Effects of tumor necrosis factor-alpha on insulin action in cultured human muscle cells.

Reported discrepancies in the effects of tumor necrosis factor (TNF)-alpha in modulating insulin sensitivity of cultured cells may relate both to cell types studied and to the time course of exposure to the cytokine. Additionally, the relationship of effects on glucose metabolism to changes in the insulin signaling pathway cannot be assumed. For in vitro study, the cell type most relevant to insulin resistance in humans is the cultured human muscle cell. In the present study, TNF brought about no change in the rate of glycogen synthesis in cultured human muscle cells unless present during differentiation. The presence of TNF (5 ng/ml) during the process of differentiation of myoblasts into mature myotubes diminished the response of glycogen synthesis to acute insulin stimulation. This finding was associated with an impairment of differentiation-dependent increases in total cellular glycogen synthase (GS) activity. Under the same conditions of TNF exposure, there was no effect on the response to acute insulin stimulation of the fractional activity of GS. Similarly, there was no effect on the insulin stimulation of protein kinase B (PKB) and inhibition of glycogen synthase kinase 3 (GSK-3). Acute insulin stimulation brought about a 4.08 +/- 0.44-fold stimulation of activity of PKB in the absence of TNF, with 4.81 +/- 0.70-fold stimulation in cells exposed to TNF. GSK-3 activity decreased to 74.0 +/- 5.8% of basal after insulin stimulation without TNF and 78.3 +/- 5.0% after TNF exposure. However, differentiation of myocytes, as defined by an increase in the acetylcholine receptor, myogenin, and mature creatine kinase isoform expression, was impaired in TNF-treated cells. These studies demonstrate that TNF, if present during differentiation, decreases insulin-stimulated rates of storage of glucose as glycogen and total GS activity but does not downregulate the insulin-signaling system to GS. More generally, TNF also inhibits differentiation of human muscle cells in culture.

Prevalence of prothrombin G20210A, factor V G1691A (Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T in seven different populations determined by multiplex allele-specific PCR.

Individuals belonging to six racial groups (African American, Asian Indian, Caucasian, Hispanic, Korean, Native American), and a seventh group comprised of referred patients with thrombosis were genotyped for the prothrombin G20210A mutation, the factor V G1691A (Leiden) mutation, and the methylenetetrahydrofolate reductase (MTHFR) C677T mutation by multiplexed allele-specific PCR. The prothrombin 20210A and factor V 1691A allele frequencies in the thrombosis patients, 3.2% and 9.5%, were significantly higher than those in the random Caucasians, 1.3% and 1.8%, (p = 0.043 and p <0.001, respectively). The relative risk of venous thrombosis was determined to be 2.4-fold for carriers of the prothrombin 20210A allele (odds ratio = 2.54; 95% confidence interval = 0.94, 6.82) and 4.5-fold for carriers of the factor V 1691A allele (odds ratio = 5.06; 95% confidence interval = 2.25, 11.36). Among the seven populations, significant differences were observed in the MTHFR 677T allele distribution, however this mutation was not determined to be a risk factor for venous thrombosis in the patient group studied, either alone or in combination with the prothrombin 20210A and/or the factor V 1691A allele(s).

Anti-F(ab')2 antibody in HIV type 1 infection: relationship to hypergammaglobulinemia and to antibody specific to the V3 loop region of glycoprotein 120.

As HIV infection and autoimmune disease share certain similarities, it has been suggested that HIV may disrupt control of humoral immunity by the antiidiotype network, and that this may be evident as increased IgG antibody to F(ab')2. When anti-F(ab')2 was quantified by ELISA in sera of randomly chosen HIV-infected versus uninfected donors, some HIV-infected sera did contain increased anti-F(ab')2, resulting in a median amount twofold higher than in uninfected sera. Moreover, when data were grouped by blood CD4 lymphocyte count, anti-F(ab')2 in HIV+ groups appeared to rise as CD4 lymphocytes declined. However, increased anti-F(ab')2 mirrored the elevation in serum IgG closely, and normalization of anti-F(ab')2 to serum IgG concentration equalized the groups so that no relationship to CD4 lymphocytes remained. Hypergammaglobulinemia is therefore strongly implicated as a cause of variation in anti-F(ab')2. After dissociation of immune complexes, anti-F(ab')2 activity per microgram of monomeric IgG was slightly increased over normal only in the HIV-infected group with fewest CD4 lymphocytes, without statistical significance. In contrast, the proportion of IgG antibody to the V3-neutralizing determinant in HIV-1 decreased significantly as disease advanced. The same was true for 12 HIV+ individuals studied longitudinally for 500-1300 days. The data suggest that measuring serum anti-F(ab')2 is misleading when immune complexes are present: apparent increases as disease progresses are due to increased IgG and, possibly, to related technical artifacts. During HIV infection, the proportion of antiidiotypic IgG in fact remains unaltered or falls, making this an unlikely cause of suppressed humoral immunity to HIV-1.

