
We find that reversion to a control diet, even with persistent maternal obesity, helps to attenuate some epigenomic and circadian perturbations. Evidence is accumulating that exposure of mammalian fetuses to environmental endocrine disruptors EEDs affects epigenetic regulation of gene expression in germline cells. Such epigenetic changes may be heritable, conveying EED effects trans-generationally. However, mechanistic bases of such EED-induced epigenetic reprogramming largely remain unexplored.
Chicken has a long history as a favored homothermic vertebrate model animal for studies in developmental biology due to its excellent accessibility to live embryos at early stages of development. In contrast to mammals whose sex is strictly determined by the XY sex chromosomes, avian phenotypic sex determined by the ZW sex chromosome system [males have homologous ZZ and females have heterologous ZW sex chromosomes] is readily reversed by fetal exposure to exogenous sex steroids or inhibitors of sex steroid synthesis during early development.
Taking advantage of the exogenous estrogen-inducible gonadal feminization of genetically male embryos, in the present study we attempted to establish an in vivo model of EED-induced changes in DNA methylation in germline and somatic cells in gonads. White Leghorn ZZ-male chicken embryos were exposed to 17alpha-ethynylestradiol EE2 during early stages of development Day , and their gonads were collected shortly before hatching Day 19 for morphological characterization, mRNA expression profiling by microarray and qPCR, and pyrosequencing determination of promoter DNA methylation.
Exposure of ZZ-male embryos to EE2 before completion of the morphological testicular differentiation Day 7. The highly reproducible and sequence-specific DNA hypomethylation of chicken embryonic CYP19A1 promoter induced by exposure to EE2 during early in ovo development may provide unique opportunities to study mechanisms of the epigenetic and trans-generational effects of endocrine disruptors.
There are numerous tissue-dependent and differentially methylated regions T-DMRs in the mammalian genome. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm TE and inner cell mass ICM in blastocyst. In this study, we aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. These two loci are hyper-methylated in TS cells and hypo-methylated in ES cells. They were in extremely hypomethylated status.
DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic cell lineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity. During mouse blastocyst formation the segregation of the inner cell mass ICM and trophoblast is governed by the mutually antagonistic effects of the transcription factors OCT4 and CDX2.
Evidence indicates that suppression of the Oct4 gene in the trophoblast is mediated by CDX2. Nonetheless, the underlying epigenetic modifiers required for CDX2-dependent repression of Oct4 are largely unknown. Using a combination of RNA interference and gene expression analysis we found that BRG1 regulates Oct4 transcription specifically at the blastocyst stage.
We found that in BRG1 deficient blastocysts Cdx2 expression is normal, yet there is widespread expression of OCT4 in the trophoblast cells. Moreover, using trophoblast stem TS cells as a model system for the blastocyst trophoblast we found that CDX2 interacts with BRG1 via co-immunoprecipitation. In toto, these findings point to a novel role for BRG1 and CDX2 in transcriptional repression of the Oct4 gene in the developing trophoblast. The epigenetic mechanisms involved in establishing and maintaining genomic imprinting are steadily being unmasked. The nucleosome remodeling and histone deacetylation NuRD complex is implicated in regulating DNA methylation and expression of the maternally-expressed H19 gene in preimplantation mouse embryos.
The NuRD complex in mammalian cells is composed of at least 7 polypeptides. We find that Mta2 is the only zygotically expressed Mta gene prior to the blastocyst stage and that RNAi-mediated knockdown of Mta2 transcript leads to biallelic H19 expression and loss of DNA methylation in the imprinting control region ICR in blastocysts. In addition, biallelic expression of the paternally-expressed Peg3 gene, but not Snrpn , is also observed in blastocysts following Mta2 knockdown. No reduction in the amount of MTA1 or MTA3 was observed following targeting of their mRNAs, precluding any insight into the role of these two genes in genomic imprinting.
Like other regulatory molecules, altered miRNA expression has been implicated in the formation of cancers. To identify putative tumor suppressor microRNAs in ovarian cancer, we used Illumina next generation sequencing technology to comprehensively profile microRNAs in human ovarian cancers, cancer cell lines, and normal ovarian surface epithelium NOSE. We found that miR was the most down-regulated miRNA, decreased an average of fold range: Likewise, miR levels were decreased fold range: Using an established bioinformatic algorithms, we identified hundreds of potential miR We followed up these findings by validating in vitro the interrelationship of the predicted upregulated target genes with the downregulation of the miR microRNA.
Overexpression of miR in the serous ovarian cancer cell line, OVCAR8, resulted in inhibition of proliferation and induction of apoptosis. Similar responses to miR were observed in a number of ovarian cancer cell lines with a dysfunctional p53 pathway i. Our results reveal a tumor suppressive effect of miR in ovarian cancer.
Further in vivo studies to explore the impact of altered miR expression are highly warranted and are being performed. The pituitary gland is the master endocrine regulator responsible for controlling such physiologic functions as fertility. To understand the cellular processes that contribute to hormonal fluctuations and feedback signaling that regulate fertility we must first understand the development of the pituitary.
