Vitrified turkey ovarian tissue cultured and assessed through gene expression patterns: A potential screening tool.

Biobanking of turkey ovarian tissue has the potential to play a crucial part in preserving female genetics. To date, ovarian tissue has only been vitrified using a standard protocol, with immediate analyses after warming, therefore, long-term cryoinjury is unknown. Long-term cryoinjury was investigated here by in-ovo culturing, fresh (non-vitrified), a purposefully suboptimal poor vitrification (PV), and the standard vitrified (StV) protocol. Assessments were performed via cellular morphological changes and mRNA gene expression differences, immediately (day 0) or after 2, 4, or 6 days of in-ovo culturing. On day 0, the mRNA levels of heat-shock protein A2 (HSPA2) were lowest in the fresh tissue, and increased 5-fold in the StV treatment, and 18-fold in the PV treatment. Whereas, by day 6, growth determining factor 9 (GDF9) mRNA levels within the fresh tissue were over 3-fold and 21-fold higher than StV and PV treatments, respectively. After 6 days of in-ovo culture the follicle density was highest in the fresh ovarian tissue (4701 ± 950 #/mm3), followed by the StV (1601 ± 300 #/mm3), with PV having the lowest density (172 ± 145 #/mm3). This shows that although the density of follicles was higher in StV versus PV, a considerable number (∼65 %) were lost compared to the fresh treatment. Additionally, the HSPA2 expression could be an early screening tool, whereas GDF9 expression could be a late screening tool, used to assess turkey ovarian tissue vitrification protocols. We conclude that the StV protocol should be further optimized to try and improve follicle numbers post-warming.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge.

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n = 6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.

Turkey hen sperm storage tubule transcriptome response to artificial insemination and the presence of semen.

Introduction: Sperm storage within the uterovaginal junction (UVJ) of avian species occurs in specialized structures termed sperm storage tubules (SSTs) and allows for prolonged storage of semen, though the molecular mechanisms involved in semen preservation are not well understood. Little work has been done examining how function of the SSTs is impacted by insemination and by semen present in the SSTs. Methods: Transcriptome analysis was performed on isolated SSTs from turkey hens receiving no insemination (control), sham-insemination, or semen-insemination at three timepoints (D1, D30, and D90 post-insemination). Bioinformatic and functional annotation analyses were performed using CLC Genomics Workbench, Database for Annotation, Visualization, and Integrated Discovery (DAVID), and Ingenuity Pathway Analysis (IPA). Pairwise comparisons and k-medoids cluster analysis were utilized to decipher differential expression profiles in the treatment groups. Results: The SST transcriptome of the semen inseminated group exhibited the greatest differences within the group, with differences detectable for up to 90 days post insemination, while control and sham-inseminated groups were more similar. In the semen-inseminated samples, upregulation of pathways relating to classical and non-classical reproductive signaling, cytoskeletal remodeling, physiological parameters of the local UVJ environment, and cellular metabolism was observed. In the sham-inseminated samples, upregulation of immune pathways and non-reproductive endocrine hormones was observed. Discussion: This work provides insights into the molecular level changes of the SST in response to insemination as well as to the presence of semen. Results from this study may have direct implications on fertility rates as well as potential strategies for avian semen cryopreservation protocols.

Transcriptome Analysis During Follicle Development in Turkey Hens With Low and High Egg Production.

Low and high egg producing hens exhibit gene expression differences related to ovarian steroidogenesis. High egg producing hens display increased expression of genes involved in progesterone and estradiol production, in the granulosa layer of the largest follicle (F1G) and small white follicles (SWF), respectively, whereas low egg producing hens display increased expression of genes related to progesterone and androgen production in the granulosa (F5G) and theca interna layer (F5I) of the fifth largest follicle, respectively. Transcriptome analysis was performed on F1G, F5G, F5I, and SWF samples from low and high egg producing hens to identify novel regulators of ovarian steroidogenesis. In total, 12,221 differentially expressed genes (DEGs) were identified between low and high egg producing hens across the four cell types examined. Pathway analysis implied differential regulation of the hypothalamo-pituitary-thyroid (HPT) axis, particularly thyroid hormone transporters and thyroid hormone receptors, and of estradiol signaling in low and high egg producing hens. The HPT axis showed up-regulation in high egg producing hens in less mature follicles but up-regulation in low egg producing hens in more mature follicles. Estradiol signaling exclusively exhibited up-regulation in high egg producing hens. Treatment of SWF cells from low and high egg producing hens with thyroid hormone in vitro decreased estradiol production in cells from high egg producing hens to the levels seen in cells from low egg producing hens, whereas thyroid hormone treatment did not impact estradiol production in cells from low egg producing hens. Transcriptome analysis of the major cell types involved in steroidogenesis inferred the involvement of the HPT axis and estradiol signaling in the regulation of differential steroid hormone production seen among hens with different egg production levels.

