Enhancement of immunogenicity of Jeg3 cells by ectopic expression of HLA-A*0201 and CD80

PROBLEM: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. METHOD OF STUDY: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. RESULTS: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. CONCLUSION: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells.     

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.     

Blinded, Externally Controlled Multicenter Evaluation of Light Microscopy and PCR for Detection of Microsporidia in Stool Specimens

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.     

20q13.2 Amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast

The 20q13 region harboring recently described putative oncogenes is frequently amplified in invasive ductal carcinoma (IDC). The aim of this study was to examine the 20q13 copy number in intraduct hyperplasia (IH), atypical duct hyperplasia (ADH), and ductal carcinoma in situ (DCIS) adjacent to IDC. In 5 patients, comparative genomic hybridization (CGH) after laser microdissection revealed 20q13 amplification in four of five cases of IH, in all of three cases of IH with atypia, all five of DCIS, and all five of IDC. Fluorescence in situ hybridization (FISH) confirmed the amplification at 20q13.2 in IH in the two specimens analyzed. The amplification rate, however, was higher in DCIS and IDC. In phenotypically normal ductal epithelium normal values were found for 20q13 copy number by FISH (n=2) and CGH (n=5). Although the number of cases presented here is small, our results suggest that mutations in the 20q13.2 region in IH may be associated with accelerated proliferation and hyperplasia of the ductal epithelium. Progression to DCIS and ICD is accompanied by a further increase in the 20q13.2 copy number. Received: 17 March 1999 / Accepted: 22 June 1999     

Testosterone suppresses protective responses of the liver to blood-stage malaria

Testosterone induces a lethal outcome in otherwise self-healing blood-stage malaria caused by Plasmodium chabaudi. Here, we examine possible testosterone effects on the antimalaria effectors spleen and liver in female C57BL/6 mice. Self-healing malaria activates gating mechanisms in the spleen and liver that lead to a dramatic reduction in trapping activity, as measured by quantifying the uptake of 3-mum-diameter fluorescent polystyrol particles. However, testosterone delays malaria-induced closing of the liver, but not the spleen. Coincidently, testosterone causes an approximately 3- to 28-fold depression of the mRNA levels of nine malaria-responsive genes, out of 299 genes tested, only in the liver and not in the spleen, as shown by cDNA arrays and Northern blotting. Among these are the genes encoding plasminogen activator inhibitor (PAI1) and hydroxysteroid sulfotransferase (STA2). STA2, which detoxifies bile acids, is suppressed 10-fold by malaria and an additional 28-fold by testosterone, suggesting a severe perturbation of bile acid metabolism. PAI1 is protective against malaria, since disruption of the PAI1 gene results in partial loss of the ability to control the course of P. chabaudi infections. Collectively, our data indicate that the liver rather than the spleen is a major target organ for testosterone-mediated suppression of resistance against blood-stage malaria.     

Silent mutations in the phenylalanine hydroxylase gene as an aid to the diagnosis of phenylketonuria.

Direct sequencing of the phenylalanine hydroxylase (PAH) gene indicated the existence of silent mutations in codons 232, 245, and 385, linked to specific RFLP haplotypes in several Caucasian populations, namely Germans, Bulgarians, Italians, Turks, and Lithuanians. All three mutations create a new restriction site and can be easily detected on PCR amplified DNA. The usefulness of the silent mutations for diagnostic purposes depends on the haplotype distribution in the target population. The combined analysis of these markers and one or two PKU mutations forms a simple panel of diagnostic tests with full informativeness in a large proportion of PKU families, which helps to avoid the problems of genetic heterogeneity and of prenatal genomic Southern blot analysis.     

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene

Haemophilia A is a X‐linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are “private”, because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype‐phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated. © 2001 Wiley‐Liss, Inc.     

Localization of corticotropin-releasing activity in the rat hypothalamus

Hypothalamic nuclei were removed from frozen sections of rat brain and examined for their corticotropin-releasing activity. The highest concentration was measured in the median eminence. In addition there was significantly more activity detected in the nuclei paraventricularis, supraopticus, suprachiasmaticus and arcuatus than in the other nuclei.     

Requirement for tumor necrosis factor receptor 2 expression on vascular cells to induce experimental cerebral malaria

Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.     

Interferon-gamma (IFN-gamma) and IL-4 expressed during mercury-induced membranous nephropathy are toxic for cultured podocytes.

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.