Transcripts In Peripheral Blood Mononuclear Cells Research Articles

Development of TaqMan probe-based RT-qPCR assays for detection of BoHV-1 latency in Bovine

Bovine herpesvirus-1 is highly contagious virus of cattle and buffaloes all over the world. It establishes lifelong latency in ganglionic neurons of the peripheral nervous system. Since, trigeminal ganglia are the main sites of latency, therefore, it is challenging to detect BoHV-1 in latently infected live animals. No research work has been done to correlate the sero-prevalence and latency in peripheral blood mononuclear cells (PBMC). The present study was designed to detect BoHV-1 latency related transcript or microRNA in peripheral blood mononuclear cells of sero-positive animals. The highly sensitive RT-qPCR assays based on TaqMan chemistry have been developed for the detection of transcripts of BoHV-1 latency. The limit of detection (LOD) of the assays for ORF-1 specific RT-qPCR and miRNA specific RT-qPCR was 460 copies and 117 copies respectively. The efficiency of the developed assays was 93.5% for ORF-1 and 97.93% for miRNA transcript. None of the PBMC samples of seropositive animals found positive for ORF-1 and miRNA transcripts in developed assays.The absence of latency specific transcripts in PBMC might be due to very low expression i.e. beyond the LOD of newly developed assays or absence of latency in PBMC of seropositive animal. However, further studies are required to establish the fact. To the best of authors knowledge, this is the first report of latency-specific RT-qPCR assay development and its application in PBMC of BoHV-1 seropositive cattle.

Innate immune response to double-stranded RNA in American heritage chicken breeds

Backyard poultry flocks that employ heritage breeds of chicken play a crucial role in the maintenance of poultry pathogens of economic and zoonotic importance. This study examined innate immunity to viral pathogens in heritage chicken breeds using a model of viral double-stranded RNA (dsRNA). Following intraperitoneal injection of high molecular weight (HMW) -poly(I:C)/Lyovec into 4-wk-old chicks, we evaluated gene expression in peripheral blood mononuclear cells (PBMCs) and splenocytes. There was a significant difference across breeds in the expression of IL-4, IL-12p40, IFNγ, and B-cell activating factor (BAFF) in the spleen. In PBMCs, a significant difference in IFN-α expression was seen across breeds. Approximately 57% of IFN-α transcripts in PBMCs was explained by levels of expression of MDA5 transcripts. Using flow cytometry, we showed that only monocytes/macrophages (KUL01+ cells) expressed the scavenger receptor CD163. Regression analysis showed that 42% of fold change in CD163 expression on PBMCs was explained by breed (P < 0.0004). In general, breeds that responded to HMW-poly(I:C) by showing higher upregulation of IFNγ, IL-1β, and IL-12p40 transcripts in the spleen, and higher IFNα transcripts in peripheral blood, expressed less CD163 on blood monocytes. These findings suggest a genetic basis for the response of chickens to double-stranded RNA. Surface expression of the scavenger receptor CD163 in PBMCs following injection of high molecular weight poly(I:C) may be a rapid method to select chickens for breeding based on innate immune response to viral dsRNA.

Differential expression and regulation of HSP70 gene during growth phase in ruminants in response to heat stress

