Latest findings on the placenta from the point of view of immunology, tolerance and mesenchymal stem cells.

OBJECTIVE: Overview of current placental findings from the point of view of immunology, tolerance and mesenchymal stem cells. TYPE OF STUDY: Review. SETTING: Medicínské centrum Praha. CONCLUSION: The placenta is an important organ that connects mother and developing fetus during pregnancy. For the uncomplicated course of pregnancy and fetal development the placental function is crucial. The placenta provides not only the replacement of breathing gases, nutrients and waste materials, but also creates an immunological interface between the mother and the fetus. Maternal tolerance towards the fetus carrying paternal antigens is induced at the fetomaternal interface due to the mutual molecular interactions. Immune tolerance at the interface between placenta and decidua is ensured mainly due to the expression of HLA-C, HLA-E, HLA-F, and HLA-G on trophoblasts and their interactions with receptors expressed on uterine NK cells. Regulatory T cells and DC-10 cells also play an important role at the fetomaternal interface on the mothers side of placenta. However, some fetal cells, such as Hofbauer cells or granulocytic myeloid-derived suppressor cells are also partially involved in inducement of maternal tolerance towards the fetus. Recently, considerable attention is also paid to mesenchymal stem cells derived from both placental and umbilical tissues. These mesenchymal stem cells play an important role in inducement of immune tolerance and exhibit better immunomodulatory properties than mesenchymal stem cells isolated from adult human tissues.

Impact of genomic risk factors on survival after haematopoietic stem cell transplantation for patients with acute leukaemia.

The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival.

Identification and validation of biomarkers associated with acute and chronic graft versus host disease.

Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number of inflammatory cytokines/chemokines are implicated in GVHD and have been identified in numerous single centre studies as potential biomarkers for acute and/or chronic GVHD. In this study, we analysed candidate inflammatory biomarkers (B-cell activating factor (BAFF), interleukin 33 (IL-33), CXCL10 and CXCL11) in a two-centre study. Biomarkers were evaluated pre-transplant and at serial time points post transplant in acute and chronic GVHD patient sera with time-matched control samples from patients without GVHD. Further validation was performed using the human skin explant assay, clinical GVHD biopsies and mRNA expression analysis. BAFF was significantly increased pre-transplant. BAFF, IL-33, CXCL10 and CXCL11 showed increased levels in acute GVHD patient sera and high protein expression in grades II-III of the in vitro skin explant graft versus host reaction (GVHR) group. BAFF, CXCL10 and CXCL11 also showed increased mRNA expression levels in clinical biopsies compared with the no/low-grade GVHD group. BAFF, CXCL10 and CXCL11 levels were increased in chronic GVHD patient sera. The results identify BAFF and CXCL10 as predictive biomarkers for acute GVHD and BAFF, CXCL10 and CXCL11 as useful diagnostic biomarkers for acute GVHD and chronic GVHD.

Predicting survival using clinical risk scores and non-HLA immunogenetics.

Previous studies of non-histocompatibility leukocyte antigen (HLA) gene single-nucleotide polymorphisms (SNPs) on subgroups of patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) revealed an association with transplant outcome. This study further evaluated the association of non-HLA polymorphisms with overall survival in a cohort of 762 HSCT patients using data on 26 polymorphisms in 16 non-HLA genes. When viewed in addition to an already established clinical risk score (EBMT-score), three polymorphisms: rs8177374 in the gene for MyD88-adapter-like (MAL; P=0.026), rs9340799 in the oestrogen receptor gene (ESR; P=0.003) and rs1800795 in interleukin-6 (IL-6; P=0.007) were found to be associated with reduced overall survival, whereas the haplo-genotype (ACC/ACC) in IL-10 was protective (P=0.02). The addition of these non-HLA polymorphisms in a Cox regression model alongside the EBMT-score improved discrimination between risk groups and increased the level of prediction compared with the EBMT-score alone (gain in prediction capability for EBMT-genetic-score 10.8%). Results also demonstrated how changes in clinical practice through time have altered the effects of non-HLA analysis. The study illustrates the significance of non-HLA genotyping prior to HSCT and the importance of further investigation into non-HLA gene polymorphisms in risk prediction.