A(1,2)BO(1,2) genotyping by multiplexed allele-specific PCR.

The ABO blood group is the most clinically important human alloantigen system in transfusion medicine. The system involves three antigens A, B and H. H antigen is converted to either A or B by the activity of alpha1-->3-N-acetyl-galactosaminyl transferase (A transferase) or alpha1-->3 galactosyl transferase (B transferase). The O phenotype is the result of an inactive glycosyltransferase, which is unable to glycosylate the H antigen. The immunological properties of the ABO system were identified at the turn of the century; however, the genetic basis of the ABO system has only recently been characterized. This has enabled the development of a number of molecular ABO typing methods. Described here is a two-reaction multiplex allele-specific PCR (ASPCR) genotyping assay for the A1, A2, B, O1 and O2 subtypes. 11 different allele-specific oligonucleotide primers were selected to detect the presence or absence of the O1 associated G--> (-) deletion at base 261, the O2 associated G --> A substitution at base 802, the B associated G --> A substitution at base 803, and finally the A2 associated C --> (-) deletion at base 1059. A total of 122 peripheral blood samples were genotyped and serologically forward and reverse typed. A concordance rate of 98.4% (120/122 samples) was observed between the actual genotype and the serologically-based predicted genotype. These results indicate that this assay provides a rapid, accurate, and simple method for A(1,2)BO(1,2) genotyping that serves as a useful supplement to standard serological ABO typing.

The thiazolidinedione insulin sensitiser, BRL 49653, increases the expression of PPAR-gamma and aP2 in adipose tissue of high-fat-fed rats.

The effects of the thiazolidinedione insulin sensitiser BRL 49653 on plasma leptin concentrations and on epididymal fat OB, PPAR-gamma and aP2 mRNA expression were examined in high-fat-fed and high-carbohydrate-fed adult Wistar rats. Diets were given for 4 weeks, with BRL 49653 (10 micromol/kg/day) administered by oral gavage for the last 4 days. Treatment with BRL 49653 reduced plasma leptin concentrations in high-fat-fed rats from 2.34 +/- 0.19 (n=9) to 1.42 +/- 0.09 (n=9) ng/ml (p<0.001). Plasma leptin was unaffected by BRL 49653 in the high-carbohydrate-fed rats. There was no difference in OB mRNA expression between high-fat-fed and high-carbohydrate-fed rats, with or without treatment. PPAR-gamma and aP2 mRNA expression were significantly increased in the high-fat-fed rats treated with BRL 49653 (p < 0.01 and p < 0.001 respectively), but not in carbohydrate-fed rats.

Identification of rhesus macaques with spontaneous endometriosis.

Rhesus macaques (Macaca mulatta) with endometriosis were identified using reproductive histories, serum levels CA-125, pelvic ultrasonography, laparoscopy, and histopathology. All animals were evaluated from a large breeding colony and had a history of infertility and/ or spontaneous abortions. Laparoscopy and ultrasonography were performed on 40 macaques: 27 macaques from the breeding colony with elevated CA-125 levels, ten macaques from the breeding colony with normal or low serum CA-125 levels, and three macaques from another facility with previously diagnosed spontaneous endometriosis. Clinical endometriosis was diagnosed by laparoscopy in 16/37 (43%) macaques from the breeding colony and was confirmed by histologic examination in all animals biopsied. The disease was classified as minimal (40%), mild (25%), moderate (10%), or severe (25%). The most common sites of endometriosis were the serosal surface of the uterus (75%) and the posterior cul-de-sac (75%). In this study, CA-125 levels were useful in identifying animals from the breeding colony with endometriosis. The rhesus macaque provides a valuable animal model to study endometriosis and potentially to assess efficacy of therapeutic agents for this disease condition.

Measurement of gene-specific transcription by nuclease protection of pulse-labeled nuclear RNA.

A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.