Heat shock proteins regulate the physiological mechanism of heat stress adaptation at cellular level. The present investigation was carried out to analyse the HSP70 gene regulation in various growth stage in ruminants in peripheral blood mononuclear cells (PBMCs). The relationship between HSP gene expression and thermotolerance in age-specific manner in ruminants has not been analysed. Therefore m-RNA HSP70 expression level was examined in different age groups of Jamunpari goat during hot climatic conditions. The experiment was carried out in 32 animals of Jamunapari goat belonging to the age groups of 3-months, 9-months, 12-months, and adults (2–3 year). Total RNA was isolated from peripheral blood mononuclear cells. The physiological response such as rectal temperature (RT), respiration rate (RR) and heart rate (HR) was used as indicator to heat stress. Temperature Humidity Index (THI) was used as an indicator of severity of environmental stress. The THI range varied from 82.00–92.08 during experimental period. The m-RNA HSP70 expression level at 9-month age of animals was up-regulated and significantly higher than other age groups. It was observed that the level of HSP70 transcripts in PBMCs was highest at 9-month age group, and age-related decline in HSP70 expression was observed in adult age. Based on the physiological response, the contrasting heat-stress phenotypes were recognised as heat stress susceptible (HSS) and heat stress tolerant (HST) individuals and the expression of m-RNA HSP70 was analysed at different ages in response to chronic heat stress. The differential mRNA expression of HSS individuals at 3 and 9-month of age showed the highest fold expression than HST. Age and phenotype had significant effect (p < 0.01) on the crossing point (CP) value. The m-RNA HSP70 gene expression in different age groups was correlated with heat stress tolerance and this could be used as biomarker for breeders to analyse the HSP response in -vivo in ruminants.

Isolation and characterization of uterine leukocytes collected using a uterine swab technique.

Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses. Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (TReg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16- CD56++ and CD16- CD56++ NK transcripts in PBMC and UBMC samples. Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45+ leukocytes. Higher percentages of CD16- CD56++ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45+ CD3- vs. 2.73%, respectively), suggesting that CD16- CD56++ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16+ CD56++ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface. Intrauterine leukocytes, especially CD16- CD56++ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16- CD56++ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.

Mechanistic differences underlying HIV latency in the gut and blood contribute to differential responses to latency-reversing agents.

While latently HIV-infected cells have been described in the blood, it is unclear whether a similar inducible reservoir exists in the gut, where most HIV-infected cells reside. Tissue-specific environments may contribute to differences in the mechanisms that govern latent HIV infection and amenability to reactivation. We sought to determine whether HIV-infected cells from the blood and gut differ in their responses to T-cell activation and mechanistically distinct latency reversing agents (LRAs). Cross sectional study using samples from HIV-infected individuals (n = 11). Matched peripheral blood mononuclear cells (PBMC) and dissociated total cells from rectum ± ileum were treated ex vivo for 24 h with anti-CD3/CD28 or LRAs in the presence of antiretrovirals. HIV DNA and 'read-through', initiated, 5' elongated, completed, and multiply-spliced HIV transcripts were quantified using droplet digital PCR. T-cell activation increased levels of all HIV transcripts in PBMC and gut cells, and was the only treatment that increased multiply-spliced HIV RNA. Disulfiram increased initiated HIV transcripts in PBMC but not gut cells, while ingenol mebutate increased HIV transcription more in gut cells. Romidepsin increased HIV transcription in PBMC and gut cells, but the increase in transcription initiation was greater in PBMC. The gut harbors HIV-infected cells in a latent-like state that can be reversed by T-cell activation involving CD3/CD28 signaling. Histone deacetylation and protein kinase B may contribute less to HIV transcriptional initiation in the gut, whereas protein kinase C may contribute more. New LRAs or combinations are needed to induce multiply-spliced HIV and should be tested on both blood and gut.

Increased AHR Transcripts Correlate With Pro-inflammatory T-Helper Lymphocytes Polarization in Both Metabolically Healthy Obesity and Type 2 Diabetic Patients