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During embryonic development, the pituitary is induced from the oral ectoderm, forming Rathke's pouch RP , a primordial structure containing proliferating progenitors. As development progresses, progenitor cells encounter a cell fate decision: Recent evidence shows that critical mediators of this decision are cyclin dependent kinase inhibitors CDKI. Increased CDKI expression is associated with a quiescent state and allows for differentiation. Disruption of this process can lead to abnormalities in pituitary development forming either a hypomorphic or hypermorphic organ, which can affect reproductive physiology.
Loss of the CDKI, p21 leads to spontaneous tumor development in mice, indicating its role as a tumor suppressor. Recent evidence from our lab has shown that mice mutant for p21 have increased numbers of cells in mitosis in embryonic development. Furthermore, loss of p21 results in incomplete RP separation from the underlying ectoderm. With these observations, we hypothesize that loss of p21 disrupts pituitary progenitor cell proliferation and can affect pituitary development. To test this hypothesis, we examined wildtype and p21 mutant pituitaries throughout embryonic life and evaluated proliferation profiles, organ morphology as well as which molecules and pathways collaborate with p RP closure, in wildtype mice, occurs between e By utilizing somite staging, we now show that RP closure is delayed in the absence of p Recent work from our lab shows that the transcriptional repressor, Hes1 , of the Notch signaling pathway is able to influence p21 expression in the developing pituitary.
Loss of Hes1 results in increased cell death, at e Interestingly, loss of p21 in Hes1 mutant pituitaries recovers the ectopic cell death. Taken together these data indicate a potential regulatory mechanism controlling p21 expression as well as linking p21 to cell death regulation. Further experiments will clarify which cell death pathway is induced by p Recently our lab has revealed a synergistic role for p21 with another CDKI, p27, of the same protein family.
Loss of p27, alone, results in pituitary hyperplasia. We now show that loss of both p21 and p27 results in hyperplastic pituitary growth as young as 21 days. This early hyperplastic phenotype may indicate that loss of CDKI action during embryonic development may contribute to adult phenotypes. Further experiments will clarify at which stage of development increased proliferation occurs.
With these studies we show that the cell cycle inhibitor p21 alone and synergistically with other molecules is necessary to maintain a balance between progenitor expansion and cellular differentiation. Full-length 52 kDa Smad3 was induced after 30 minutes of activin treatment, whereas the smaller Smad3 species accumulated slightly later, between 30 minutes and 1 hour, and then persisted beyond 2 hours. Activin-induced phosphorylation of both large and small phospho-Smad3 proteins was blocked by follistatin treatment or transfection of siRNA directed against Smad3.
We determined that the small Smad3 protein was neither a proteolytic- nor a proteasome-processed form of Smad3, but a transcription product limited to the gonadotrope. To test the biological function of the small Smad3 protein, we constructed a constitutively active C-terminal fragment of Smad3 protein. These glycoforms are distinct from isoforms , which reflect poorly-defined properties related to the amount, type, and charge of carbohydrate, not the number of protein sites glycosylated. Assaying a number of human pituitaries, we have demonstrated that in younger, reproductively active women, hFSH-di is present at higher levels than hFSH-tetra.
In peri-menopausal women, the levels of the FSH glycoforms are roughly equal. Owing to the apparent age-related shift from hFSH-di dominance to hFSH-tetra dominance, we set out to compare their relative bio-potencies in a series of in vitro assays. In these cells, purified hFSH-di was of similar potency to that of equine FSH, which exists mostly as a tri-glycosylated hormone. Our results suggest that as women age, they produce a less potent form of FSH. Moreover, we have determined that commercially available hFSH preparations utilized for controlled ovarian hyperstimulation COH , regardless of whether they are recombinant or menopausal preparations, consist almost entirely of hFSH-tetra.
Whether the increased activity of hFSH-di relative to hFSH-tetra observed in vitro extends to similar activity in vivo represents a potentially important therapeutic question that is currently being investigated. In sheep, prenatal exposure to testosterone during a critical period for differentiation can masculinize both the external genitalia of fetuses and the ovine sexually dimorphic nucleus oSDN of the hypothalamus-preoptic area of fetuses.
The present study investigated the possible independent control of these two anatomical end points, as revealed by their developmental history. Specifically, we tested the hypothesis that separate critical periods exist for androgenization of the genitalia and the brain. As a control C pregnant ewes were treated with oil during the same gestational periods.
Fetuses were weighed and their crown-rump length and ano-genital distance measured. Their brains were then immersion fixed for subsequent volumetric analysis. The oSDN was identified by its characteristic expression of aromatase mRNA using in situ hybridization and by thionin staining. Female fetuses from the Early TP group possessed a penis, scrotum devoid of testes, uterus and ovaries, whereas the external and internal genitalia of females from the Late TP and Control groups were normal. Neither period of exposure to TP grossly affected the genitalia of male fetuses.
