Circulating tumor DNA methylation marker MYO1-G for diagnosis and monitoring of colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising diagnostic and prognostic marker for many cancers and has been actively investigated in recent years. Previous studies have already demonstrated the potential use of ctDNA methylation markers in the diagnosis and prognostication of colorectal cancer (CRC). This retrospective study validated the value of methylation biomarker MYO1-G (cg10673833) in CRC diagnosis and disease monitoring using digital droplet PCR (ddPCR), a biomarker selected from our previous study due to its highest diagnostic efficiency. METHODS: Blood samples of CRC and control samples from tumor-free individuals at two institutions were collected to quantify the methylation ratio using ddPCR. Area under curve (AUC) was calculated after constructing receiver operating characteristic curve (ROC) for CRC diagnosis. Sensitivity and specificity were estimated and comparisons of methylation ratio in different groups were performed. RESULTS: We collected 673 blood samples from 272 patients diagnosed with stage I-IV CRC and 402 normal control samples. The methylation biomarker discriminated patients with CRC from normal controls with high accuracy (area under curve [AUC] = 0.94) and yielded a sensitivity of 84.3% and specificity of 94.5%. Besides, methylation ratio of MYO1-G was associated with tumor burden and treatment response. The methylation ratio was significantly lower in patients after their radical operation than when compared with those before surgeries (P < 0.001). Methylation ratio was significantly higher in patients with disease progression than those with stable disease (P = 0.002) and those with complete response or partial response (P = 0.009). CONCLUSIONS: Together, our study indicated that this methylation marker can serve as a potential biomarker for diagnosing and monitoring CRC.

The effects of folate and iron deficiency followed by supplementation on blood morphology and inflammation biomarkers in rats.

BACKGROUND: Little is known about the relation between iron and folic acid (FA) supplementation and inflammation. The aim of this study was to evaluate the effects of iron and folate deficiency and supplementation on blood morphology parameters, and to assess the role of iron and folate transporters in inflammation. METHODS: A four-week period of FA and iron deficiency in Wistar rats was followed by randomization into a group fed with a diet deficient in FA and supplemented with Fe (DFE), a group fed a diet deficient in Fe and supplemented with FA (DFOL), a group fed a diet supplemented with Fe and FA (FEFOL), a group fed a diet deficient in Fe and FA (D), and a group fed a control diet (C). The blood Crp concentration and blood count were determined. The expression of SLC11A2, SLC46A1, SLC19A1, and TFR2 proteins was assessed using the western blot method. RESULTS: After ten days on the experimental diets, the rats in the DFOL group had a 21% higher concentration of white blood cells (WBC) than the FEFOL group did (p < 0.05). We did not observe any differences between the groups in terms of C-reactive protein (Crp) concentration. We also did not find any other differences between the groups in other morphological parameters. Analysis of the correlation between blood count parameters and the expression of iron and folate transporters gave conflicting results. CONCLUSIONS: To conclude, iron and folate supplementation may affect WBC concentration in the blood.

Serum amino acid concentrations are modified by age, insulin resistance, and BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms in subjects with obesity.

BACKGROUND & AIMS: The amino acid profile of young adults is modified by sex, body mass index (BMI) and insulin resistance (IR). However, we do not know if age or the presence of specific polymorphisms in the genes of BCAT2 and BCKDH contribute to changes in the amino acid profile, especially in subjects with obesity. Therefore, we have evaluated the effect of age, the presence of IR and the polymorphisms of BCAT2 rs11548193 and BCKDH rs45500792 on the concentration of amino acids in subjects with obesity. METHODS: This was a cross-sectional study conducted with 487 subjects with obesity. Participants underwent a physical examination in which their clinical history was obtained and a blood sample was taken for biochemical, hormonal, and DNA analysis. RESULTS: Adults <30 years old with obesity had higher levels of alanine, arginine, aspartate, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and valine than adults ≥30 years old. Interestingly, regardless of age, we found that arginine, aspartate, serine decreased, while proline and tyrosine increased in the presence of IR; tyrosine and sum of branched-chain amino acids (∑BCAA) were the amino acids that increased in the presence of BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms. CONCLUSIONS: We found that the amino acid profiles of subjects with obesity are differentially modified by age, the presence of IR, and the presence of the BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms.