[Extracellular chromosome 21--derived microRNAs in maternal circulation: evaluation of their diagnostic potential for screening of Down syndrome]. / Chromozom 21 - specifické mikroRNA v materské cirkulaci: zhodnocení jejich významu pro screening Downova syndromu u plodu.

OBJECTIVE: Initially, we focused on the detection of extracellular microRNAs in maternal circulation, whose genes are located on human chromosome 21 (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802). Subsequently, we studied if plasmatic concentrations and/or expression profile of extracellular chromosome 21-derived microRNAs would distinguish between pregnancies bearing euploid foetuses and those affected with Down syndrome. DESIGN: Pilot study. SETTING: Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague. METHODS: 12 women with normal course of gestation (mean 16.4 weeks, median 16.0 weeks), 12 pregnancies bearing Down syndrome foetus (mean 18.2 weeks, median 18.5 weeks) and 6 non-pregnant individuals were involved in the retrospective study. RNA enriched for small RNAs (including microRNAs) was isolated from 1ml of plasma sample. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay. RESULTS: Commercial systems enabled reliable detection of 4 out of 5 extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155). Expression profile of extracellular miR-99a, miR-125b-2 and miR-155 was significantly higher in the cohort of pregnant women than in non-pregnant individuals. Also plasmatic levels of miR-99a and miR-125b-2 were significantly increased in pregnant women. Unfortunately, the concentrations and gene expression of extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. CONCLUSION: Analysis of extracellular chromosome 21-derived microRNAs does not distinguish between pregnancies with euploid and aneuploid foetuses and has no benefit for screening programmes.

Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls.

OBJECTIVE: Heat shock proteins (Hsps) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this work was to study Hsp mRNA and protein levels to determine whether they can be used to differentiate between RA, osteoarthritis (OA), and healthy controls. METHODS: Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 mRNA expression was analysed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 24 RA, 11 OA, and 21 healthy controls. Hsp70 and HspBP1 protein levels were measured in serum using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Hsp gene expression profiles differ significantly between inflammatory (RA) and non-inflammatory (OA) joint diseases, showing significantly increased Hsp27 and Hsp90α mRNA levels in RA synovial tissues. Up-regulated Hsp60 and Hsp90α together with down-regulated Hsp70 and elevated HspBP1/Hsp70 mRNA ratios can be used to differentiate between RA patients and healthy individuals through analysis of peripheral blood samples. Despite increased HspBP1 levels in RA sera, Hsp70 levels and the HspBP1/Hsp70 protein ratio remained identical in the RA patients and healthy individuals, which may contribute to the inhibition of Hsp70 anti-apoptotic activity. CONCLUSION: Hsp gene expression analysis can be implemented as a new diagnostic approach to facilitate differentiation between RA, OA, and healthy controls.

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma--next step toward reliable non-invasive prenatal diagnostics.

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n=51) or female (n=19) fetus sampled throughout gestation and absent in non-pregnant individuals (n=29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ=0.66, P<0.001), total RASSF1A and GLO (ρ=0.65,P<0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ=0.62, P<0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.

[Detection of placenta-specific microRNAs in maternal circulation]. / Detekce placentárne specifických mikroRNA v materské cirkulaci.