The volumes of the oSDN between early and late control fetuses were not different within sexes and thus were combined for comparison with the TP-treated groups. These results demonstrate that the critical period for sexual differentiation of the oSDN occurs between Days 60 and 90 of gestation indicating that masculinization of the oSDN can be separated from that of the external genitalia, which occurs between Days 30 and 60 of gestation. The observation that exposure of male fetuses to TP from Day 30 to 60 of gestation blocked masculinization of the oSDN suggests that there may be de-masculinizing effects of early exposure to exogenous androgen in the male fetal brain.
The objective of this study is to determine if there are neuroendocrine correlates associated with male parental behavior. Besides humans, the common marmoset is one of only a few species of primates to show extensive paternal care. We designed the study to evaluate hormone release over time from hypothalamic explants collected from male marmosets in two groups: Ten male marmosets were used in this study. Five of the males were experienced fathers who had at least two sets of offspring prior to the study and were between eight and eleven years of age. Induced RNA from positive wells was sequenced by NGS in pol and gp41 regions to determine the most prominent viral strains in the well.
Immunosuppressive regimens were per standard of care, and for kidney recipients included antithymocyte globulin ATG for induction. IPPM increased over time in 7 patients ranging fold with 4 remaining elevated higher than baseline for 26 weeks or more. Viral clones were maintained in the LR for all four patients, and in 3 patients there was an emergence of previously unseen viral variants post-transplant.
Using QVIA we measured an increase in size of the LR in several patients post-transplant, an unexpected result in a cohort of patients receiving lymphocyte-depleting ATG treatment. The reservoir of HIV infected cells in antiretroviral treated individuals represents a challenging hurdle for cure of infection. In infected individuals, clonally expanded, HIV-infected cells persist and comprise a significant fraction of the reservoir.
Defining HIV integration sites IS could provide insight into genes that may be modulated by the provirus, which may contribute to the persistence of these clones. Sequencing of HIV IS can be challenging and costly due to the relative rarity of infected cells in treated individuals, especially in those who initiate ART during primary infection.
However, HIV infection of astrocytes in vitro is inefficient. We investigated if HIV strains from the central nervous system were able to infect astrocytes. An HIV isolate was characterized in cell lines and primary astrocytes. These observations indicate that this isolate was an X4-tropic virus. Its X4 tropism was further confirmed by sequencing the env gene and prediction based on its V3 loop. When the virus stock was initially incubated with primary fetal astrocytes, no infection was detected. However, the virus could infect astrocytes via a transwell culture system where HIV-infected JKT cells were loaded on the top chamber and only HIV particles could go through its membrane and reach astrocytes in the bottom wells of plate.
This is consistent with prior observations that immature HIV particles released from the infected lymphocytes were able to directly bind to CXCR4 on astrocytes in the absence of CD4 triggering virus-cell fusion and leading to the infection. Single genome amplification of HIV and sequencing was performed in CSF cells in a follow-up visit, but only defective genome containing gag was obtained.
Despite adequate antiretroviral therapy, low level HIV may be still present in the CSF that may predict viral reservoir in the brain. The ability to determine if a curative intervention reduces the reservoir or if a latency reversing agent is effective will depend on access to ultrasensitive, high-throughput measurements of residual viremia. Current viral quantification methods are limited by lack of sensitivity or the need for specialized, lengthy processing. Dilutions of the 4 samples calculated to range from 9 to 0. An additional 36 reps were performed on a subset of 19 samples, 7 of which were initially undetectable.
Four of seven initially undetectable samples had positive results when tested with the additional reps. Given the assay's performance characteristics, its lack of reliance on specialized specimen handling and the highly automated approach, this assay is well-suited to early- and late-phase clinical trials of HIV curative interventions. While the mortality associated with HIV-1 infection has decreased, the incidence of neurocognitive impairment NI has increased.
Previous studies have detected the HIV-1 protein Tat within the periphery, CSF, and brain of infected patients, even those adherent to antiretroviral therapy. Current studies seek to identify and characterize predominant genetic variations within Tat that correlate with NI and examine their functional impact on intracellular promoter transactivation and extracellular secretion, neurotoxicity functions of the protein. Results have demonstrated no difference in LTR transactivation in these patient-derived Tats.
Logistic regression models have predicted that specific amino residues correlated with the NI status of a given patient. These Tat variants are being produced and examined for alterations in transactivation of the viral LTR or host genes and impact on secretion and for extracellular activity in an animal model to assess alterations in behavior caused by injection of extracellular Tat within the prefrontal cortex PFC.
Results have demonstrated certain amino acid variants in Tat to reduce LTR transactivation. Amino acids associated with Tats from NI patients have the potential to differentially alter selected protein functions and may be involved in determining whether individuals develop HAND. A major barrier to developing HIV cure interventions is the lack of validated assays that reliably quantify HIV in plasma below the limit of detection of current clinical assays.
We sought to objectively assess the performance characteristics of newer, more sensitive assays to quantify plasma viremia. Results were analyzed for sensitivity, specificity, reproducibility, and ability to accurately quantify HIV in standards. Data from five laboratories using 6 assays were included in this analysis. Ultrasensitive p24 Ag assays were not able to quantitatively measure HIV in the diluted standards.