Systemic transcriptome comparison between early- And late-onset pre-eclampsia shows distinct pathology and novel biomarkers.

OBJECTIVES: Pre-eclampsia is a leading cause of morbidity and mortality during pregnancy. Although the two forms of this disorder, early- (EOPE) and late-onset of pre-eclampsia (LOPE) are different, the underlying pathology remains elusive. We aim to unravel the difference and to identify novel biomarkers for EOPE and LOPE. MATERIALS AND METHODS: A complete comparison of both placental and peripheral blood transcriptomes was performed to investigate the pathology of pre-eclampsia. Single-cell transcriptomics of the maternal-fetal interface were integrated to identify novel biomarkers for EOPE and LOPE which were further verified at protein or mRNA level in patients. RESULTS: We found that the transcriptomes of placentae from EOPE, but not LOPE, were significantly different from their respective controls. Conversely, the transcriptomes of peripheral blood from LOPE were more different from their controls than EOPE. Importantly, we identified that several classical biomarkers of pre-eclampsia were expressed specifically in extravillous trophoblast and syncytiotrophoblast and only upregulated in EOPE, suggesting they should not be applied to all pre-eclampsia patients in general. We further identified novel biomarkers for EOPE and LOPE from differentially expressed genes (DEGs) of placental and peripheral blood, respectively. The new biomarkers EBI3, IGF2, ORMDL3, GATA2 and KIR2DL4 were experimentally verified with patient blood samples. CONCLUSION: Our data demonstrate distinct pathology of EOPE and LOPE, and uncover new biomarkers that can be applied in diagnosis for pre-eclampsia.

Independent and Interactive Influences of Environmental UVR, Vitamin D Levels, and Folate Variant MTHFD1-rs2236225 on Homocysteine Levels.

Elevated homocysteine (Hcy) levels are a risk factor for vascular diseases. Recently, increases in ultraviolet radiation (UVR) have been linked to decreased Hcy levels. This relationship may be mediated by the status of UVR-responsive vitamins, vitamin D and folate, and/or genetic variants influencing their levels; however, this has yet to be examined. Therefore, the independent and interactive influences of environmental UVR, vitamin D and folate levels and related genetic variants on Hcy levels were examined in an elderly Australian cohort (n = 619). Red blood cell folate, 25-hydroxyvitamin D (25(OH)D), and plasma Hcy levels were determined, and genotyping for 21 folate and vitamin D-related variants was performed. Erythemal dose rate accumulated over six-weeks (6W-EDR) and four-months (4M-EDR) prior to clinics were calculated as a measure of environmental UVR. Multivariate analyses found interactions between 6W-EDR and 25(OH)D levels (pinteraction = 0.002), and 4M-EDR and MTHFD1-rs2236225 (pinteraction = 0.006) in predicting Hcy levels. The association between 6W-EDR and Hcy levels was found only in subjects within lower 25(OH)D quartiles (<33.26 ng/mL), with the association between 4M-EDR and Hcy occurring only in subjects carrying the MTHFD1-rs2236225 variant. 4M-EDR, 6W-EDR, and MTHFD1-rs2236225 were also independent predictors of Hcy. Findings highlight nutrient-environment and gene-environment interactions that could influence the risk of Hcy-related outcomes.

CpG-SNP site methylation regulates allele-specific expression of MTHFD1 gene in type 2 diabetes.