OBJECTIVE: We focused on the detection of microRNAs in maternal circulation during the course of physiological pregnancy. We tested initially microRNAs (mir-135b and miR-517a) which presence in maternal circulation had been previously demonstrated and those microRNAs (miR-518b and miR-517a) with up-regulated expression profile in placentas derived from pregnancies during the onset of preeclampsia. Further we selected those microRNAs, which were reported to be placenta specific according to the miRNAMap database (these microRNAs were significantly expressed in the placenta and simultaneously showed no or minimal expression in other tissues). SETTING: Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague. METHODS: RNA enriched for small RNAs (including microRNAs) was isolated from 1 ml of maternal plasma during the 12th, 16th and 36th week of gestation and 200 microl of peripheral blood derived from healthy non-pregnant women. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay. RESULTS: From the cohort of tested microRNAs we excluded those ones, which were not detectable in maternal circulation during pregnancy (miR-136 and miR-519a) and/or were demonstrated in peripheral blood of healthy non-pregnant women (miR-34c, miR-224, miR-512-5p, miR-515-5p, miR-516-5p, miR-518f*, miR-519d, miR-519e). CONCLUSION: On the base of the current study results we finally selected 6 most suitable microRNAs (miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) for subsequent studies concerning the follow-up of placenta specific microRNAs in maternal circulation during pregnancy and the differentiation between normal and pathologic pregnancies (preeclampsia, IUGR) within the same gestational age.

Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis.

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.

Peripheral blood mononuclear cell responses to heat shock proteins and their derived synthetic peptides in juvenile idiopathic arthritis patients.

BACKGROUND: Sequence homology and cross reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsps might be involved in the etiopathogenesis of autoimmune diseases. OBJECTIVE: In our study we stimulated peripheral blood mononuclear cells (PBMC) of patients with juvenile idiopathic arthritis (JIA) and healthy controls with various hsp-derived peptides together with the whole molecules of corresponding hsp. METHODS: PBMC were cultured with recombinant human hsp60 (rh-hsp60), rh-hsp70, Mycobacterium bovis hsp65 (M.bovis hsp65), P562-571 human hsp60, P180-188 M. bovis hsp65, P450-463 human hsp70 and P545-554 cytokeratin derived synthetic peptides. Cell responses were measured after incorporation of (3)H-thymidine and expressed as stimulation indices. RESULTS AND CONCLUSION: We found elevated proliferative response to rh-hsp60, M. bovis hsp65 and P562-571 human hsp60 derived peptide in patients with JIA polyarthritis. Significantly elevated proliferation to P180-188 M. bovis hsp65 was found in JIA lasting more than 2 years. None of the particular clinical characteristics (RF, ANA, HLA B27 and disease activity) seemed to be associated with hsp or hsp-derived synthetic peptide proliferative response in the JIA cohort.

Heat shock protein 70 membrane expression on fibroblast-like synovial cells derived from synovial tissue of patients with rheumatoid and juvenile idiopathic arthritis.

OBJECTIVE: To screen fibroblast-like synovial cells derived from synovial tissue of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) patients for the membrane expression of the heat shock protein Hsp70. METHODS: We performed flow cytometric (fluorescence-activated cell sorting, or FACS) analysis on fibroblast-like synovial cells of 15 RA patients and three JIA patients to investigate Hsp70 membrane expression. Skin fibroblasts derived from the operation wound (n = 4) and peripheral blood mononuclear cells (PBMC) of seven RA and three JIA patients were also tested. Peripheral blood lymphocytes (PBL) and skin fibroblasts of 10 healthy individuals were used as negative controls. RESULTS: A significantly higher percentage of Hsp70 membrane expression was found on fibroblast-like synovial cells derived from arthritis-affected joints in RA patients (mean 47.7%) when compared with autologous skin fibroblasts (mean 9.5%, p < 0.001) and control skin fibroblasts (mean 5.6%, p < 0.001) or autologous PBL (mean CD45/Hsp70-positive 10.4%, p < 0.001) and control PBL (mean CD45/Hsp70-positive 7.7%, p < 0.001). A high percentage of Hsp70 membrane expression was also observed on fibroblast-like synovial cells derived from three patients with JIA (mean 35.2%) when compared with autologous PBL (mean CD45/Hsp70-positive 10.4%). Synovial cells derived from non-affected joints in a patient with RA who underwent synovectomy for trauma showed low expression of Hsp70 (10.9%). CONCLUSION: Fibroblast-like synovial cells derived from patients with severe course of RA and JIA are strongly positive for membrane-expressed Hsp70.