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose transcription activity is regulated by small compounds provided by diet, xenobiotics, and metabolism. It has been proven to be involved in energy homeostasis and inflammation in most recent years. Epidemiologically, exposure to xenobiotic AHR ligands contributes to obesity and type 2 diabetes (T2D). AHR is also the critical transcription factor determining the lineage commitment of pro-inflammatory Th17 and Th22 cells from naïve CD4+ T lymphocytes. It has been well-illustrated in animal models that IL-22, the major effector cytokine of Th17 and Th22 cells, played a major role in the interaction of metabolism and gut microbiota. But there were still missing links between gut microbiota, IL-22, and metabolism in humans. Our previous findings indicated that elevated circulating levels of IL-22 and frequencies of Th22 cells were associated with insulin resistance in both patients with obesity and T2D. Additionally, the hyperactive Th17 and Th22 cells phenotype also correlate with islets β-cell dysfunction in T2D. In this study, we made efforts to determine AHR expressions in peripheral blood mononuclear cells (PBMCs) from patients with T2D and metabolically healthy obesity (MHO). Correlation analyses were conducted to assess the possible link between AHR and the metabolic and inflammatory context. We revealed that mRNA expression of AHR was up-regulated and correlated with the percentage of Th17, Th22 as well as Th1 cells. Elevated plasma levels of IL-22 and IL-17 also correlated with increased AHR transcripts in PBMCs from both MHO and T2D patients. The transcription factor AHR may thus have a plausible role in the interaction between metabolism and pro-inflammatory status of patients in the development of obesity and T2D.

Recipient interleukin 6 gene polymorphism and expression predict HCV recurrence post liver transplantation.

Liver transplantation (LTX)is a lifesaving- effective protocol for patients suffering end stage liver disease (ESLD) and its complications post HCV infection. Recurrence of disease is a frequent clinical complication that is observed in patients undergoing LTX. Cytokines play a central role in the immunological events occurring after the surgery. Using Allelic Discrimination PCR, the allelic variation G174C of IL-6 gene was investigated. The abundance of IL6- mRNA and plasma IL6 cytokine levels were evaluated by using qRT-PCR in peripheral blood mononuclear cells (PBMCs) and enzyme-linked immunosorbent assay (ELISA) respectively in 76 liver transplant recipients recruited from Al Sahel teaching hospital, Ministry of Health and Population Cairo Egypt within the period between June 2015 and October 2017. The frequencies of IL-6 GG genotype and the G allele were significantly detected more in LTX recipients who experienced HCV recurrence versus those who did not suffer recurrence when compared to healthy controls (P=0.001) and (P=0.006), respectively. On the contrary, levels of IL-6 related transcripts in PBMC's of recurrent patients were indifferent from non-recurrent patients and healthy controls (P≥0.124). Interestingly, the circulating IL-6 protein in plasma was significantly elevated in recurrent as compared to the non-recurrent recipients (P=0.002). HCV recurrence post liver transplantation occur more frequently in patients with -174 G/G IL-6 genotype and elevated plasma IL-6 levels.

Whole blood vs PBMC: compartmental differences in gene expression profiling exemplified in asthma

BackgroundBlood has proven to be a useful resource for molecular analysis in numerous biomedical studies, with peripheral blood mononuclear cells (PBMCs) and whole blood being the major specimen types. However, comparative analyses between these two major compartments (PBMCs and whole blood) are few and far between. In this study, we compared gene expression profiles of PBMCs and whole blood samples obtained from research subjects with or without mild allergic asthma.MethodsWhole blood (PAXgene) and PBMC samples were obtained from 5 mild allergic asthmatics and 5 healthy controls. RNA from both sample types was measured for expression of 730 immune-related genes using the NanoString nCounter platform.ResultsWe identified 64 uniquely expressed transcripts in whole blood that reflected a variety of innate, humoral, and adaptive immune processes, and 13 uniquely expressed transcripts in PBMCs which were representative of T-cell and monocyte-mediated processes. Furthermore, analysis of mild allergic asthmatics versus non-asthmatics revealed 47 differentially expressed transcripts in whole blood compared to 1 differentially expressed transcript in PBMCs (FDR < 0.25). Finally, through simultaneous measurement of PBMC proteins on the nCounter assay, we identified CD28 and OX40 (TNFRSF4), both of which are critical co-stimulatory molecules during T-cell activation, as significantly upregulated in asthmatics.ConclusionsWhole blood RNA preserved in PAXgene tubes is excellent for producing gene expression data with minimal variability and good sensitivity, suggesting its utility in multi-centre studies requiring measurement of blood gene expression.

HIV elite control is associated with reduced TRAILshort expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) dependent apoptosis has been implicated in CD4 T-cell death and immunologic control of HIV-1 infection. We have described a splice variant called TRAILshort, which is a dominant negative ligand that antagonizes TRAIL-induced cell death in the context of HIV-1 infection. HIV-1 elite controllers naturally control viral replication for largely unknown reasons. Since enhanced death of infected cells might be responsible, as might occur in situations of low (or inhibited) TRAILshort, we tested whether there was an association between elite controller status and reduced levels of TRAILshort expression. Cohort study comparing TRAILshort and full length TRAIL expression between HIV-1 elite controllers and viremic progressors from two independent populations. TRAILshort and TRAIL gene expression in peripheral blood mononuclear cells (PBMCs) was determined by RNA-seq. TRAILshort and TRAIL protein expression in plasma was determined by antibody bead array and proximity extension assay respectively. HIV-1 elite controllers expressed less TRAILshort transcripts in PBMCs (P = 0.002) and less TRAILshort protein in plasma (P < 0.001) than viremic progressors. Reduced TRAILshort expression in PBMCs and plasma is associated with HIV-1 elite controller status.

Genetic and epigenetic regulation of major histocompatibility complex class I gene expression in bovine trophoblast cells.

ProblemThe regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC‐Ia) and non‐classical MHC class I (MHC‐Ib) genes are poorly understood.Method of studyQuantitative reverse transcription‐ polymerase chain reaction (PCR) was used to compare the abundance of MHC‐I transcripts and related transcription factors in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Methylation of MHC‐I CpG islands was detected by bisulfite treatment and next‐generation sequencing. Demethylation of PBMC and PTC with 5′‐aza‐deoxycytidine was used to assess the role of methylation in gene regulation.Results MHC‐I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator (CIITA), and STAT1. The MHC‐Ia genes and BoLA‐NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of BoLA‐NC2, ‐NC3, and ‐NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC‐Ib by 2.8‐ to 6‐fold, whereas MHC‐Ia transcripts were elevated less than 2‐fold.Conclusion DNA methylation regulates bovine MHC‐Ib expression and is likely responsible for the different relative levels of MHC‐Ib to MHC‐Ia transcripts in PBMC and PTC.

Transcriptomes of whole blood and PBMC in chickens

Global transcriptome analysis of chicken whole blood to discover biomarkers of different phenotypes or physiological disorders has never been investigated so far. Whole blood provides significant advantages, allowing large scale and non-invasive sampling. However, generation of gene expression data from the blood of non-mammalian species remains a challenge, notably due to the nucleated red blood cells, hindering the use of well-established protocols. The aim of this study was to analyze the relevance of using whole blood cells (WB) to find biomarkers, instead of Peripheral Blood Mononuclear Cells (PBMC), usually chosen for immune challenges. RNA sources from WB and PBMC was characterized by microarray analysis. Our results show that the quality and quantity of RNA obtained from WB was suitable for further analyses, although the quality was lower than that from PBMC. The transcriptome profiling comparison revealed that the majority of genes were expressed in both WB and PBMC. Hemoglobin subunits were the major transcripts in WB, whereas the most enriched biological process was related to protein catabolic process. Most of the over-represented transcripts in PBMC were implicated in functions specific to thrombocytes, like coagulation and platelet activation, probably due to the large proportion of this nucleated cell type in chicken PBMC. Functions related to B and T cells and to other immune functions were also enriched in the PBMC subset. We conclude that WB is more suitable for large scale immunity oriented studies and other biological processes that have been poorly investigated so far.

Differential expression of interferon alpha inducible genes in peripheral blood mononuclear cells from patients chronically infected with hepatitis C virus and healthy donors.

The impact of exposure to interferon-alpha (IFN-α) on gene expression in peripheral blood mononuclear cells (PBMC) from hepatitis C virus (HCV)-infected and healthy individuals was investigated to recognize whether their PBMC differ in expression of IFN-inducible genes (ISGs) following treatment with IFN-α2b. PBMC obtained from healthy and treatment-naïve HCV-infected patients were cultured with IFN-α2b for 30min, 2h, 4h and 72h, and gene expression was analyzed using mRNA microarray technology. IFN-α caused differential up-regulation of many known ISGs in PBMC from both HCV-infected and healthy subjects. In comparison to untreated controls, the highest augmentation in PBMC ISG expression occurred after 4-hour exposure to IFN-α2b in both groups. The analysis identified 84 transcripts, representing 64 known and 2 unknown genes, that were up-regulated by at least 5-fold in PBMC from infected and uninfected individuals. However, the expression of IFN-α inducible genes was impaired in the PBMC from HCV-infected individuals compared to healthy controls. This was due to an increased baseline expression of the transcripts in PBMC of HCV-infected patients. These findings expand our understanding of IFN-responses in HCV-infected individuals and suggest that functions of PBMC, which include immune effector cells, are altered in patients chronically infected with HCV.

Increased interleukin-17 transcripts in peripheral blood mononuclear cells, a link between T-helper 17 and proinflammatory responses in bladder cancer.

Background:Interleukin (IL)-17-producing CD4+ T helper (Th17) cells thatare known by producing IL-17 have recently been defined as a unique subset of proinflammatory helper cells. IL-17 is an inflammatory cytokine with robust effect on many cells and it can play important roles in pathogenesis of diverse groups of immune-mediated diseases.Objectives:The aim of this case-control study was to determine the gene expression of IL-6, IL-17, and transforming growth factor beta (TGF-β) in Iranian patients with bladder cancer.Patients and Methods:Blood samples were collected from 37 patients with bladder cancer and 37 healthy individuals with no history of malignancies or autoimmune disorders, based of simple sampling. The expression of IL-6, IL-17, and TGF-β were measured by quantitative real-time polymerase chain reaction (qRT-PCR).Results:The mean of IL-17 transcripts was significantly higher in patients with bladder cancer compared with healthy individuals (0.33 ± 0.06 vs. 0.42 ± 0.14, ) (P = 0.04), but their TGF-β was lower (12.53 ± 8.41 vs. 54.94 ± 17.95, ) (P = 0.04). However, the IL-6 transcripts level was similar in both groups (5.34 ± 2.40 vs. 8.07 ± 3.28, ) (P > 0.05) and there was not any significant difference between the noted cytokines expressions among patients with different stages and grades.Conclusions:As most of the cases studied in this investigation were in stages I and II, IL-17 as a prominent proinflammatory cytokine may play an important role in recruiting and infiltrating of antitumor immune responses in early stages of bladder cancer. Furthermore, it can be used as predictor for the clinical stage and prognosis of cancers such as bladder carcinoma.

Levels of inflammatory cytokines, adrenal steroids, and mRNA for GRα, GRβ and 11βHSD1 in TB pleurisy

Our previous work on the immune-endocrine features of patients with pulmonary tuberculosis (TB) showed markedly decreased plasma levels of dehydroepiandrosterone (DHEA) together with augmented concentrations of Cortisol and pro- and anti-inflammatory cytokines. Studies in peripheral blood mononuclear cells (PBMC) indicated a lower mRNA α/β ratio of glucocorticoid receptors -GR- together with a higher 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) mRNA expression in cases with severe pulmonary TB. Since Pleural TB (PLTB) is a rather benign manifestation of TB, we now analyzed the systemic and local immune-endocrine profile as well as the GRα, GRβ, 11βHSD1 and 11βHSD2 transcripts in PBMC and pleural effusion mononuclear cells (PEMC) of patients with PLTB. PLTB patients had increased levels of IL-1β, IL-6 and IFNγ together with reduced Cortisol and DHEA concentrations in pleural fluids. Also, a significantly increased expression of 11βHSD1 and GRα was found in PEMC compared to PBMC. Findings point out to an appropriate immune response and a substantial inflammatory reaction, wherein the low Cortisol concentrations may be equally effective, because of the increased expression of GRα and 11βHSD1 transcripts which may optimize the immunomodulatory properties of Cortisol.

Biologically relevant doses of mixed aflatoxins B and G up-regulate MyD88, TLR2, TLR4 and CD14 transcripts in human PBMCs

Context: Aflatoxins (AFs) are highly hazardous carcinogenic mycotoxins originated from very common fungi present in the environment. Their effect on key immune-surveillance molecules is unclear.Objective: We aimed to examine the effect of mixed AFs on immunologically relevant molecules and on viability in human peripheral blood mononuclear cells (PBMCs), in conditions similar to those occurring naturally, i.e. using a mixture of environmentally relevant levels of AFB1, AFB2, AFG1 and AFG2.Materials and methods: We evaluated the mRNA expression of MyD88, toll-like receptor (TLR)-2, TLR4 and CD14, in human PBMCs treated with a mixture of AFB1, AFB2, AFG1 and AFG2 at different doses for 2, 12 and 24 h. We used qRT–PCR to assess changes in transcripts of MyD88, TLR2, TLR4 and CD14 in PBMCs. We also evaluated the viability of PBMCs exposed to AFs.Results: Biologically relevant levels of mixed AFs elicited early immune modulation in human PBMCs. qRT–PCR results showed several folds increase of MyD88, TLR2, TLR4 and CD14 transcripts in PBMCs as early as 2 h post-exposure to mixed AFs. Kinetics and dose–response of the up-regulation differed for mentioned gene transcripts. Further, prolonged exposure to mixed AFs decreased PBMCs viability.Conclusion: Immunotoxicity of AFs on PBMCs may be mediated by up-regulation of key immune-surveillance molecule transcripts. The description of these effects induced by AFs on PBMCs are novel and should be taken into account when considering AF-related infectious and noninfectious diseases in areas highly exposed to AFs.

AB0066 Expression of NLRP3-inflammasome-related transcripts in peripheral blood of rheumatoid arthritis patients treated with infliximab

BackgroundThe assembly of a molecular scaffold termed the NLRP3-inflammasome complex, in monocytes, macrophages and dendritic cells, leads to the cleavage of pro-interleukin-1β(pro-IL-1β) into active IL-1β, a proinflammatory cytokine involved in...

High Levels of FCγR3A and PRF1 Expression in Peripheral Blood Mononuclear Cells from Patients with Primary Biliary Cirrhosis

Innate immunity plays an important role in the pathogenesis of primary biliary cirrhosis (PBC) that needs to be characterized. Levels and clinical relationship of Fc gamma receptor III-A (FcγR3A), tyrosine kinase binding protein (TYROBP) and perforin-1 (PRF1), important genes for nature killer (NK) cells, were analyzed in PBC patients. The purpose of this study was to explore the expression levels of the above-mentioned genes in peripheral blood mononuclear cells (PBMCs) from PBC patients. A total of 102 PBC patients and 85 healthy controls (HC) were recruited. The relative levels of FCγR3A, TYROBP, and PRF1 mRNA transcripts in PBMCs were determined by RT-PCR. The percentages of peripheral blood NK, natural killer T (NKT), FCγR3A(+) or PRF1(+) NK cells and PRF1(+) NKT cells in PBC patients and HC were also characterized by flow cytometry analysis. The potential associations of the percentages of NK and NKT cells with clinical indexes were analyzed. The relative levels of FCγR3A, TYROBP, and PRF1 mRNA transcripts and the percentages of PRF1(+) NK and NKT cells in PBC patients were significantly higher than that in HC. Moreover, the percentages of PRF1-expressing NK and NKT cells in PBC patients were negatively associated with the levels of serum gamma-glutamyltransferase (GGT) and Mayo risk scores, and the relative levels of FCγR3A expression in NK cells of PBC patients were positively associated with the levels of serum GGT. FCγR3A and PRF1 may participate in the pathogenesis and progression of PBC.