However, whether these cells are latently infected or support low levels of viral production is still unclear. We evaluated the relative contributions of latency and viral production in LN from individuals on suppressive ART. The frequency of Tfh cells was measured by flow cytometry in all samples. Our results indicate that residual levels of HIV transcription decrease with the time spent on suppressive ART and that the contribution of Tfh cells to the HIV reservoir may diminish after several years of viral suppression. Some subjects on antiretroviral therapy have detectable levels of HIV-1 in their cerebrospinal fluid CSF despite having undetectable levels in their plasma i.
In addition, single genome amplification SGA was used to generate full-length env clones from these viral populations and their macrophage tropism was determined based on their ability to enter Affinofile cells expressing a low density of CD4, similar to the level expressed on macrophage. Deep sequencing analyses revealed that the CSF escape virus from the two time-points formed a lineage that was diverse, genetically distinct from virus found in the plasma pre-therapy, and partially drug-resistant. Genetic analyses of viral populations in plasma at three time-points after ART initiation, but prior to plasma viral suppression, revealed emergence of a viral lineage that was genetically similar to the CSF escape virus population.
Entry assays revealed that the CSF escape virus had an enhanced ability to utilize a low level of CD4 for entry. Our results indicate that CSF escape is a variable condition with different origins. Current antiretroviral therapy allows the sustained control of viremia in most HIV-infected patient.
Treatment interruptions almost invariably result in a rapid rebound of viremia to pre-therapy levels. Here we characterized clonal viruses from the reservoir of two patients. Viral emergence was monitored by p24 quantification in the culture supernatant. Individual clonal viruses were isolated by short term culture, and their replication kinetics and per-particle infectivities were determined.
The near full-length genome of individual clones was sequenced. Clonal replication-competent viruses from 12 wells from each patient were investigated. Within each patient, clonal viruses displayed different replication kinetics generating peaks between day 4 and 9 patient 1 , and between day 9 and 14 patient 2. Also, for viruses reaching the peak on the same day, the amount of virus produced in the supernatant varied extensively.
Single-cycle per-particle infectivities differed by 4-fold patient 1 and 2-fold patient 2. Near full-length genome comparison will be used to identify genetic determinants of the observed variances, taking advantage of the close phylogenetic relatedness of individual clones from each patient. This work highlights differences in the genotypic and phenotypic properties of reservoir viruses from treated patients. The characterization of replication competent viruses is crucial for the design of strategies aiming the reduction of the HIV reservoir or the prevention of virus rebound.
HIV integration is a key step in the viral replication cycle. Prior studies disagree on whether HIV preferentially integrates in oncogenes leading to clonal proliferation. Among a total of 31, detected integration sites, there was a high degree of similarity in integration site loci 7, sites and frequency between clinical and humanized mouse samples.
The majority of insertions occurred in genes that may serve to promote viral latency, supported by the observation that a higher number of unique integration sites correlated with lower levels of residual viral transcription, and the observation that integrations were enriched in genes associated with cell cycle, response to DNA damage, and viral transport. Upon initiating ART, plasma viremia undergoes multiphasic decay kinetics reflecting loss of infected cells. HIV infected cells also decline but kinetics of decay during first weeks of ART have not been well characterized.
In particular, it is not known what proportion of infected cells contribute to viremia. To address this issue, we developed sensitive and accurate multiplexed droplet digital approaches ddPCR to quantify early HIV-1 decay kinetics. During second phase decay, virus production declined to c. After initiating ART, HIV reservoirs responsible for persistent viremia are relatively limited and exhibit substantial variation in virus production. Genetic diversity and immune escape within the replication-competent HIV reservoir are barriers to cure; our knowledge of these parameters remains incomplete, particularly among infected youth.
We additionally investigate possible vaccine-induced selective pressures altering reservoir genetic composition. Inferred CTL escape mutation burden varied within and between hosts. All but one participant exhibited at least one example of susceptible and escaped forms of the same CTL epitope co-existing within their reservoir. Results suggest that HIV vaccines may have perturbed the reservoir in a subset of participants.
The higher burden of Pol escape in perinatal infection suggests that reservoir escape burden in conserved, rather than in the variable, HIV regions may correlate with untreated infection time as the former regions escape more slowly. Targeting reactivated latent reservoir cells with HIV vaccines will require consideration for the complexity of within- and between-host reservoir diversity and escape burden.
Longitudinal samples were available at 1, 6, and month post-detection. Given the continuous nature of within-host HIV evolution and reservoir establishment, and the long-lived nature of latently-infected cells, the reservoir should comprise a genetically heterogeneous archive of within-host HIV evolution. This heterogeneity could complicate immune-based cure strategies but our understanding in this area remains limited, in part because methods to infer latent HIV sequence ages are lacking.
We developed a phylogenetic method to reconstruct HIV integration dates and applied it to date reservoir sequences in persons with long-term viremia suppression. The method involves inference and optimal rooting of a maximum-likelihood phylogeny from longitudinal within-host plasma HIV-RNA and reservoir sequences, followed by calibration of a linear model relating root-to-tip distances of the former to their sampling dates. The model is then used to convert reservoir root-to-tip distances to their establishment integration dates.