The interaction of genetic and epigenetic mechanisms is one of the underlying causes of phenotypic variability in complex diseases such as type 2 diabetes (T2D). To explore the influence of genetic and epigenetic changes in T2D, we examined the effect of methylation of CpG-SNP sites on allele-specific expression (ASE) in one-carbon metabolism pathway genes in T2D. Case-control study was conducted on 860 individuals (430 T2D and 430 controls). CpG-SNPs shortlisted through in silico analysis were genotyped using tetra ARMS PCR and validated using Sanger DNA sequencing. Global DNA methylation was carried out using RP-HPLC. Promoter DNA methylation and CpG site-specific methylation were carried out using bisulfite sequencing. mRNA expression and ASE were examined by SYBR green and TaqMan assay, respectively. Four exonic CpG-SNPs of MTHFD1, MTRR, and GGH genes were identified in folate pathway genes. Among these, MTHFD1 rs2236225 showed significant association with T2D independent of obesity, displayed ASE, and correlated with CpG-SNP site-specific methylation when compared with controls. Our results demonstrate that SNP rs2236225 in the CpG site of MTHFD1, which regulates allele-specific gene expression in PBMCs is methylation dependent and may perturb one-carbon metabolism pathway in T2D subjects.

Prognostic impact of genetic variants of CYP19A1 and UGT2B17 in a randomized trial for endocrine-responsive postmenopausal breast cancer.

Polymorphisms of genes involved in estrogen synthesis have been linked to breast cancer risk, prognosis, and treatment response. We investigated the prognostic impact of a deletion spanning the entire UGT2B17 gene (UGT2B17*2) and genetic variants of the aromatase CYP19A1 and estrogen receptor α (ESR1) in 125 postmenopausal women with ER-positive breast cancer enrolled in a randomized pre-surgical trial. The UGT2B17*2 was estimated by copy number variation assays and the CYP19A1 rs10046/rs4646 and ESR1 rs2077647/rs2234693/rs9340799 by TaqMan allelic discrimination assays. Serum exemestane/17-hydroxy exemestane were determined by MS and estrone (E1)/estradiol (E2)/ by GC-MS/MS. The association of genetic polymorphisms with "any event" was assessed by the Cox proportional hazards models adjusted for confounders. The UGT2B17*2 was associated with higher levels of 17-hydroxy exemestane (P = 0.04) and better prognosis (HR = 0.45; 95% CI: 0.20-1.01; P = 0.05) compared with homozygote UGT2B17 wt. The CYP19A1 rs10046 A and rs4646 C alleles were associated with higher estrogen levels: rs10046 AA vs. AG/GG genotypes had median E1 of 35.9 vs. 27.4 pg/mL (P = 0.05) and E2 of 7.57 vs. 3.9 pg/mL (P < 0.004). After a median follow-up of 7 years, women carrying the "low estrogen" alleles rs10046 G and rs4646 A had a better prognosis compared with homozygote wt for both polymorphisms (HR = 0.40; 95% CI: 0.17-0.93; P = 0.03). Our analysis points to an impact of UGT2B17 and CYP19A1 in postmenopausal endocrine responsive breast cancer. Carriers of UGT2B17*2 and CYP19A1 low estrogen variants may have better prognosis, supporting studies addressing the role of these polymorphisms in optimizing endocrine therapy. Trial registration: http://www.isrctn.com/ISRCTN86894592.

Acute-phase protein-like properties of endoplasmic reticulum aminopeptidase 1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi-functional enzyme. In this study, we analysed its role in lipopolysaccharide-induced inflammatory response in wild-type and ERAP1-knockout mice. Following lipopolysaccharide injection, ERAP1 was secreted into the blood, increasing leucine aminopeptidase activity and NO synthesis therein. Among the amino acids tested, arginine concentration was significantly increased in wild-type mice compared to ERAP1-knockout mice. These results suggest that ERAP1 behaves similar to acute-phase proteins, which are secreted into the blood in response to infectious/inflammatory stimuli and are involved in enhancing NO synthesis as a host defense mechanism.