Characterization of hypothalamo-pituitary-thyroid axis gene expression in the hypothalamus, pituitary gland, and ovarian follicles of turkey hens during the preovulatory surge and in hens with low and high egg production.

Dysregulation of the preovulatory surge (PS) leads to lowered egg production. The hypothalamo-pituitary-thyroid (HPT) axis has been shown to influence plasma progesterone levels and follicle ovulation. The presence of thyroid hormone receptors (THR) in the reproductive axis suggests possible effects of thyroid hormone. To further understand the potential role of thyroid hormone on the PS, HPT axis plasma hormone concentrations and gene expression were characterized surrounding the PS in average egg producing hens (AEPH), low egg producing hens (LEPH), and high egg producing hens (HEPH) (n = 3 hens/group). Data were analyzed using the mixed models procedure of SAS, with significance indicated at P < 0.05. Average egg producing hens and HEPH displayed lower levels of triiodothyronine (T3) and higher levels of thyroxine (T4) inside of the PS, whereas LEPH showed inverse T3 and T4 levels relative to the PS. Expression of mRNA for hypothalamic thyrotropin-releasing hormone (TRH), pituitary thyrotropin (TSHB), and the main thyroid hormone metabolism enzyme (DIO2) were downregulated during the PS in AEPH and HEPH. Low egg producing hens displayed higher expression of mRNA for hypothalamic TRH as well as pituitary TSHB and DIO2 compared with HEPH. Average egg producing hens expression of THR mRNAs was upregulated during the PS in the hypothalamus but downregulated in the pituitary. High egg producing hens showed decreased expression of THR mRNAs in both the hypothalamus and pituitary when compared with LEPH. In ovarian follicles, THR mRNAs were more prevalent in the thecal layer of the follicle wall compared with the granulosa layer, and expression tended to decrease with follicle maturity. Minimal differences in follicular THR expression were seen between LEPH and HEPH, indicating that THR expression is unlikely to be responsible for steroid hormone production differences occurring between LEPH and HEPH. Generally, downregulation of the HPT axis was seen during the PS in AEPH and HEPH, whereas upregulation of the HPT axis was seen in LEPH. Further studies will be required to clarify the role of the HPT axis in the regulation of ovulation and egg production rates in turkey hens.

Differences in invitro responses of the hypothalamo-pituitary-gonadal hormonal axis between low- and high-egg-producing turkey hens.

Low-egg-producing hens (LEPH) ovulate less frequently than high-egg-producing hens (HEPH) and exhibit differences in mRNA levels for components of the hypothalamo-pituitary-gonadal (HPG) axis, suggesting differential responsiveness to trophic stimulation. Ovulation frequency is governed by the production of the pituitary gonadotropins and feedback of the ovarian follicle steroid hormones, which are regulated by HPG axis stimulation and inhibition at the hypothalamic level. The pituitary and follicle cells from LEPH and HEPH were subjected to in vitro hormonal treatments to stimulate or inhibit the HPG axis, followed by expression analysis of mRNA levels for HPG axis genes and radioimmunoassays for steroid hormone production. Statistical analysis was performed using the mixed models procedure of SAS. The pituitary cells from HEPH showed upregulation of genes associated with ovulation stimulation, whereas cells from LEPH showed upregulation of genes associated with inhibition of ovulation. High-egg-producing hens' follicle cells displayed a higher sensitivity and responsiveness to gonadotropin treatment. Level of egg production impacted ovulation-related gene expression in the pituitary cells as well as steroid hormone production in the follicle cells, with HEPH displaying a greater positive response to stimulation. These findings indicate that differences in egg production among turkey hens likely involve differential responsiveness of the cells within the HPG axis.

Effects of heat stress on performance, blood chemistry, and hypothalamic and pituitary mRNA expression in broiler chickens.