Putative reservoir sequences were interspersed throughout both within-host phylogenies and exhibited comparable diversity to pre-cart plasma RNA sequences sampled over 10 years. Historic within-host genetic bottlenecks were also recorded in the reservoir. Inferred proviral integration dates were consistent with the reservoir harboring both ancestral and recent lineages, with the oldest dating to 20 years prior to sampling.
Sensitivity analyses confirmed that the method can be applied to as few as two plasma HIV-RNA timepoints and that it is robust to rooting approach, thus broadening its applicability. Our method for reservoir dating provides a novel and powerful addition to the HIV persistence research toolkit and reveals a genetically diverse. Although antiretroviral therapy ART can limit HIV replication to below the limit of detection of clinical viral load assays, individuals experience residual viremia. We hypothesized that infected proliferating cells that persist during prolonged ART will consist primarily of effector memory T Tem cells and that these cells contribute to the monotypic residual viremia.
Sequences were assembled into a maximum-likelihood phylogenetic tree. HIV IS were identified from sorted cells using multiple displacement amplification-integration site looping assay. The proviral C2V5 env sequences adjacent to IS were used to compare to plasma C2V5 sequences to identify the potential cellular origin s of plasma virus.
Although from a single case, we found peripheral Tem may contribute to persistent monotypic plasma virus during ART. Ongoing studies will examine the genome integrity of this provirus. Further analysis of integration sites in immune cells may elucidate the potential biological pathways manipulated by HIV to allow persistence during ART. Differences between methods were calculated with a generalized linear model. To elucidate the mechanisms behind these effects, we measured the virus diversity and reservoir size in patients treated with temporary ART during PHI.
No significant difference was observed in the HIV nucleotide diversity of these markers between the treated and untreated patients. The HIV T-cell reservoir harbors only a small percentage of intact HIV genomes, but little is known about the functional capacity of viral proteins expressed by these sequences. We have focused here on expression and function of the HIV envelope glycoprotein, a major target for the host immune response and a strong effector of HIV cytopathicity. After in vitro T-cell stimulation, Env sequences were obtained from two sources: Env sequences were cloned in a Rev-independent expression vector.
Env expression was examined quantitatively by flow cytometry, Western Blot and immunofluorescence. Env function was tested using a quantitative cell-cell fusion assay and a pseudotype infectivity assay. Only clones with intact sequences were tested. As expected, clusters of identical sequences were seen in all 4 patients and Env diversity was widest in patients with longest HIV history. Fusogenicity was significantly lower among mRNA-derived Envs: Similar findings were observed in a pseudotype infectivity assay, with a significant correlation.
The discovery of apparently intact, yet partially or fully defective, Env sequences in the HIV reservoir has two implications. First, Env fusogenicity and cytopathicity could be one important factor driving selection of defective HIV genomes in the reservoir. A sanctuary likely contains long-lived cells, has limited drug penetration, and is an evolutionary compartment prior to cART; all features consistent with the brain. Reads were aligned using MAFFT and manually edited for a sequence total of 52, s and 34, s Maximum likelihood phylogenies were inferred using PhyML.
Charge, length, and number of glycosylation sites were calculated for env variable regions. Brain-derived sequences showed significant population structure compared to non-brain. At the lowest clustering threshold 0. At higher thresholds, additional LN and colon sequences also grouped with the P sequences. Different charge and glycosylation site patterns characterized brain and non-brain tissues. Brain-derived virus is a distinct population with potential functional differences that may impact persistence.
HIV-DNA decreased after 48 weeks of dolutegravir-based regimen in all individuals except for switch cases. Although HIV infection promotes several macrophage MO -associated comorbidities, the viral mechanism driving these specific processes remains unknown. The HIV Nef protein is maintains a variety of functional domains. We hypothesized that subtle changes within these domains stimulate MOs towards specific disease pathways. We used nonlinear models trained using machine learning to distinguish Nef sequences from patients with either HAND or cancer, based on slight physicochemical shifts within functional domains.
For 23 subjects, the primary pathology, other than HIV infection, was known. Brain sequences were available from 10 subjects with HAND. Evolved neural networks ENNs were applied to training and testing data sets to classify 1 brain from non-brain sequences and 2 cancer from non-cancer sequences. The best ENN was assayed for performance on the training, testing, and a third validation dataset of sequences with unknown etiology.
Each sequence was scored and a binary threshold was used to discriminate samples into one of the two classes.
Nef sequences from subjects with either HAND or cancers at death were classified with Our approach elucidated structure-function properties of disease specific Nef that were not discernable from primary sequence information alone. Modifications in several functional domains were associated with brain-derived Nef; whereas the AP2 binding domain alone strongly associated with cancer-derived Nef.
Underlying physical and chemical differences of Nef are associated with specific pathologies, suggesting that a Nef-associated mechanism may promote MO-associated disease pathways. Disease-associated Nef will be useful as a biomarker and in biological assays to design novel drug targets to treat HIV-associated comorbidities. The impact of antiretroviral therapy ART including novel integrase inhibitors on HIV reservoir is not clearly characterized. A total of participants achieved or maintained virological control on DBR.
According to the clinical stage of HIV infection, participants were assigned to different groups: Though, dolutegravir failed at reducing HIV reservoir levels when introduced as a switching regimen. Beside hemostasis, human platelets carry important immunological functions by interacting with immune cells. Furthermore, platelets interact with infectious pathogens and internalize HIV in vitro.
Human primary macrophages were co-cultured with PRP without or with the platelet activation-blocker Abciximab to assess capacity of platelet-containing HIV to transfer and propagate infection. The presence of HIV in platelets was correlated with patient clinical status. Platelets from cART-treated patients with detectable but also undetectable viral loads enclose infectious viruses as detected consistently by viral RNA, p24 expression, and localization within platelet internal compartment.
Functionally, platelet sheltering HIV can propagate productive infection to macrophages in-vitro. Blocking platelet activation with the therapeutical agent Abciximab prevents this transfer. This study not only sheds new light on the biology of HIV pathogenesis, describing an alternative pathway for viral dissemination in which platelets act as carriers of infectious viruses and as new players in cell-to-cell HIV transmission, but also suggests new diagnostic and therapeutic perspectives to improve immunological recovery in cART-treated patients.
HIV eradication efforts have been unsuccessful due to virus persistence in cellular and tissue reservoirs. These cells control the magnitude and specificity of the GC response and like Tregs are essential for the maintenance of self-tolerance and immune homeostasis. However, the immunosuppressive role of TFR cells in HIV infection and their contribution to viral control is not completely understood, particularly in clade C infection.
Thus, we set out to investigate TFR cells using LN and peripheral blood PB samples and further determine the effect of early treatment on the frequency and function of this cell subset. This result was confirmed by IHC. These results could have implications for immunotherapeutic interventions aimed at using TFR and TFH cells as potential targets. The eradication of the latent virus reservoir is a key barrier to an HIV-1 cure, but the molecular signatures and pathways of latently infected cells not completely understood.
Identification of biomarkers enriched in latently infected cells will allow for the monitoring and targeted of the latent reservoir for eradication. Here, we utilize single-cell RNA-seq to determine the transcriptomes of immune cells from HIV-infected individuals before and after ART to identify transcriptionally unique clusters of cells that may express candidate biomarkers. Single-cell transcriptome analysis demonstrated that immune cells from infected individuals are significantly altered during infection when compared to seronegative individuals.
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ART initiation restores some of the molecular pathways to that of seronegative individuals, but many immune cells remain altered during ART when compared to seronegative individuals. We have established high-throughput RNA-seq at single-cell resolution. Single-cell resolution has also determined biomarkers of HIV-infected cells within the same donors.
Future studies using this approach on cell populations enriched in the latent pool may yield novel biomarkers for the identification of the HIV latent reservoir. Treatment of a primary cell model of latency model Cultured TCM model , with either bryostatin alone or bryostatin with venetoclax, led to significant decreases in HIV DNA and replication competent reservoir. Further study is needed to explore venetoclax as an adjunct to kick-and-kill therapies. In addition, these cells display poor killing capacity in a redirected cell lysis assay.
Instead, they possess high levels of non-cytolytic polyfunctional responses directed against HIV peptides. Such responses were low or absent in non-controllers or those on ART. During established infection, HIV appears to be controlled in tissues by non-cytolytic rather than cytolytic mechanisms. Knowledge regarding how HIV is controlled in this setting should be used to inform the identification and development of potentially curative interventions. HIV-specific broadly neutralizing antibodies bnAbs , defined by unusually high neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, targeting them for immune clearance.
Few studies have assessed the reactivity of bnAbs against viruses reactivated from reservoirs. A panel of bnAbs were tested for surface binding to cells infected with 36 reservoir isolates by flow cytometry, and for neutralizing activity against the same viruses using a TZM-bl assay. Combination of two bnAbs, e. We observed substantial heterogeneity in the binding and neutralization profiles of different bnAbs to reactivated reservoir viruses.
Our results provide guidance on the selection of bnAbs for interventional cure studies. Current HIV cure studies are focused on the eradication of the viral reservoir but are limited by low sensitivity and poor reproducibility. This is partly due to the low frequency of cells harboring latent replication competent virus. Furthermore, the process of reactivation of the latent reservoir remains entirely stochastic resulting in an underestimation of the size of the viral reservoir.
Finally, the in vivo administration of RA to macaques with low viral loads led to a moderate increase in plasma viral loads. Infected cells that persist are thought to be invisible to the immune system, but if there is intermittent antigen expression and recognition, HIV antibody Ab and T cell responses may correlate with measures of HIV persistence. These findings suggest that the total frequency of HIV-infected cells HIV DNA may be a better marker of antigen expression that drives immune responses on ART than CA RNA in blood or residual viremia, which reflect activity of only a small fraction of proviruses that can be induced to express antigen.
These results suggest the immune system is sensing infected cells; tracking immune responses may be a method of assessing the impact of reservoir-reducing strategies. SAMHD1 is subject to regulation by multiple cyclin-dependent kinases, which inactivate the enzyme by phosphorylation at a specific threonine residue T We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons IFN and pharmacologic agents to modulate this process.
