Intestinal peptide transporter PepT1 is over-expressed during acute cryptosporidiosis in suckling rats as a result of both malnutrition and experimental parasite infection.

Cryptosporidium parvuminfection induces amino acid malnutrition leading to growth retardation in children. Owing to the nutritional efficiency of peptides compared to free amino acids and the resistance of the di-tripeptide transporter PepT1 to mucosal injury, we analyzed the intestinal expression of PepT1 during experimental acute cryptosporidiosis in suckling rats from day 4 to day 50. PepT1 mRNA levels were increased at the peak of infection (day 10) all along the small intestine and normalized after spontaneous clearance of the parasite (day 21). Immunolocalization of PepT1 showed that its expression was maintained in the brush border membrane of enterocytes in infected rats from day 4 to day 50 all along the small intestine. Our results suggest a transcriptional up-regulation during acute cryptosporidiosis in response to both C. parvum-induced malnutrition and parasite implantation. As no treatment is available, a semi-elemental diet should be considered part of the treatment of cryptosporidiosis.

Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta.

Trophoblasts of the human placenta differentiate along two pathways to give either extravillous cytotrophoblasts (EVCT) with invasive properties and that are implicated in the implantation process, or villous cytotrophoblasts (VCT) that by cell fusion form multinucleated syncytiotrophoblasts. We report the first isolation and purification of these two cell types from the same chorionic villi of first trimester human placenta. We also studied their differentiation in vitro. Electron microscopy showed that in contrast to VCT, EVCT had no microvilli but contained large fibrinoid inclusions. EVCT cultures required a matrix to invade, and as previously established, VCT cultured on plastic dishes aggregated and fused to form syncytiotrophoblasts. These differentiation processes were characterized by a particular pattern of gene expression as assessed by real-time PCR and confirmed by immunocytochemical analysis of the corresponding proteins. EVCT cultured in vitro expressed high levels of HLA-G, c-erbB2, human placental lactogen, and very little human chorionic gonadotropin. Interestingly, TGFbeta2 was a marker of EVCT in vitro and in situ. These data offer a new tool for cell biologists to study the molecular mechanisms involved in human placental development and its pathology.

Inflammatory arthropathy in MRL hematopoietic chimeras undergoing Fas mediated graft-versus-host syndrome.

OBJECTIVE: To determine whether overexpression of the Fas ligand (FasL) on activated lpr T lymphocytes could induce arthritic lesions when grafted into syngeneic wild-type MRL mice expressing normal Fas receptor levels. METHODS: Lethally irradiated MRL+/+ mice were reconstituted with congenic MRL/lpr bone marrow cells and splenocytes overexpressing FasL. Fas-mediated cytotoxic properties of repopulating lpr splenic lymphocytes were evaluated in vitro. Simultaneously, the hind paw ankles of the hematopoietic chimeras were histologically examined. RESULTS: The lpr lymphocytes repopulating the spleen overexpressed FasL and had in vitro Fas-mediated cytotoxic activity. Simultaneously, in vivo, articular (synovitis, pannus) and periarticular (periostitis) inflammation with bone resorption were observed. CONCLUSION: Arthritic lesions may be induced in Fas-expressing recipients by persistent engrafted syngeneic lymphocytes overexpressing FasL.

Cryptosporidium parvum infection in suckling rats: impairment of mucosal permeability and Na(+)-glucose cotransport.