This study was conducted to evaluate potential hormonal mechanisms associated with the stress response, thermoregulation, and metabolic changes of broiler chickens exposed to high environmental temperature. Nine hundred 1-day-old male broiler chicks (Ross 708) were placed in floor pens and raised to 24 d. At 24 d, chicks were randomly assigned to 1 of 2 treatments, heat stress (HS) or no HS, and allocated into battery cages in 8 batteries (10 birds per cage, 2 cages per battery). On day 31, blood was collected prior to HS and analyzed using an iSTAT analyzer. Half of the batteries were then moved into 2 rooms with an elevated ambient temperature (35°C) for 8 h. The remaining batteries stayed in the thermoneutral rooms with an ambient temperature of 22°C. Beginning at 5 h after the initiation of HS, blood was collected and analyzed using an iSTAT analyzer, birds were euthanized, and hypothalamus and pituitary samples were collected (16 birds per treatment), flash frozen, and stored at -80°C until RNA extraction. Reverse transcription-quantitative PCR was used to compare mRNA levels of key corticotropic and thyrotrophic genes in the hypothalamus and pituitary. Levels of mRNA for each target gene were normalized to PGK1 (pituitary) and GAPDH (hypothalamus) mRNA. Differences were determined using mixed model ANOVA. HS decreased (P < 0.05) feed intake, BW, bicarbonate, potassium, CO2, and triiodothyronine, while it increased mortality, glucose, pH, plasma thyroxine, and corticosterone. Expression of pituitary corticotropin-releasing hormone receptor 1 was downregulated (P < 0.001), while corticotropin-releasing hormone receptor 2 mRNA levels were higher (P = 0.001) in HS birds. HS increased expression of thyroid hormone receptor ß (P = 0.01) (2.8-fold) and thyroid stimulating hormone ß (P = 0.009) (1.4-fold). HS did not affect levels of mRNA of genes evaluated in the hypothalamus. Results showed that HS significantly affected both the thyrotropic and corticotropic axes. Understanding the role and regulation of these pathways during HS will allow researchers to better evaluate management strategies to combat HS.

Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production.

BACKGROUND: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. RESULTS: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells. CONCLUSIONS: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens.

Characterization of the hypothalamo-pituitary-gonadal axis in low and high egg producing turkey hens.

Variation in egg production exists in commercial turkey hens, with low egg producing hens (LEPH) costing more per egg produced than high egg producing hens (HEPH). Egg production correlates with ovulation frequency, which is governed by the hypothalamic-pituitary-gonadal (HPG) axis. Ovulation is stimulated by a preovulatory surge (PS) of progesterone and luteinizing hormone, triggered by gonadotropin releasing hormone release and inhibited by gonadotropin inhibiting hormone. Differences between LEPH and HEPH were characterized by determining HPG axis plasma hormone profiles and mRNA levels for key genes, both outside and inside of the PS (n = 3 per group). Data were analyzed with a 2-way ANOVA using the mixed models procedure of SAS. In the HPG axis, plasma progesterone levels were not affected by egg production level but were elevated during the PS. In contrast, plasma estradiol levels were higher in HEPH than in LEPH but were not associated with the PS. LEPH exhibited decreased gene expression associated with ovulation stimulation and increased gene expression associated with ovulation inhibition in the hypothalamus and pituitary. In ovarian follicle cells, LEPH displayed decreased gene expression associated with progesterone, androgen, and estradiol production in the F1 follicle granulosa cells, F5 theca interna cells, and small white follicle cells, respectively. Different degrees of stimulation and inhibition within all tissues of the HPG axis were noted between LEPH and HEPH turkey hens, with HEPH showing higher expression of genes related to ovulation and steroidogenesis.

Characterization of gene expression in the hypothalamo-pituitary-gonadal axis during the preovulatory surge in the turkey hen.