Gene-specific modulation of RNA synthesis and degradation by extremely low frequency electromagnetic fields.

Pulse-labeling studies from our laboratory and others have shown that extremely low frequency (ELF) electromagnetic fields can produce a transient increase in gene transcription. In this study, the synthesis, degradation and processing, and steady state levels of specific RNA species during exposure to ELF radiation were determined in human leukemia HL-60 cells. The overall steady state RNA levels, assessed by continuous and equilibrium labeling with 3H-uridine, were not affected by ELF exposure. Northern blot analysis using probes specific for c-myc, beta-actin, and 45S ribosomal RNA gene products revealed that ELF did not alter the steady state levels of these RNAs. Examination of gene-specific transcription by a novel nuclease protection assay revealed that while ELF did not substantially alter the transcription rates for c-myc and beta-actin, transcription of the 45S ribosomal RNA gene was increased by 40-50%. To explain the observed increase in the synthesis of 45S ribosomal RNA without an associated increase in its steady state level, the degradation and processing of the ribosomal gene transcript in the presence and absence of an ELF field were followed by pulse-chase 3H-uridine labeling. This revealed that ELF radiation accelerated both the processing and degradation of the ribosomal RNA transcript. During ELF exposure, the half-life of the 45S ribosomal RNA was decreased from 115 min. to 85 min. These results show that ELF can selectively affect RNA levels by modulating either the transcription rate and/or RNA post-transcriptional processing and turnover.

Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys.

Simian hemorrhagic fever (SHF) virus and a new strain of Ebola virus were isolated concurrently in recently imported cynomolgus monkeys (Macaca fascicularis) being maintained in a quarantine facility. Ebola virus had never been isolated in the U.S. previously and was presumed to be highly pathogenic for humans. A chronology of events including measures taken to address the public health concerns is presented. The clinicopathologic features of the disease were abrupt anorexia, splenomegaly, marked elevations of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, with less prominent elevations of blood urea nitrogen, creatinine, and other serum chemistry parameters. Histologically, fibrin deposition, hemorrhage, and necrosis of lymphoid cells and reticular mononuclear phagocytes were present in the spleens of SHF and of Ebola virus-infected animals. Intravascular fibrin thrombi and hemorrhage were also present in the renal medulla and multifocally in the gastrointestinal tract. Necrosis of lymphoid and epithelial cells was occasionally noted in the gastrointestinal tract. The histopathologic findings considered specific for Ebola virus infection include hepatocellular necrosis, necrosis of the zona glomerulosa of the adrenal cortex, and interstitial pneumonia, all of which were generally associated with the presence of 1 to 4 mu intracytoplasmic amphophilic inclusion bodies. The disease spread within rooms despite discontinuation of all direct contact with animals, and droplet or aerosol transmission was suspected. Antibody to Ebola virus developed in animal handlers but no clinical disease was noted, suggesting a less virulent strain of virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Panhypogammaglobulinaemia caused by gold therapy.

Gold compounds have been successfully used for over 50 years in the treatment of rheumatoid arthritis, but their mechanism of action is unknown. The main disadvantage is the frequent occurrence of side effects which often necessitate discontinuation of therapy. Recently, there have been reports of reduction in immunoglobulin levels in patients on gold treatment; we report a further case of hypoglobulinaemia associated with gold therapy and recommend that immunoglobulins be monitored prior to and during treatment.

Evaluation of a radioimmunoassay for neuron specific enolase in small cell lung cancer.

A radioimmunoassay for neuron specific enolase (NSE), a marker of neuroendocrine differentiation, has been evaluated in small cell lung cancer (SCLC). In untreated patients 25/38 (68%) with localized SCLC had raised blood levels of NSE (greater than 13 ng ml-1), in extensive disease 34/39 (87%) patients had raised NSE levels. In patients with non-small cell lung cancer (NSCLC) the serum levels were raised in 16/94 (17%). In extensive tumours of non-pulmonary origin NSE levels were increased in 24/116 (20%) patients. Longitudinal studies indicated a good correlation between the response to chemotherapy and fall of NSE levels. Tumour progression was accompanied by a rising NSE in 25/29 patients, with doubling times of 7-90 days. In patients with progression with a normal NSE the recurrence was a NSCLC. Cerebral metastases occurring as the only recurrence during clinical complete remission were not accompanied by a rise of NSE. Serum NSE levels provides a valuable monitor for SCLC during and after chemotherapy.