Impact of the UGT2B17 polymorphism on the steroid profile. Results of a crossover clinical trial in athletes submitted to testosterone administration.

This article studies the genetic influence of polymorphism of the UGT2B17 gen on the urinary steroid profile and its implications for the anti-doping field. The study presents the results of a triple-blind randomized placebo-controlled crossover trial with healthy athletes submitted to a single dose of 250 mg of testosterone cypionate. Forty urine samples were collected from each participant. Mass spectrometry-based techniques commonly used in Anti-Doping laboratories, were employed to measure the urinary concentration and the Δδ13C values of a selection of target compounds for testosterone (T) administration together with LH. Twelve volunteers were included in the study; the polymorphism was evenly distributed among them. After T administration, the most meaningful change affected the Testosterone/Epitestosterone ratio (T/E) and the urinary concentration of LH. In relation with T/E, the wild type homozygous (ins/ins) group there was a mean relative increase of 30 (CI 95%: 25.2 to 36.7); in the heterozygous mutant (del/ins) group it was 19.8 (CI 95%:15.9 to 24.7); and in the homozygous mutant (del/del) group it was 19.7 (CI 95% 14.9 to 26.2). In the case of LH, it́s observed how LH values decrease significantly after the administration of Testex homogeneously among the three groups. The main outcome was related to the (del/del) group (homozygous mutant), where due to the depressed basal level of the steroid profile, if the longitudinal steroid profile of the athlete was not available, the analysis by GC/MS would not produce an "atypical" result according to the WADA TD2016EAAS despite the T administration. However, the genotyping of the UGT2B17 polymorphism, the follow up of LH and the use of GC-C-IRMS makes it possible to identify most of these samples as Adverse.

Diminished circulating concentration of interleukin-35 in Helicobacter pylori-infected patients with peptic ulcer: Its association with FOXP3 gene polymorphism, bacterial virulence factor CagA, and gender of patients.

BACKGROUND: IL-35 modulates immune and inflammatory responses during infections. Here, we investigated IL-35 levels and a single nucleotide polymorphism, rs3761548, in FOXP3 gene in Helicobacter pylori-infected patients with peptic ulcer (PU), to clarify possible associations. MATERIALS AND METHODS: This study includes 100 H. pylori-infected PU patients, 100 H. pylori-infected asymptomatic subjects (AS), and 100 noninfected healthy subjects (NHSs). Serum IL-35 levels and the genotyping were determined using ELISA and RFLP-PCR methods, respectively. RESULTS: In PU patients, the IL-35 levels were lower than AS and NHS groups (P < .001). The IL-35 levels in CagA+ H. pylori-infected participants from PU and AS groups were lower than individuals infected with CagA- strains (P < .02 and P < .04, respectively). Women had higher IL-35 levels than men among PU, AS, and NHS groups (P < .0001). In PU patients, AA genotype and A allele at rs3761548 were more frequent than total healthy subjects (AS + NHS groups) and associated with an increased PU risk (AA genotype: OR = 5.51, P < .0001; A allele: OR = 3.857, P < .002). In PU and AS groups, IL-35 levels were lower in subjects displaying AA genotype or A allele than subjects displaying CC genotype or C allele, respectively (P < .0001 and P < .03 for PU patients; P < .001 and P < .02 for AS group, respectively). CONCLUSIONS: Decreased IL-35 levels could be involved in PU development in H. pylori-infected individuals. IL-35 levels are affected by CagA status of H. pylori, participants gender, and genetic variations at rs3761548. The AA genotype and A allele at rs3761548 could represent a risk factor for PU development.

Association between interleukin-35 polymorphisms and coronary heart disease in the Chinese Zhuang population: a case-control study.