A preovulatory surge (PS) of luteinizing hormone (LH) and progesterone triggers follicle ovulation, which is the first step of egg production and is orchestrated by the hypothalamo-pituitary-gonadal (HPG) axis. In the HPG axis, hypothalamic peptides, gonadotropin releasing hormone, and gonadotropin inhibitory hormone, control the production of follicle stimulating hormone and LH by the pituitary, which subsequently regulate ovarian production of estradiol and progesterone, respectively. The goal of this study was to characterize the HPG axis function of average egg producing hens by assessing plasma hormone profiles and hypothalamic, pituitary, and follicle gene expression outside and during the PS (n = 3 per group). Results were analyzed by a one-way ANOVA using the mixed models procedure of SAS. Plasma estradiol was not affected by the PS (P > 0.05), but plasma progesterone levels increased 8-fold during the PS when compared to basal progesterone levels (P < 0.05). HPG axis gene expression related to ovulation stimulation (e.g., GNRH, GNRHR, and LHB) was down-regulated during the PS; whereas gene expression related to follicle development (e.g., FSHB) was up-regulated during the PS. Additionally, in the hypothalamus and pituitary, estradiol receptor expression was up-regulated during the PS, whereas progesterone receptor expression was down-regulated during the PS. In the follicle cells, gene expression pertaining to progesterone (e.g., STAR), androgen (e.g., HSD17B1), and estradiol (e.g., CYP19A1) production was up-regulated during the PS. Prior to this study, the HPG axis had yet to be characterized during the PS in the turkey hen. This study showed that the PS significantly impacted gene expression in the hypothalamus, pituitary, and ovarian follicles. These results provide a foundation for further research into the regulation of ovulation and egg production in turkey hens.

Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis.

BACKGROUND: Previously we reported that mice deficient in toll-like receptor 4 (TLR-4) signalling were protected from diet-induced non-alcoholic steatohepatitis (NASH). Another member of the toll-like receptor family, TLR-2, has been shown to play a role in lipid trafficking via uptake of diacylated lipoproteins. However, a role for TLR-2 in NASH has not been elucidated. The objectives of the current study were to examine the influence of dietary fat quality and TLR-2 on NASH pathogenesis. METHODS: Steatohepatitis was induced in male Db, C57BL/6 and TLR-2(-/-) mice by feeding an L-amino acid-defined diet that was deficient in methionine and choline (MCDD). Mice fed the base diet supplemented with methionine and choline (control diet; CD) were used as controls. To determine the role of fat quality, MCDD was enriched with polyunsaturated corn oil (PUFA) or coconut oil that is comprised mostly of saturated fat (SAFA); the total amount of each fat was 112.9 g/kg of diet. After 8 weeks of feeding CD or MCDD, hepatic steatosis, inflammation and necrosis were evaluated in histological sections. Total RNA was extracted from frozen liver samples and mRNA expression of TNFalpha, collagen alpha1, IL-10, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), TLR-4, and CD14, was analyzed via real-time PCR. Protein levels of TLR-2 were analyzed by western blot. RESULTS: Panlobular macrovessicular steatosis and diffuse leukocyte infiltration were noted in PUFA-fed Db mice. Histological scores demonstrated significantly less steatosis, inflammation and necrosis in SAFA-fed mice of all mouse strains. However, compared to wild type mice, hepatocellular damage was notably more severe in TLR-2(-/-) mice. Consistent with histological findings, mRNA expression of TNFalpha was elevated by approximately 3-fold in TLR-2(-/-) mice; PPAR-gamma expression was blunted in this strain compared to wild type. Expression of the matrix protein collagen alphaI was also significantly higher in TLR-2(-/-) mice, indicating a pro-fibrogenic state. Sensitivity to steatohepatitis due to dietary fat or TLR-2 deficiency correlated significantly with alterations in the expression of TLR-4 as well as the co-receptor CD-14. CONCLUSIONS: Our findings suggest that dietary saturated fat plays a protective role against MCDD-induced steatohepatitis, whereas TLR-2 deficiency exacerbated NASH. The mechanism underlying the response to dietary fat and TLR-2 likely involves altered signalling via the TLR-4 pathway.

Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

Is it wraparound yet? Setting quality standards for implementation of the wraparound process.

The wraparound process has increasingly been used as a mechanism to plan and coordinate services for children with behavioral health needs and their families. This has led to growing interest in assessing wraparound implementation against standards for quality. However, there has been little consideration of how best to establish such benchmarks or guidelines. Using both a norm-referenced and criterion-referenced approach, this study established preliminary criteria for assessing the adequacy of wraparound implementation using the Wraparound Fidelity Index, version 3, a multi-informant interview that assesses conformance to wraparound principles. The evaluation system was then applied to ten wraparound programs and 11 different study samples assessed via the Wraparound Fidelity Index version 3 in research studies. The system was constructed to discriminate different wraparound conditions assessed in research studies while still being attainable by the ten established wraparound programs. Implications for evaluating wraparound programs and for setting fidelity benchmarks in behavioral health services research are discussed.