Mature, adherent macrophages were incubated with various interferons or pharmacologic agents, then analyzed for SAMHD1 protein and T phosphorylation or infected with HIV The viral restriction enforced via interferons or Dasatinib could be overcome by either incubation with excess deoxynucleosides or by addition of the SIV accessory protein Vpx in trans. Furthermore, we show that the levels of SAMHD1 phosphorylation determine macrophage susceptibility to infection in the absence of stimuli.
Here, we propose a common mechanism through which IFNs exert their potent anti-viral effects, and provide an explanation for the extreme donor-to-donor variability of macrophage susceptibility to HIV A loss in proliferative capacity is a key feature of exhaustion, but little is known about how this capacity is regulated in these cells.
Therapeutic immunization with a DNA vaccine expressing conserved elements CE can redirect and broaden T-cell responses towards conserved regions of virus. However, immune suppression may limit the efficacy of such responses. Myeloid-derived suppressor cells MDSC are a suppressive subset of immature myeloid cells activated and expanded during inflammatory cytokine elevation.
Additional studies are in progress to evaluate gMDSC in animals receiving CE-vaccination in combination with T-cell protection strategies, latency reversal and exhaustion blocking agents. Our results to date suggest that gMDSC can suppress T-cell responses to viral antigens in vivo and reduce the efficacy of therapeutic vaccines.
Furthermore, the remarkable increase in gMDSC following ATI is a concern as these responses may limit efficacy by suppressing the ability of vaccine-induced T-cell responses to control viral rebound. During chronic infection, one potential mechanism for this failure is relatively low levels of virus-specific CD8 T cells in B cell follicles, where virus is most concentrated, permitting ongoing virus replication. It is not known whether this phenomenon also occurs during early infection.
These data suggest that during early stages of infection, low levels of follicular SIV-specific CD8 T cells may permit ongoing viral replication, similar to what we previously reported in chronic disease. These findings provide important insights into SIV immunopathogenesis and may help inform future cure strategies. Combination anti-retroviral therapy cART is effective in maintaining low viral loads, restoring CD4 T cell counts, and prolonging life. However, even individuals with durably suppressed viral load under cART experience residual immune dysfunction. Though some CD8 T cell function is restored under cART, cells retain an effector-like phenotype and exhibit impaired antigen-specific proliferation.
Measures to ameliorate this residual dysfunction, such as IL therapy, could be used to boost HIV cure and therapeutic vaccination strategies. The success of the shock and kill strategy depends both on the reactivation of the latent reservoir and on the ability of cytotoxic T cells CTLs to recognize and kill HIV infected cells. Although several agents efficiently reactivate latently infected cells in vitro , they do not induce a measurable decrease of the HIV reservoir in vivo and are limited by systemic toxicity. Studies on the functional quality of CTL clonotypes are needed to elucidate what characteristics are required to eliminate the HIV reservoir.
Characterizing the function of different CTL clonotypes might help define parameters indicative of efficient reservoir killing in vivo. We gated on two T-cell populations: However, the combination of CTL exhaustion and the establishment of CTL escape variants represent major hurdles towards this goal. Recent work suggests that SIV-infected macrophages are relatively resistant to CTL-mediated killing, but the mechanism behind their differential susceptibility is unknown.
Imaging flow cytometry was used to assess effector-target conjugates while cytokine-bead arrays characterized pro-inflammatory chemokines released by macrophages. These results suggest that inefficient CTL-mediated killing of macrophages may contribute to reservoir persistence and chronic inflammation in HIV infection. An improved understanding of the precise mechanisms underlying the observed resistance to target cell killing and resulting hypersecretion of pro-inflammatory cytokines and chemokines will be necessary to develop approaches capable of efficiently eliminating infected macrophages and hampering chronic inflammation.
HIV-infected people are not all equal towards HIV virus, at least regarding clinical and biological progression. Rather rare cases of individuals that do not progress or spontaneously control viral replication after a period of ART treatment are intriguing. Our objective was to compare different clinical phenotypes between them and with healthy donors using discriminating gene analysis, in order to identify family of genes that may distinguish them.
We performed a Gene Set Enrichment Analysis. Using curated gene sets from the Reactome Pathway Database, we detected upregulated gene sets in each of the two phenotypes. We performed hierarchical clustering of the patients to identify clusters of patients with similar gene expression profiles. Using classical multidimensional scaling, we projected the patients on four-dimensional space. We performed a pilot study including two treated post-treated aviremic seroconverters DE, MEZI , three non treated chronic HIV-infected patients H1 to H3 , one viremic seroconverter TW , one viremic patient that does not progress and consequently does not need any treatment CD and four healthy donors DS1 to DS4 , comparing their transcriptome profiles.
This work suggests correlates of immune protection induced by early treatment in specific profiles of HIV-infected patients through Transcriptome analysis. Our analysis may allow better understanding causes of non activation of the immune system, as well as innate factors contributing to viral control. However, immune exhaustion can also be detected on other immune cells eg.
