Three-dimensional in vitro model of adipogenesis: comparison of culture conditions

In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat. Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis. Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated. Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates. At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions. Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs. Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.     

Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

An outbreak of aseptic meningitis with a rubella-like rash probably caused by ECHO virus type 4

Summary An outbreak of aseptic meningitis in association with ECHO virus type 4 is described. This virus was isolated in 8 out of 12 cases with this syndrome from stool specimens and in two cases also from spinal fluids and furthermore from healthy family contacts. Neutralizing antibodies in the patients sera could not be demonstrated with the conventional tube tests while complement fixing antibodies with increasing titer against ECHO-4 occurred. There is an extensive heterotypic complement fixing antibody response in patients sera within the ECHO virus group between this group and the Coxsackie or the poliomyelitis group as well as beetwen the Coxsackie and the poliomyelitis groups.The choice of tissue for isolation of enteroviruses is discussed. Trypsinized human embryonic kidney seems to provide a suitable medium for this group of viruses.A rubella-like exanthem was seen in about two thirds of the virus positive patients. Some cases had diphasic course.     

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.     

Comparison of GB virus C, HIV, and HCV infection markers in hemophiliacs exposed to non-inactivated or inactivated factor concentrates.

BACKGROUND: Until the mandatory introduction of viral inactivation techniques of blood plasma products in the early 1980s many recipients of these products were infected with various viral pathogens. OBJECTIVES: To determine the rate of transmission of GB virus C/hepatitis G virus (GBV-C/HGV) HCV, and HIV through non-virus-inactivated clotting factor concentrates in hemophiliacs, as well as the relation between amount of administered clotting factor and risk for GBV-C/HGV infection. STUDY DESIGN: In this cross-sectional study, we determined retrospectively the rates of infection markers for GBV-C/HGV, HCV, and HIV in a German cohort of hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor concentrates (group A) and in a second group of hemophiliacs who were treated exclusively with virus-inactivated clotting factor (group B). The presence of anti-virus antibodies was determined by ELISA. Viral RNA was detected by RT-PCR. Markers for viral infections were compared to amounts of administered non-virus-inactivated clotting factor. RESULTS: Among hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor the prevalence for GBV-C/HGV, HCV, and HIV was 40.3%, 98.6%, and 56.3%, respectively. In contrast to HIV, the rate of GBV-C/HGV infections did not increase with increasing amounts of consumed non-inactivated clotting factor. Even in the subgroup of heavily treated hemophiliacs the rate of GBV-C/HGV infection markers did not exceed 45%. CONCLUSIONS: The amount of non-virus-inactivated clotting factor is not predictive for the risk of GBV-C/HGV infection in hemophiliacs. Despite repeated parenteral exposure more than 55% of hemophiliacs were not infected with GBV-C/HGV. Our findings indicate a high frequency of host factors preventing parenteral transmission of GBV-C/HGV.     

CD1d-restricted natural killer T cells are potent targets for human immunodeficiency virus infection

Invariant human natural killer T cells (NKT) express a restricted T-cell receptor (TCR) Valpha24Vbeta11 repertoire. These cells share both phenotypic and functional similarities between NK and T cells. Given the emerging role of NKT cells as critical cells in bridging the gap between innate and adaptive immunity, we examined their susceptibility to productive human immunodeficiency virus (HIV) infection by T-tropic, M-tropic, and primary isolates of HIV. We generated three human NKT cell clones (CA5, CA29, and CA31). Phenotypic characterization of these Valpha24+ Vbeta11+ clones indicated that they were predominately positive for CD4, CD161, HLA-DR, CD38, CD45RO, and CD95 expression. The NKT cell clones expressed significantly more surface CCR5 molecules/cell and lower CXCR4 molecules/cell than phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMC). Consistent with the surface expression of CCR5 and CXCR4, the NKT clones were also selectively susceptible to HIV M-tropic, T-tropic, and primary isolate infection, as evaluated by both HIV p24 enzyme-linked immunosorbent assay and intracellular staining of HIV proteins. The amount of p24 production was dependent on the NKT clone studied and the HIV strain used. Clones CA29 and CA31 were also susceptible to HIV IIIB infection. The virions produced by these clones were able to productively infect PHA-stimulated PBMCs with the same kinetics as for primary infection of CD4+ blast. Collectively, this data demonstrates that NKT cells can be a target for productive HIV infection but with a lag in the time to peak p24 production.     

Semiautomated quantification of hepatitis B virus DNA in a routine diagnostic laboratory

The Cobas Amplicor HBV Monitor test for quantitative determination of hepatitis B virus (HBV) DNA in serum has recently been introduced. To evaluate the performance of this assay in a routine diagnostic laboratory, reproducibility of results was determined with the First European Union Concerted Action HBV Proficiency Panel and the Accurun 325 HBV DNA Positive Control, Series 300. Results for 270 routine serum samples were additionally evaluated. To avoid the retesting of a large number of samples due to titers exceeding the upper limit for the linear range of the assay, sera of patients with chronic hepatitis B (CHB) were diluted prior to the assay to 10(-4) in normal human plasma, which is included in the assay. The mean coefficient of variation was 22.9% for all input HBV DNAs. Of 270 routine serum samples, 182 (150 sera from transplant donors and 32 sera from patients who had recovered from CHB) tested negative. Eighty-six sera were found to be HBV DNA positive; in six sera, HBV DNA levels were found to exceed the upper limit for the linear range of the assay and had to be retested. In the remaining two sera, inhibition occurred. The semiautomated Cobas Amplicor HBV Monitor test showed sufficient reproducibility and helped in avoiding human error. The relatively narrow linear range of detection is a limitation of the new assay.     

Non-A, non-B hepatitis in persistent carriers of hepatitis B virus

There are reports in the literature that infection with hepatitis A virus in hepatitis B carriers can result in resolution of the carrier state. In an attempt to induce clearance of the carrier state of hepatitis B virus in two persistently infected chimpanzees, the chimpanzees were infused with documented non-A, non-B infectious material. Biochemical and histopathological evidence of hepatitis was accompanied by the unique abnormalities of endoplasmic reticulum associated with non-A, non-B hepatitis in the chimpanzees. Elevation of alanine aminotransferase was accompanied by fourfold reduction in one chimpanzee and sixfold reduction in the other in the plasma levels of HBV-associated DNA polymerase activity and simultaneously by twofold reduction in the concentration of hepatitis B surface antigen in both chimpanzees. A mediator may account for these changes in markers of hepatitis B virus infection, and this mechanism may also explain the occurrence of spontaneous regression in some persistently infected carriers. The significance of transient red cell anaemia in non-A, non-B hepatitis, which was observed in one of the chimpanzees, is yet to be established.