Relations between program and system variables and fidelity to the wraparound process for children and families.

OBJECTIVE: Past research and experience have indicated that characteristics of organizations and service systems influence the quality of mental health services delivery. The study aimed to illuminate such associations by examining the relationship between a set of program- and system-level variables and fidelity to the wraparound model for children and families. METHODS: A measure of fidelity to the wraparound process for children and families (the Wraparound Fidelity Index) was administered to families receiving services in eight mental health agencies across the United States. Program administrators at each agency also completed by telephone a standardized interview for assessing program and system conditions hypothesized to influence quality of implementation (Program Administrator version of the Wraparound Fidelity Index). Regression analyses were used to assess the relationship between program and system variables and fidelity. RESULTS: Results of interviews with program administrators revealed substantial variation across programs in the number and type of program and system supports for wraparound implementation. Regression analyses found a significant association between the number of organizational and system supports and Wraparound Fidelity Index scores. CONCLUSIONS: Results support the hypothesized relationship between program and system conditions and the quality of service delivery. Results also highlight the importance of engineering the system and organizational context (maintaining low caseload sizes, establishing interagency partnerships, and implementing accountability mechanisms) in order to provide a solid foundation for high-quality wraparound to children and families.

Fluctuations of functionally distinct CD8+ T-cell clonotypes demonstrate flexibility of the HIV-specific TCR repertoire.

T-cell receptor (TCR) diversity of virus-specific CD8+ T cells likely helps prevent escape mutations in chronic viral infections. To understand the dynamics of the virus-specific T cells in more detail, we followed the evolution of the TCR repertoire specific for a dominant HLA-B*08-restricted epitope in Nef (FLKEKGGL) in a cohort of subjects infected with HIV. Epitope-specific CD8+ T cells used structurally diverse TCR repertoires, with different TCRbeta variable regions and with high amino acid diversity within antigen recognition sites. In a longitudinal study, distinct Vbeta populations within the HIV-specific TCR repertoire expanded simultaneously with changes in plasma viremia, whereas other Vbeta populations remained stable or even decreased. Despite antigenic variation in some subjects, all subjects had the consensus sequence present during the study period. Functional analysis of distinct Vbeta populations revealed differences in HIV-specific IFN-gamma secretion ex vivo as well as differences in tetramer binding, indicating functional heterogeneity among these populations. This contrasts with findings in a subject on antiretroviral therapy with suppression of viremia to less than 50 copies/mL, where we observed long-term persistence of a single clonotype. Our findings illustrate the flexibility of a heterogeneous HIV-1-specific CD8+ TCR repertoire in subjects with partial control of viremia.

Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape.

Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.

Analysis of the TCR beta variable gene repertoire in chimpanzees: identification of functional homologs to human pseudogenes.

Chimpanzees are used for a variety of disease models such as hepatitis C virus (HCV) infection, where Ag-specific T cells are thought to be critical for resolution of infection. The variable segments of the TCR alphabeta genes are polymorphic and contain putative binding sites for MHC class I and II molecules. In this study, we performed a comprehensive analysis of genes that comprise the TCR beta variable gene (TCRBV) repertoire of the common chimpanzee Pan troglodytes. We identified 42 P. troglodytes TCRBV sequences representative of 25 known human TCRBV families. BV5, BV6, and BV7 are multigene TCRBV families in humans and homologs of most family members were found in the chimpanzee TCRBV repertoire. Some of the chimpanzee TCRBV sequences were identical with their human counterparts at the amino acid level. Notably four successfully rearranged TCRBV sequences in the chimpanzees corresponded to human pseudogenes. One of these TCR sequences was used by a cell line directed against a viral CTL epitope in an HCV-infected animal indicating the functionality of this V region in the context of immune defense against pathogens. These data indicate that some TCRBV genes maintained in the chimpanzee have been lost in humans within a brief evolutionary time frame despite remarkable conservation of the chimpanzee and human TCRBV repertoires. Our results predict that the diversity of TCR clonotypes responding to pathogens like HCV will be very similar in both species and will facilitate a molecular dissection of the immune response in chimpanzee models of human diseases.