NK cells , although less is known about the functional consequences. Here we characterized the expression of exhaustion markers on NK cells during HIV infection and evaluated their role in NK cell function. On the contrary, PD-1 was upregulated in NK cells from viremic individuals. NK cells from virally suppressed HIV patients have similar expression of markers of exhaustion, and similar function to that of HIV-negative donors, while these markers are altered in viremic donors. TIM-3 and TIGIT expression was associated with better function of NK cells, and thus further investigation should be conducted to evaluate if NK cells with upregulation of these markers have enhanced immunotherapeutic, antiviral activity in vivo.
Understanding the nature of the latent reservoir in tissue sanctuary sites is critical to the success of HIV cure strategies. A few human and non-human primate studies have reported that HIV-1 continues to replicate in lymph nodes LN during therapy while other studies have found no evidence of ongoing replication across multiple tissue compartments. Here, we used excisional LN and paired blood samples from hyperacute HIV infected subjects who initiated cART in Fiebig stage I to determine if there is persistent virus replication and identify cell subsets that harbor residual virus during cART.
Flow cytometry was used to phenotypically define germinal center GC Tfh subsets. HIV cure strategies should take into consideration the influence of potential ongoing virus replication in santuary sites during ART. To abrogate these effects, immunomodulatory agents IMA like ruxolitinib or rapamycin have been used in combination with PKCa. The hydrolytic activities of the degradation machinery are tunable by various stimuli, causing changes in protein degradation. The generation of known epitopes and potential MHC-binders was affected in a drug- and sequence-dependent manner.
Treatment with Bryostatin partly changed the MHC-peptidome of PBMCs, showing common as well as some unique peptides coming from different locations within their source proteins. Changes in peptidase activities and degradation patterns induced by PKCa or IMA are not necessarily linked to the activation status of the cells and are specific to the drug used. The modulation in antigen processing and peptide presentation caused by drug treatment diversifies the range of CD8 responses needed for clearance of reactivated HIV-infected cells.
Early initiation of antiretroviral therapy ART in vertically HIV-infected children provides an opportunity to limit the size of reservoir, but whether and how the time of ART treatment initiation can durably impact host immune responses associated with HIV infection is still unknown. However ET showed a better quality of these cells demonstrated by a higher proportion of cytokine producing T cells compared to LT.
On the contrary, LT response was paucifunctional. Our results suggest that time of ART initiation in HIV-infected children has a long-term impact on the quality but not the quantity of the host HIV-specific T-cell immune responses and also reinforced the importance of early treatment initiation. Larger studies are warranted to confirm these characteristics of host immunity and whether they can be targeted in functional cure approaches.
Strikingly, none of the PR tested showed impairment in trans infection either prior to or after initiation of cART. This mechanism could limit cART effectiveness. There is no cure for HIV-1, largely because HIV establishes a small but sustained pool of latently infected cells that are not cleared by antiretroviral therapy. We and others are investigating strategies to firstly reactivate the latent HIV reservoir and then use T cell immunotherapy to clear reactivated cells.
Currently, the level of pre-existing virus escape in the reservoir is unclear. In order to design effective T cell immunotherapies to boost and, or induce de novo T-cell responses, we investigated the landscape of T cell responses in durably suppressed HIV-1 infected participants. Power calculations derived from these data suggest that group sizes as low as six are sufficient to examine the immunogenicity of T cell vaccines. We detected a wide responses breadth of T cell responses across our cohort, with all HIV-1 proteins targeted. We also identified escape variants in the latent reservoir.
On-going studies are focusing on estimating the level of pre-existing escape and examining patterns of escape in the reservoir, which in turn will inform the design of T cell immunotherapies. How the enhanced transcriptional strength of the promoter-variant strains of HIV-1C modulates viral latency is the primary aim of the present study. Furthermore, a positive correlation between the Tat transcript levels and the rapidity of GFP switch off in the stronger viral promoters is indicative of the Tat-mediated positive feedback playing a critical role in regulating viral latency.
Allogeneic stem cell transplantation allo-SCT in HIV-infected subjects with severe hematological malignancies is the only described strategy capable to dramatically reduce HIV latent reservoir. Whether this putative eradication strategy is associated with seroreversion has not been established yet. Surprisingly, in two cases we found an undetermined Pt 19 and Pt 28 western blot.
These levels started to decrease directly after allo-SCT. We have observed evidence of seroreversion a few years after allo-SCT. At the initiation of ART, the viral load rapidly declines to very low levels, but integrated HIV DNA measurements show only minor changes over time, suggesting the reservoir is relatively stable. However, DNA measures may mask dynamic changes in thereplication-competent reservoir due to the large excess of defective proviruses.
Proviral integrity was determined through annotation of viral ORFs and stem loops. Write a customer review. Amazon Giveaway allows you to run promotional giveaways in order to create buzz, reward your audience, and attract new followers and customers. Learn more about Amazon Giveaway. Set up a giveaway. Feedback If you need help or have a question for Customer Service, contact us. Would you like to report poor quality or formatting in this book? Click here Would you like to report this content as inappropriate? Click here Do you believe that this item violates a copyright?
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