Non-invasive fetal RHD and RHCE genotyping from maternal plasma in alloimmunized pregnancies.

BACKGROUND: In this prospective study, we assessed the feasibility of fetal RH genotyping by analysis of DNA extracted from maternal plasma samples of alloimmunized pregnant women using real-time PCR and primers and probes targeted toward RHD (exon 7 and exon 10) and RHCE (intron 2 and exon 5) genes. METHODS: We analysed 23 alloimmunized pregnant women (16 anti-D, 5 anti-D + C, 2 anti-E) at risk of haemolytic disease of the newborn (HDN) within 11th and 37th week of pregnancy and correlated the results with serological analysis of cord blood. RESULTS AND CONCLUSION: Detection of the presence of the RHD gene, the C and/or E alleles of the RHCE gene in maternal plasma samples is highly accurate and enables implementation in a clinical diagnostic algorithm for following pregnancies at risk for HDN. The absence of RHD gene, the C and/or E alleles of RHCE gene in the current pregnancy excludes the risk of HDN caused by anti-D, anti-C and/or anti-E alloantibodies and the performance of invasive fetal-blood sampling.

In vitro autoreactivity against skin and synovial cells in patients with juvenile idiopathic and rheumatoid arthritis.

In the present study we compared specific lysis of various autologous target cells in patients with juvenile idiopathic arthritis JIA; n = 8) or rheumatoid arthritis RA; n = 17) with those of healthy controls (n = 15). (51)Cr-release cytotoxic assay with autologous peripheral blood mononuclear cells as effector cells was used. When compared with controls, effector cells of patients with JIA or RA were found to lyse significantly autologous synovial cells (p < 0.0005) and epidermal keratinocytes (p < 0.0005), however, no difference was found for autologous dermal fibroblasts.

[Medical genetics in reproductive medicine]. / Vyuzití lékarské genetiky v reprodukcní medicíne.

Reproductive genetics (RG) is another new field of medical genetics, integrated with reproductive medicine, assisted reproduction and developmental genetic. RG is closely linked to the perioconceptional prevention, perinatology, ultrasound and biochemical screening in the end of the first and beginning of the second trimesters. RG is based on the system of specialized genetic counseling, clinical cytogenetics, molecular cytogenetics and molecular genetics to provide prefertilization, preimplantation and classical prenatal diagnosis in the Ist to IIIrd trimesters. Thus, RG is part of the fetal medicine and therapy. The six years experience with RG is summarized. A system of the specialized health care, organized, if possible in one integrated center of RG and reproductive medicine (RM) is presented. Reproductive medicine provides all necessary clinical gynecological and andrological surveillance, with assisted reproduction and further obstetrical ultrasound examinations, including nuchal translucency measurements and 2D, 3D ultrasound, echocardiography examinations, if indicated, as well as the invasive method of prenatal diagnosis and perinatology care. Specialized genetic counseling and cytogenetic analysis, if indicated, should be offered to all partners with reproductive disorders as well as to oocyte donors. Chromosome anomalies are disclosed in 6% of men with abnormal sperm analysis as well as in women with severe reproductive disorders. In males with severe oligo, azoospermia, the sperm aneuploidy analysis by molecular cytogenetic methods is recommended. Advised is also the molecular genetic detection of Y chromosome microdeletions, which is detected in 9% of our azoospermic men with deletions in AZFb region. CFTR gene mutations and intron 8 and 10 polymorphism examination is provided not only in men with obstructive azoospermia (CBAVD), but also if severe oligospermy with less than 1 x 10(6) sperm/ml is detected. Molecular genetic analysis of thrombophilic mutations of factor II., V. (Leiden) and MTHFR gene in unexplained recurrent abortions and in cases with unsuccessful IVF is part of the diagnostic strategy. The population frequencies of carriers of mutations of factor II. (2.3%), factor V.-Leiden (5.7%) and MTHFR gene (38%) were determined. The laser biopsy of the first polar body and of blastomeres was introduced for FISH analysis of chromosome aneuploidies. Quantitative fluorescent PCR (QFPCR) detection is used for testing of the most frequent delta F508 CFTR gene mutation and the most frequent aneuploidies of chromosome 13, 18, 21, X and Y. QFPCR was successfully tested for male fetal sex examination from partially purified fetal cells in the maternal blood. The first trimester ultrasound and biochemical screening is recommended to all successful pregnancies after different IVF methods. If borderline levels of first trimester biochemical screening of PAPP-A protein and beta hCG are detected without pathological ultrasound findings, classical triple test of biochemical screening in 16th week of gestation is recommended. If pathological results of ultrasound and biochemical screening are disclosed, invasive prenatal genetic diagnosis is indicated as well as in pregnancies after ICSL, if there is not any obstetrical contraindication.