Na(+)-glucose transport and transepithelial permeability were investigated during symptomatic acute cryptosporidiosis in newborn rats. The infection resulted in a significant (P < 0.01) decrease in the ileal short-circuit current and a nonsignificant fall in the transepithelial potential difference and conductance. In glucose-stimulated conditions, the rise in ileal short-circuit current and transepithelial permeability were significantly lower in Cryptosporidium parvum-infected rats than in controls (delta Isc = 3.24 +/- 1.21 microA.cm-2 vs delta Isc = 5.09 +/- 2.23 microA.cm-2 in infected and control animals, respectively; P < 0.001; delta PD = -0.35 +/- 0.13 mV vs delta PD = -0.44 +/- 0.14 mV for infected and control animals, respectively; P < 0.01). Electrical parameters were not affected by addition of the cyclooxygenase inhibitor indomethacin in either Cryptosporidium-infected newborn rats or controls. Horseradish peroxidase and mannitol flux studies demonstrated a significant decrease (P < 0.05) in transepithelial molecular permeability in infected enterocyte rats, HRP flux = 380, range 68-5570 ng.cm-2, and mannitol flux = 1.06, range, 0.34-1.44%.cm-2.min-1, compared with controls rats, HRP flux = 4446 range, 1121-124,363 ng.cm-2, and mannitol flux = 1.99, range, 0.57-5.09%.cm-2.min-1; P < 0.05. These effects could originate from C. parvum-induced alteration of intracellular trafficking of pinocytosis vesicles and therefore account for the decrease in permeability to solute and macromolecules, together with impaired transcellular nutrient transport, in suckling rats.

Investigation of the effects of 50 Hz magnetic fields on purified human hematopoietic progenitors.

Epidemiological reports suggest a possible association between exposure to extremely low frequency electromagnetic fields (ELF-EMFs) and the frequency of leukemia in men working in the field of electricity or in children living near power lines. At present, there is no experimental evidence for such an association. In this study we investigated the effects of 50 Hz EMFs (sinusoidal EMF of 10 microT or 1 mT) on human purified hematopoietic progenitor cells which are the first targets of a leukemogenic process. The results failed to reveal any significant changes in cell proliferation, cell kinetics, ultrastructure or clonogenic potential of these progenitors which could be related to a leukemogenic effect.

Use of a renal tubule cell line (LLC-PK1) to study the nephrotoxic potential of a kappa-type Bence-Jones protein.

The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing a model for studying the potential nephrotoxicity of these proteins. The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye uptake and measurement of cellular enzyme activities) and morphological electron microscopy. After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference compared to treatment with bovine albumin in the same conditions. The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1 cells. Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic effect to be distinguished from that of beta-lactoglobulin. This isolate could serve as a "moderate control" for a later study with a BJP having caused acute renal failure.

Epstein-Barr virus-related localized hepatic lymphoproliferative disorders after liver transplantation.

BACKGROUND: Localized hepatic post-transplant lymphoproliferative disease is uncommon. In such cases, lymphocyte Epstein-Barr virus (EBV) infection may promote an intrahepatic B-lymphocyte monoclonal expansion. METHODS: From 1990 to 1991, 149 patients underwent liver transplantation for various liver failures. Immunosuppressive therapy was azathioprine, cyclosporine-A, and methylprednisolone. Rejection episodes were treated by methylprednisolone bolus injection with or without OKT3 therapy. Three patients (2%), aged 38, 50, and 47 years, developed lymphoproliferative disease localized in the transplanted livers within 5 months of liver transplantation (a patient had been immunosuppressed for 3 years before the lymphoproliferative disease occurred within the third allografted liver). Diagnoses were obtained by fine needle aspiration. In situ hybridizations were performed with the kappa/lambda mRNA-kit FITC DAKO (DAKO Corporation, Carpenteria, CA) and the early mRNA-EBER oligonucleotide FITC DAKO: RESULTS: Lymphoproliferative diseases were all classified as diffuse polymorphic large cell lymphomas in the working formulation and considered as lymphoproliferative disorders with polymorphic large cells in the Frizzera classification. All large cells were CD20-positive, CD45-positive and CD45RO-negative. In situ mRNA light chain hybridization demonstrated monoclonality in two cases. In all three cases, EBV mRNA was detected in large cells by early mRNA-EBV (EBER) in situ hybridization. Patients were treated with doxorubicin, cyclophosphamide, vincristine, and VM26. Two patients maintained a complete remission 3 years after six cycles of chemotherapy, whereas one died of an early opportunistic infection. CONCLUSION: Epstein-Barr virus may play a special role in the pathogenesis of lymphoproliferative disorders that develop in patients who have undergone liver transplantation.

Effects of chronic dietary fructose with and without copper supplementation on glycaemic control, adiposity, insulin binding to adipocytes and glomerular basement membrane thickness in normal rats.

Sucrose feeding over a long period has been reported to induce glomerular basement membrane (GBM) thickening and insulin resistance in normal rats. These effects are attributed to the fructose moiety of the sucrose molecule, to Cu deprivation or both. Consequently, our aim was to evaluate the long-term effects of fructose feeding with normal or high amounts of Cu on body weight, plasma lipids, blood glucose regulation, GBM thickening and insulin binding to adipocytes. Four groups of eight Sprague-Dawley rats were fed for 10 weeks on a diet containing 570 g carbohydrate/kg supplied either as starch (S), dextrose (D), fructose (F) or fructose-starch (1:1, w/w; FS), and an adequate amount of Cu (12 micrograms Cu/g diet). A fifth group was fed on diet F supplemented with 24 micrograms Cu/g diet (FCu). After 10 weeks the epididymal adipose tissue and kidney weights expressed per 100 g body weight (relative weight) were heaviest in the F and FCu groups (P < 0.0001, ANOVA). The GBM thickness was within the normal range in the five groups but significantly higher in group D (1.95 (SE 0.04) nm and lower in group FS (1.79 (SE 0.02) nm when compared with group S (1.85 (SE 0.03) nm; P < 0.05). Insulin binding to adipocytes (expressed per cell) was lowest in the F and FCu groups, intermediate in groups D and FS and highest in group S (P < 0.05). Fasting plasma insulin level was higher in group F than in the FCu and FS groups (P < 0.05), whereas fasting plasma glucose, total cholesterol and triacylglycerol levels remained within the normal range in all groups. We conclude that in normal rats a 10-week fructose-rich diet with an adequate amount of Cu produced deleterious metabolic effects on adipose tissue, insulin binding to adipocytes, and plasma insulin, but not on GBM thickening even though kidney weight was significantly increased. However, a moderate fructose intake mixed with other sugars did not have adverse effects.

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: a multiparametric study involving transmission electron microscopy and fetal DNA amplification.

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free intervillous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.

[Rapid diagnosis of cytomegalovirus hepatitis after liver transplantation. Use of monoclonal antibody E13 directed against an early viral antigen]. / Diagnostic rapide des hépatites à cytomégalovirus après transplantation hépatique. Utilisation de l'anticorps monoclonal E13 dirigé contre un antigène viral précoce.

Liver biopsies obtained from 10 liver transplant patients for whom a cytomegalovirus (CMV) hepatitis was suspected on the basis of clinical and biological abnormalities have been studied by an immunohistological method using the monoclonal antibody E-13 directed against an early viral antigen. Biopsies were performed between the 30th and the 77th day after transplantation. In 7 of the 10 cases, CMV hepatitis was diagnosed by histological examination because of the association of lobular cytolysis, inflammatory infiltrate with neutrophils and intranuclear inclusions. Ten cases were positive with the E-13 antibody including the 3 cases in which histological examination was not conclusive. In all cases, positive results gave a nuclear staining pattern in hepatocytes (63%) or endothelial cells (40%), independently of the presence of inclusions. Rapid diagnosis of CMV hepatitis is of significant importance since anti-viral drugs, such as DHPG (Ganciclovir), are now efficient. The immunohistochemical method with E-13 could permit easy and rapid detection of CMV in liver specimens.

Persistence of Trypanosoma cruzi antigens in the inflammatory lesions of chronically infected mice.

Different tissues and organs of mice infected with Trypanosoma cruzi trypomastigotes have been examined for the presence of parasites and parasitic antigens during both the acute and the chronic phases of infection. Specimens of skeletal and cardiac muscles, spleen, liver, brain and sciatic nerves were studied by histological and immunological methods. During the acute phase of infection, the parasites were commonly observed in these tissues. In the chronic phase of the experimental infection, pseudocysts filled with amastigotes were seen in less than 1% of the tissue sections, while immunohistological methods showed that T. cruzi antigens were present in 11% of the inflammatory infiltrates. These findings suggest that antigenic stimulation persists throughout the chronic phase, even though the parasites are not morphologically detectable.

Correlated ultrastructural and immunological findings in CD8+ and CD4+ peripheral T-cell lymphomas.

Ultrastructural features of CD8+ and CD4+ peripheral T-cell lymphomas were studied and the results revealed distinct traits correlated with phenotype. CD8+ peripheral T-cell lymphomas were characterized by a rich organular pattern with a well-developed Golgi apparatus, dense granules, numerous and atypical giant mitochondria. CD4+ peripheral T-cell lymphomas were characterized by wide cytoplasmic areas devoid of organelles. Since similar ultrastructural features differentiated normal and pathological CD8+ and CD4+ peripheral T-lymphocytes, we conclude that under both normal and pathological conditions the expression on the part of the peripheral T-lymphocytes of CD8+ and CD4+ phenotypes is associated to morphological features which distinguish the two subsets.

LAV-like viral particles in lymph node germinal centers in patients with the persistent lymphadenopathy syndrome and the acquired immunodeficiency syndrome-related complex: an ultrastructural study of 30 cases.

The detection of LAV- or HTLV III-type viral particles in lymph node germinal centers from patients with the persistent lymphadenopathy syndrome (LAS) or the acquired immunodeficiency syndrome (AIDS)-related complex (ARC) is an important diagnostic factor in the prodromal stages of AIDS. These particles, the morphology of which is defined, are situated solely in the extracellular spaces delimited by cytoplasmic extensions of the dendritic reticular cells. Often few in number, they were found in 26 of the 30 lymph nodes studied, selected uniquely on the basis of light microscopic criteria (predominantly follicular lymphoid hyperplasia). The four negative nodes contained no, or fewer than two, germinal centers in the samples taken for ultrastructural study. The diagnosis of the LAS or the ARC was always confirmed clinically and biologically. Thus, lymph node biopsy and the corresponding ultrastructural study are important steps in the diagnosis of AIDS.

[Ultrastructural markers in LAV-HTLV III virus infection: study of the rectal mucosa in 16 patients]. / Marqueurs ultrastructuraux au cours de l'infection par le virus LAV-HTLV III: étude de la muqueuse rectale chez 16 patients.

Ultrastructural study of rectal mucosa was performed in 6 patients with AIDS related complex (ARC) and in 10 patients with AIDS. There were 16 men (mean age: 39.1 years): 8 homosexuals, 3 Haitians, 2 Africans and 3 IV drug abusers, all having significant titers of LAV antibodies. Two types of ultrastructural markers were observed: tubuloreticular structures (TRS) were found in endothelial cells, lymphocytes and macrophages in 1/6 ARC patients and 10/10 AIDS patients. TRS have already been described in various pathological situations and seem to be related to alpha-interferon, test tube and ring shaped forms (TRF) were observed in lymphocytes and macrophages in 0/6 ARC patients and 6/10 AIDS patients. TRF have been rarely reported previously. The 6 AIDS patients with TRF were 4 homosexuals and 2 Haitians. In two of three of these patients in whom repeated ultrastructural studies were performed, the same markers were found. All AIDS patients with TRF died within a mean time of ten months after the diagnosis had been established, whereas 1/4 AIDS patients without TRF died within seven months and the three others survived for more than 14 months. These data suggest that the association of TRS and TRF in the rectal mucosa could be specific of AIDS. In LAV/HTLV III retro-virus infection, this association is an aggravating factor.