Profiling the expression of interleukin (IL)‐28 and IL‐28 receptor α in systemic lupus erythematosus patients

Interleukin (IL)-28 is an interferon-λ-family member involved in immunity against viral infection and tumour. We here determined the expression profiles of IL-28 and IL-28 receptor α (IL-28RA) in patients with systemic lupus erythematosus (SLE) to evaluate the possibility that IL-28 is linked to the pathogenesis of SLE. The serum IL-28 protein levels were determined by ELISA, and the IL-28 and IL-28RA transcript levels in peripheral blood mononuclear cells (PBMCs) and peripheral blood T cells were determined by RT-PCR. The levels in patients with SLE with the active disease activity were statistically compared with those in normal controls. IL-28 protein in sera and IL-28 transcripts in PBMCs and unactivated T cells were detectable only in some individuals, and IL-28 transcripts in T cells were induced by cell activation with anti-CD2, anti-CD3 and anti-CD28 antibodies. However, compared with normal controls, patients with SLE more frequently had detectable IL-28 protein in serum and had the higher IL-28 transcript levels in activated CD4(+) T cells, but not activated CD8(+) T cells. Two IL-28RA transcripts isoforms were detected in PBMCs and T cells, and their levels in patients with SLE were comparable with those in normal controls. The expression of IL-28, a T-cell autocrine factor, is dysregulated in patients with SLE, supporting the possibility that IL-28 may contribute to some of the SLE pathogenesis.

Administration of peripheral blood mononuclear cells into the uterine horn to improve pregnancy rate following bovine embryo transfer

Embryo transfer (ET) has been used to improve reproductive efficiency and genetic make-up in bovine species. However, the success rate of ET has not been improved since its inception. Here we examined whether administration of autologous peripheral blood mononuclear cells (PBMCs) into the uterine horn can improve pregnancy rates following bovine ET. First we determined that the abundance of interleukin (IL)-1α, IL-1β and IL-8 transcripts in PBMCs was greatest after 24 h of culture. PBMCs that had been cultured for 24 h were gently administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle. On day 7, the ET was carried out and the pregnancy rate in the PBMC-treated group was compared with that in the non-treated group. The pregnancy rate on day 60 in the PBMC-treated group (76.7%, 56/73) was significantly higher than that in the non-treated group (59.7%, 43/72, p < 0.05). These results indicate that administration of autologous PBMCs into the uterine horn improves pregnancy rates following bovine ET.

Absence of Tyrosinase‐Related Protein‐2/Dopachrome Tautomerase Transcripts in PBMCs from Vitiligo Patients

Tolerance is achieved by mechanisms occurring in both the thymus and periphery. Several reports have shown that presence of an antigen in the peripheral circulation results in tolerance induction. These reports imply that absence of a self-antigen can lead to induction of autoimmunity. Here, we show that tyrosinase-related protein 2 (TRP-2) transcript is not detected in the peripheral blood mononuclear cells (PBMC) of vitiligo patients but is detected in healthy controls. Our result indicates that probably due to lack of expression in the PBMC, TRP-2 is not available for induction and maintenance of peripheral tolerance in vitiligo patients. It is also reported by others that co-stimulatory molecules are required for the initiation of autoimmune diseases in experimental models. We therefore analysed the transcript levels of these costimulatory molecules in vitiligo patients and healthy controls. We observed that the transcripts of B7.2 and CD40 molecules are more or less similar in both patients and controls. We could not detect B7.1 in any of the human subjects. Thus, we conclude that the antigen presenting cells (APC) are not in an activated state and that constitutively activated APC are possibly not required for the progression of the disease once it has been initiated.