The detection of apoptosis in a human in vitro skin explant assay for graft versus host reactions.

AIMS: Keratinocyte apoptosis is a major pathogenic mechanism in dermal complications, such as graft versus host disease (GVHD), after allogeneic bone marrow transplantation. However, the mechanisms by which recipient target cells undergo apoptosis in GVHD are still unclear, but may result from DNA damage caused by chemotherapeutic agents and/or by direct cytokine action. The basis of this investigation was to correlate keratinocyte apoptosis with (1) the severity of graft versus host reactions (GVHR) in vitro and (2) the clinical grade (0--III) of GVHD. METHODS: Skin sections generated from an in vitro skin explant model for detecting experimental or clinically relevant GVHR were investigated for the detection of apoptotic nuclei using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique. This investigation also aimed to establish whether the TUNEL assay could be used as an additional, predictive method for the severity of GVHD before transplantation in potential patient/donor pairs given standard GVHD prophylaxis (cyclosporin A and methotrexate). RESULTS: By comparing mean values of apoptosis for each GVHR grade in a cohort of 83 retrospective skin sections it was shown that as the severity of GVHR increased there was a parallel increase in the percentage of apoptotic cells (p < 0.0001). However, the correlation between clinical GVHD grade II--III and overall keratinocyte apoptosis (> 2.6%) did not reach this degree of significance (chi(2): 4.2; degrees of freedom, 1; p = 0.04; Fisher's exact test: p = 0.06). CONCLUSIONS: The detection of apoptosis correlated with degree of GVHR using an in vitro assay and a higher degree of apoptosis tended to correlate with more severe GVHD. Further studies in a larger cohort of patients, using other methods to detect apoptosis in conjunction with the TUNEL assay, may give additional insight into the complex immunopathophysiology of GVHD.

In vitro autoreactivity against skin in rheumatoid arthritis: are peripheral blood mononuclear cells of patients with rheumatoid arthritis able to lyze autologous keratinocytes?

An in vitro skin explant model was originally developed to predict the occurrence and severity of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplants. In previous studies we reported that peripheral blood mononuclear cells of patients with rheumatoid arthritis were able to induce graft-versus-host-like histopathological changes when co-cultured in vitro with autologous skin explants. The aim of the present study was to verify if observed skin damage was really of autoimmune origin. Using a 51chromium release cytotoxic assay we found that peripheral blood mononuclear cells of patients lyzed autologous keratinocytes (n=5 patients with rheumatoid arthritis) but not autologous lymphoblasts (n=4 with rheumatoid arthritis, n=8 patients with juvenile idiopathic arthritis). No specific lysis of keratinocytes or lymphoblasts was observed in healthy controls (n=15). We hypothesize that autologous peripheral blood mononuclear cells might recognize similar autoantigen(s) expressed on epidermal cells, which gives rise to an autoimmune response in the synovium.