OBJECTIVES: Inflammatory cytokines play an important role in the pathogenesis of cardiovascular disease. Few studies have investigated the association between interleukin-35 (IL-35) genetic variants and the risk of coronary heart disease (CHD). We examined the association between IL-35 polymorphisms and CHD in the Chinese Zhuang population. PATIENTS AND METHODS: A total of 707 CHD patients and 707 age-matched and sex-matched controls were enrolled in this case-control study. Genotypes of the single nucleotide polymorphisms (SNPs) of IL-35, including rs428253, rs6613, rs9807813, rs2243115, rs568408, rs582054, rs583911, rs4740, and rs393581, were examined by MassArray. Plasma IL-35 level was measured using an enzyme-linked immunosorbent assay. The multivariate logistic regression model was used to evaluate the association between the SNPs and the risk of CHD. RESULTS: In the Chinese Zhuang population, compared with the GG genotype of EBI3 rs428253, individuals with the CC genotype had a 2.02-fold (95% confidence interval: 1.07-3.84, P=0.031) higher risk of CHD. Further adjustment for potential risk factors did not alter the positive association (CC vs. GG, odds ratio=2.30, 95% confidence interval: 1.16-4.54, P=0.042). SNPs rs4740, rs2243115, rs568408, and rs582054 were not statistically related to the risk of CHD. The plasma IL-35 levels showed a marginally significant difference between rs428253 genotypes [GG: 13.39 (7.89-19.25) vs. CC+GC: 17.53 (8.98-22.56) pg/ml, P=0.057]. CONCLUSION: The EBI3 rs428253 CC genotype was associated with an increased risk of CHD in the Chinese Zhuang population, although no significant difference in IL-35 levels was observed between genotypes in healthy controls.

Lower Plasma Levels of IL-35 in Patients with Primary Biliary Cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease. Its histological characteristics, such as progressive intrahepatic bile duct destruction, cholestasis, and liver cirrhosis, are caused by the body's autoimmune disorders. Interleukin (IL)-35 has two subunits (p35 and Ebi3) and is a member of the IL-12 family of heterodimeric cytokines. IL-35 has immunosuppressive functions and plays an important role in many autoimmune diseases. In this study, we compared plasma levels of IL-35 and relative mRNA expression levels of p35 and Ebi3 in peripheral blood mononuclear cells (PBMCs) from 70 PBC patients and 70 healthy individuals. The results showed that the relative expression levels of Ebi3 mRNA were lower in PBMCs from PBC patients than in PBMCs from healthy individuals, whereas the levels of p35 mRNA were similar in both groups. Plasma IL-35 concentrations were lower in patients with PBC than in healthy individuals. Plasma levels were higher in PBC patients at an advanced stage compared to patients at an early stage. Variable plasma levels with different stages were also found in transforming growth factor beta (TGF-ß), which is mainly produced by regulatory T cells (Tregs). IL-35 and TGF-ß levels were positively correlated with each other, and IL-35 was capable of promoting the inhibitory functions of Tregs in PBC patients at both the early and late stages of disease. Lower plasma IL-35 levels were accompanied by higher levels of typical clinical parameters, such as alkaline phosphatase, or of proinflammatory cytokines, such as interferon-gamma (IFN-γ), in PBC patients (P < 0.05 for each). We propose that IL-35 may be involved in the pathogenesis of PBC and could be a potential biomarker for diagnosing this disease.

Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence.

Promyelocytic leukemia nuclear bodies (PML-NB) are sub-nuclear organelles that are the hub of numerous proteins. DNA/RNA viruses often hijack the cellular factors resident in PML-NBs to promote their proliferation in host cells. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. The association of PML-NB proteins and HBV is being addressed in a number of recent studies. Here, we report that the PML-NB protein Speckled 110 kDa (Sp110) is SUMO1-modified and undergoes a deSUMOylation-driven release from the PML-NB in the presence of HBV. Intriguingly, Sp110 knockdown significantly reduced viral DNA load in the culture supernatant by activation of the type I interferon-response pathway. Furthermore, we found that Sp110 differentially regulates several direct target genes of hepatitis B virus protein X (HBx), a viral co-factor. Subsequently, we identified Sp110 as a novel interactor of HBx and found this association to be essential for the exit of Sp110 from the PML-NB during HBV infection and HBx recruitment on the promoter of these genes. HBx, in turn, modulates the recruitment of its associated transcription cofactors p300/HDAC1 to these co-regulated genes, thereby altering the host gene expression program in favor of viral persistence. Thus, we report a mechanism by which HBV can evade host immune response by hijacking the PML-NB protein Sp110, and therefore, we propose it to be a novel target for antiviral therapy.

Heroin use is associated with lower levels of restriction factors and type I interferon expression and facilitates HIV-1 replication.

Heroin use is associated with increased incidence of infectious diseases such as HIV-1 infection, as a result of immunosuppression to a certain extent. Host restriction factors are recently identified cellular proteins with potent antiviral activities. Whether heroin use impacts on the in vivo expression of restriction factors that result in facilitating HIV-1 replication is poorly understood. Here we recruited 432 intravenous drug users (IDUs) and 164 non-IDUs at high-risk behaviors. Based on serological tests, significantly higher prevalence of HIV-1 infection was observed among IDUs compared with non-IDUs. We included those IDUs and non-IDUs without HIV-1 infection, and found IDUs had significantly lower levels of TRIM5α, TRIM22, APOBEC3G, and IFN-α, -ß expression than did non-IDUs. We also directly examined plasma viral load in HIV-1 mono-infected IDUs and non-IDUs and found HIV-1 mono-infected IDUs had significantly higher plasma viral load than did non-IDUs. Moreover, intrinsically positive correlation between type I interferon and TRIM5α or TRIM22 was observed, however, which was dysregulated following heroin use. Collectively, heroin use benefits HIV-1 replication that may be partly due to suppression of host restriction factors and type I interferon expression.

Soluble serum VCAM-1, whole blood mRNA expression and treatment response in natalizumab-treated multiple sclerosis.

BACKGROUND: Natalizumab reduces disease activity in multiple sclerosis (MS). Natalizumab binds to the very late antigen-4 and inhibits vascular cell adhesion molecule-1 (VCAM-1)-mediated transmigration of immune cells across the blood-brain-barrier. This is associated with decreased serum concentrations of soluble (s)VCAM-1 and an altered composition of immune cell-subsets in the blood. OBJECTIVE: We aimed to examine if sVCAM-1 serum concentrations and whole blood mRNA expression levels of immune activation biomarkers is associated with disease activity in natalizumab-treated MS-patients. METHODS: sVCAM-1 serum concentrations and whole blood mRNA expression were measured in blood samples from untreated RRMS-patients and from two independent groups of natalizumab-treated patients. RESULTS: sVCAM-1 serum concentrations and whole blood expression of HLX1 and IL1B mRNA were lower, whereas expression of EBI3 mRNA was higher in natalizumab-treated MS-patients. Five genes were differentially expressed in clinically unstable natalizumab-treated MS-patients in the discovery but not in the validation group. CONCLUSION: Decreased serum concentrations of sVCAM-1 and altered whole blood mRNA expression levels of a panel of immunomarkers, associated with natalizumab-treatment, are not sensitive markers of MS disease activity. However, decreased expression of pro-inflammatory HLX1 and IL1B and increased expression of immunoregulatory EBI3 may indicate a less pathogenic immune activation status in natalizumab-treated MS.

Association between single nucleotide polymorphisms in prospective genes and susceptibility to ankylosing spondylitis and inflammatory bowel disease in a single centre in Turkey.

