The source of SYBR green master mix determines outcome of nucleic acid amplification reactions

<p>Numerous real-time PCR devices and master mixes are available on the market. To perform reliable high-quality data, PCR master mix, and equipment need to be optimal. However, general lab optimized protocols are widely used for different gene targets and performed diversely between conditions. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay. This was tested across various PCR machines and amplification protocols for assessment of mRNA transcript levels, DNA copy numbers, and DNA genotypes.</p

Editorial: Immunological challenges around pregnancy complications associated with failures of maternal tolerance to the fetus

Obstetric

Inflammatory placental lesions are specifically observed in healthy oocyte donation pregnancies with extreme fetal-maternal incompatibility

Introduction: Oocyte donation (OD) pregnancy is a risk factor for pre-eclampsia (PE). Due to a higher extent of fetal-maternal human leukocyte antigens (HLA) mismatching in OD pregnancies compared to naturally conceived (NC) and in vitro fertilization (IVF) pregnancies, the immune response in OD placentas is probably divergent and affects clinical outcomes. We hypothesized that placental pathology varies among diverse pregnancy conditions and is related to fetal-maternal HLA incompatibility. Methods: Placental lesions were scored in four patient groups: OD-PE (n = 16), OD-healthy (n = 37), NC-PE (n = 45), and IVF-healthy (n = 17). All combinations were genotyped for HLA-A, -B, -C, -DR, and -DQ to calculate fetal-maternal HLA mismatches. Placentas showing chronic deciduitis with plasma cells were immunofluorescently stained with CD138 and the anti-inflammatory cytokine interleukin-10 (IL-10).Results: The distribution and severity of placental lesions varied among groups. The OD-healthy group had the highest inflammation score and greatest extent of chronic deciduitis with plasma cells (p &lt; 0.05). However, the majority of CD138+ plasma cells (90%) in OD-healthy group expressed IL-10, in contrast to the OD-PE group (58%). The OD-healthy group was separated into semi-allogeneic (≤5 HLA mismatches) and fully allogeneic (&gt;5 mismatches) subgroups. The elevated inflammatory pathology score and chronic deciduitis with plasma cells were found more often in the HLA-class-I fully allogeneic OD-healthy group than the IVF-healthy group (p &lt; 0.05). Discussion: Placental inflammatory lesions are most often present in uncomplicated OD pregnancies. Immune cells that infiltrate these lesions might play an immunosuppressive role to protect OD pregnancies from complications when facing a higher extent of fetal-maternal HLA mismatching.</p

Non-invasive Biomarkers of Acute Rejection in Kidney Transplantation: Novel Targets and Strategies

Kidney transplantation is considered the favored treatment for patients suffering from end-stage renal disease, since successful transplantation is associated with longer survival and improved quality of life compared to dialysis. Alloreactive immune responses against the donor kidney may lead to acute rejection of the transplant. The current diagnosis of renal allograft rejection mainly relies on clinical monitoring, including serum creatinine, proteinuria, and confirmation by histopathologic assessment in the kidney transplant biopsy. These parameters have their limitations. Identification and validation of biomarkers, which correlate with or predict the presence of acute rejection, and which could improve therapeutic decision making, are priorities for the transplantation community. There is a need for alternative, less invasive but sensitive markers to diagnose acute graft rejection. Here, we provide an overview of the current status on research of biomarkers of acute kidney transplant rejection in blood and urine. We specifically discuss relatively novel research strategies in biomarker research, including transcriptomics and proteomics, and elaborate on donor-derived cell-free DNA as a potential biomarker

Circulating Levels of Anti-C1q and Anti-Factor H Autoantibodies and Their Targets in Normal Pregnancy and Preeclampsia

Preeclampsia (PE) generally manifests in the second half of pregnancy with hypertension and proteinuria. The understanding of the origin and mechanism behind PE is incomplete, although there is clearly an immune component to this disorder. The placenta constitutes a complicated immune interface between fetal and maternal cells, where regulation and tolerance are key. Stress factors from placental dysfunction in PE are released to the maternal circulation evoking the maternal response. Several complement factors play a role within this intricate landscape, including C1q in vascular remodeling and Factor H (FH) as the key regulator of alternative pathway complement activation. We hypothesize that decreased levels of C1q or FH, or disturbance of their function by autoantibodies, may be associated with PE. Autoantibodies against C1q and FH and the concentrations of C1q and FH were measured by ELISA in maternal sera from women with preeclamptic and normal pregnancies. Samples originated from cohorts collected in the Netherlands (n=63 PE; n=174 control pregnancies, n=51 nonpregnant), Finland (n=181 PE; n=63 control pregnancies) and Norway (n=59 PE; n=27 control pregnancies). Serum C1q and FH concentrations were higher in control pregnancy than in nonpregnant women. No significant differences were observed for serum C1q between preeclamptic and control pregnancy in any of the three cohorts. Serum levels of FH were lower in preeclamptic pregnancies compared to control pregnancies in two of the cohorts, this effect was driven by the early onset PE cases. Neither anti-C1q autoantibodies nor anti-FH autoantibodies levels differed between women with PE and normal pregnancies. In conclusion, levels of anti-C1q and anti-FH autoantibodies are not increased in PE. C1q and FH are increased in pregnancy, but importantly, a decrease in FH concentration is associated with PE.Peer reviewe