Histological correlation between different centers using the skin explant model to predict graft-versus-host disease following bone marrow transplantation.

Acute graft-versus-host disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT) with an incidence of 40-60% and a mortality of up to 50%. Several assays have been developed to try to predict the development of GVHD including the mixed lymphocyte culture reaction, cytotoxic and helper T lymphocyte precursor frequency assays. In the Northern region of England we have used an in vitro skin explant model for predicting GVHD in MHC compatible bone marrow transplant recipients since 1988. The aims of the present study was to test the reproducibility of the model in two other bone marrow transplant centers in Europe. The assay consists of incubating patient skin explants with effector cells from mixed donor versus recipient lymphocyte cultures and the subsequent detection of graft-versus-host reactions by histopathological grading (0-IV) of the skin explants. 503 slides from 134 patients were evaluated. All were graded for negative GVHR grade 0-I or positive grade II-IV. Results from control and test slides significantly correlated between centers to the p value of 0.0001 by Fisher's exact probability test. These results show that the skin explant assay is reproducible between centers and supports the continued use of the assay to predict GVHD in allogeneic stem cell transplant recipients.

Anti-keratin antibodies in patients with juvenile idiopathic arthritis.

OBJECTIVE: We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). METHODS: An indirect immunofluorescence test with rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients'sera. RESULTS: Overall 30/60 patients with JIA had sera positiveforAKA (50%, p=0.0005) ranging from 1:20 to 1:160 dilutions. Using the classification criteria for childhood idiopathic arthritis, AKA occurred in 2/7 patients with systemic disease (28.6%), in 13/30 patients with RF negative polyarthritis (43.3%, p=0.008) and in 12/18 RF positive polyarthritis (66.7%, p=0.002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence ofAKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 16/29 patients (55.2%) with severe JIA and in 11/26 patients (42.3%) with non-severe disease. We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 44.4% patients with active JIA and in 45.9% patients in the complete or near remission. CONCLUSION: Our data suggest that AKA are present in patients with JIA. However no correlation with severity or disease activity was observed.

An in vitro skin explant assay as a predictive assay for graft-versus-host disease in a cohort of pediatric transplants.

Severe acute graft-versus-host disease (GvHD) remains a serious complication of allogeneic stem cell transplantation. An in vitro skin explant assay was used to predict the occurrence and severity of acute GvHD in a cohort of 30 pediatric patients undergoing human leucocyte antigen (HLA)-matched sibling transplants (20 patients) and matched or one antigen mismatched unrelated donor transplants (10 patients). In the cohort of HLA-matched sibling transplants, the result appeared to reflect the degree of GvHD prophylaxis. The skin explant assay correlated with GvHD outcome in 12 of 20 children, but this did not reach statistical significance (chi-square 0.95, d.f.=1, p=0.32). These results support previous observations. In this present cohort, patients were treated either with cyclosporin A (CsA) monotherapy (n=7) or with CsA plus additional methotrexate (MTX) (n=13). We have previously demonstrated that the skin explant assay was not as predictive in patients receiving CsA plus additional MTX compared to cohorts treated with CsA alone. In the group of patients treated with CsA alone, four of five patients (80%) predicted to develop GvHD developed acute GvHD of grade II or above; of two patients predicted to develop only grade 0-I GvHD, one patient developed no GvHD and the other grade II GvHD. In the CsA plus MTX group, nine patients were predicted to develop GvHD. Five of nine (55%) developed acute GvHD of grade II or above, while three of four with grade 0 or I skin explant assay results developed only grade 0-I GvHD. In a cohort of 10 patients who received unrelated donor transplants, the skin explant assay correlated with GvHD outcome in all 10 patients (Fisher's exact test p=0.008). Hence, the skin explant assay is a pretransplant in vitro GvHD predictive test that predicts the occurrence and severity of acute GvHD in pediatric unrelated donor transplants and to varying degrees, depending on the GvHD prophylaxis protocols, in HLA-matched sibling cohorts.