BACKGROUND/AIMS: To establish the prevalence of the single nucleotide polymorphisms (SNPs) of endoplasmic reticulum aminopeptidase 1 (ERAP1), IL-23 receptor (IL-23R), signal transducer and activator of transcription 3 (STAT-3) and Janus kinase 2 (JAK-2) in ankylosing spondylitis (AS) and inflammatory bowel disease (IBD) in a Turkish population. MATERIALS AND METHODS: A total of 562 subjects who presented at the Ankara University internal medicine departments of rheumatology and gastroenterology outpatient clinics were recruited in this study, including 365 patients with AS, 197 patients with IBD and 230 healthy controls. ERAP1, IL-23R, STAT-3 and JAK-2) were genotyped in competitive allele-specific polymerase chain reactions. RESULTS: The ERAP1 (rs26653) polymorphism was found to increase the disease risk in patients with AS and IBD compared with the control group (p=0.02 and p=0.01, respectively). In addition, this polymorphism revealed a significant relationship with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the Bath AS Functional Index (BASFI) in patients with AS (r=0.829, p < 0.001 and r=0.731, p < 0.001, respectively). CONCLUSION: The ERAP1 gene polymorphism might be a risk factor in the pathogenesis of AS and IBD. In contrast, IL-23R gene polymorphisms may serve a protective role in AS and IBD.

Fetal-specific CD8+ cytotoxic T cell responses develop during normal human pregnancy and exhibit broad functional capacity.

Tolerance of the semiallogeneic fetus presents a significant challenge to the maternal immune system during human pregnancy. T cells with specificity for fetal epitopes have been detected in women with a history of previous pregnancy, but it has been thought that such fetal-specific cells were generally deleted during pregnancy as a mechanism to maintain maternal tolerance of the fetus. We used MHC-peptide dextramer multimers containing an immunodominant peptide derived from HY to identify fetal-specific T cells in women who were pregnant with a male fetus. Fetal-specific CD8(+) T lymphocytes were observed in half of all pregnancies and often became detectable from the first trimester. The fetal-specific immune response increased during pregnancy and persisted in the postnatal period. Fetal-specific cells demonstrated an effector memory phenotype and were broadly functional. They retained their ability to proliferate, secrete IFN-γ, and lyse target cells following recognition of naturally processed peptide on male cells. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy and that unlike reports from some murine models, fetal-specific T cells are not deleted during human pregnancy. This has broad implications for study of the natural physiology of pregnancy and for the understanding of pregnancy-related complications.

Concurrent detection of circulating minor histocompatibility antigen-specific CD8+ T cells in SCT recipients by combinatorial encoding MHC multimers.

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8⁺ T cells. Therefore, monitoring of multiple MiHA-specific CD8⁺ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8⁺ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8⁺ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8⁺ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8⁺ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8⁺ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients.

Transfusion of minor histocompatibility antigen-mismatched platelets induces rejection of bone marrow transplants in mice.

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients.

Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1.

Umbilical cord blood transplantation is applied as treatment for mainly pediatric patients with hematologic malignancies. The clinical results show a relatively low incidence of graft-versus-host disease and leukemia relapse. Since maternal cells traffic into the fetus during pregnancy, we questioned whether cord blood has the potential to generate cytotoxic T cells specific for the hematopoietic minor histocompatibility (H) antigen HA-1 that would support the graft-versus-leukemia effect. Here, we demonstrate the feasibility of ex vivo generation of minor H antigen HA-1-specific T cells from cord blood cells. Moreover, we observed pre-existing HA-1-specific T cells in cord blood samples. Both the circulating and the ex vivo-generated HA-1-specific T cells show specific and hematopoietic restricted lysis of human leukocyte antigen-A2(pos)/HA-1(pos) (HLA-A2(pos)/HA-1(pos)) target cells, including leukemic cells. The cord blood-derived HA-1-specific cytotoxic T cells are from child origin. Thus, the so-called naive cord blood can comprise cytotoxic T cells directed at the maternal minor H antigen HA-1. The apparent immunization status of cord blood may well contribute to the in vivo graft-versus-leukemia activity after transplantation. Moreover, since the fetus cannot be primed against Y chromosome-encoded minor H antigens, cord blood is an attractive stem cell source for male patients.