A possible role for HLA-G in development of uteroplacental acute atherosis in preeclampsia

HLA-G, a non-classical HLA molecule expressed by extravillous trophoblasts, plays a role in the maternal immune tolerance towards fetal cells. HLA-G expression is regulated by genetic polymorphisms in the 3' untranslated region (3'UTR). Low levels of HLA-G in the maternal circulation and placental tissue are linked to preeclampsia. Our objective was to investigate whether variants of the 3'UTR of the HLA-G gene in mother and fetus are associated with acute atherosis, a pregnancy specific arterial lesion of the decidua basalis that is prevalent in preeclampsia. Paired maternal and fetal DNA samples from 83 normotensive and 83 preeclamptic pregnancies were analyzed. We sequenced the part of the HLA-G 3'UTR containing a 14-bp insertion/deletion region and seven single nucleotide polymorphisms (SNPs). Associations with acute atherosis were tested by logistic regression. The frequency of heterozygosity for the 14-bp polymorphism (Ins/Del) and the +3142 SNP (C/G) variant in the fetus are associated with acute atherosis in preeclampsia (66.7 % vs. 39.6 %, p = 0.039, and 69.0 % vs. 43.4 %, p = 0.024). Furthermore, the fetal UTR-3 haplotype, which encompasses the 14-bp deletion and the +3142G variant, is associated with acute atherosis in preeclampsia (15 % vs. 3.8 %, p = 0.016). In conclusion, HLA-G polymorphisms in the fetus are associated with acute atherosis. We hypothesize that these polymorphisms lead to altered HLA-G expression in the decidua basalis, affecting local feto-maternal immune tolerance and development of acute atherosis

Identification of a unique intervillous cellular signature in chronic histiocytic intervillositis

Introduction: Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta characterized by an infiltrate of CD68+ cells in the intervillous space. CHI is associated with adverse pregnancy outcomes such as miscarriage, fetal growth restriction, and (late) intrauterine fetal death. The adverse pregnancy outcomes and a variable recurrence rate of 25–100% underline its clinical relevance. The pathophysiologic mechanism of CHI is unclear, but it appears to be immunologically driven. The aim of this study was to obtain a better understanding of the phenotype of the cellular infiltrate in CHI. Method: We used imaging mass cytometry to achieve in-depth visualization of the intervillous maternal immune cells and investigated their spatial orientation in situ in relation to the fetal syncytiotrophoblast. Results: We found three phenotypically distinct CD68+HLA-DR+CD38+ cell clusters that were unique for CHI. Additionally, syncytiotrophoblast cells in the vicinity of these CD68+HLA-DR+CD38+ cells showed decreased expression of the immunosuppressive enzyme CD39. Discussion: The current results provide novel insight into the phenotype of CD68+ cells in CHI. The identification of unique CD68+ cell clusters will allow more detailed analysis of their function and could result in novel therapeutic targets for CHI

Evaluating the role of paternal factors in aetiology and prognosis of recurrent pregnancy loss: Study protocol for a hospital-based multicentre case-control study and cohort study (REMI III project)

Introduction Recurrent pregnancy loss (RPL) is defined as the spontaneous demise of two or more pregnancies before the fetus reaches viability. Despite investigation of multiple known maternal risk factors, in more than 50% of couples, this condition remains unexplained. Studies focusing on paternal factors in RPL are scarce, and therefore, paternal evaluation in RPL is currently very limited. However, regarding single miscarriage, there are multiple publications suggesting a contributive role of paternal factors. In this project, we aim to identify paternal factors associated with RPL and to improve couple-specific prediction of future pregnancy outcomes by developing a prediction model containing both maternal and paternal factors. Methods and analysis In a case-control design, the relation between unexplained RPL and paternal age, lifestyle factors, sperm DNA damage and immunomodulatory factors in peripheral blood and semen will be studied. Prospectively, 135 couples with naturally conceived unexplained RPL (cases) and 135 fertile couples without a history of pregnancy loss (controls) will be included, with collection of paternal blood and semen samples and documentation of clinical and lifestyle characteristics. In addition, 600 couples from both groups will be included retrospectively. To adjust for confounders, multivariate logistic regression will be used. The predictive value of paternal and maternal factors will be studied in the total RPL cohort consisting of approximately 735 couples. The primary outcome of the cohort study is live birth within 5 years after initial visit of the clinic. Secondary outcomes are ongoing pregnancy, time interval until next pregnancy and pregnancy complications. Ethics and dissemination This project is approved by the Medical Research Ethics Committee

Soluble HLA in the aqueous humour of uveal melanoma is associated with unfavourable tumour characteristics

A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour's HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses