CTLA4 Autoimmunity-Associated Genotype Contributes to Severe Pulmonary Tuberculosis in an African Population

The gene of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4), a negative regulator of T lymphocytes, contains a single-nucleotide polymorphism (SNP) at position +6230A->G (ct60A->G), which has been found associated with several autoimmune diseases and appears to reduce T-cell inhibitory activity. In Ghana, West Africa, we compared the frequencies of CTLA4 +6230 A/G and 6 haplotype-tagging SNPs in 2010 smear-positive, HIV-negative patients with pulmonary tuberculosis (TB) and 2346 controls matched for age, gender and ethnicity. We found no difference in allele frequencies between cases and controls. However, +6230A and a distinct CTLA4 haplotype and a diplotype comprising the +6230A allele were significantly less frequent among cases with large opacities in chest radiographs compared to those with small ones (P_{corrected [cor]} = 0.002, P_cor = 0.00045, P = 0.0005, respectively). This finding suggests that an increased T-cell activity associated with the CTLA4 +6230G allele contributes to pathology rather than to protection in pulmonary TB.     

Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.     

Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

The Sabin live poliovirus vaccination trials in the USSR, 1959.

Widespread use of the Sabin live attenuated poliovirus vaccine has had tremendous impact on the disease worldwide, virtually eliminating it from a number of countries, including the United States. Early proof of its safety and effectiveness was presented in 1959 by Russian investigators, who had staged massive trials in the USSR, involving millions of children. Their positive results were at first viewed in the United States and elsewhere with some skepticism, but the World Health Organization favored proceeding with large-scale trials, and responded to the claims made by Russian scientists by sending a representative to the USSR to review in detail the design and execution of the vaccine programs and the reliability of their results. The report that followed was a positive endorsement of the findings and contributed to the acceptance of the Sabin vaccine in the United States, where it has been the polio vaccine of choice since the mid-1960s.     

DNA recovery and direct detection of Tn5 sequences from soil

Specific Tn5 sequences inserted in the genome of Enterobacter agglomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed.     

Haemostatic alterations in malaria correlate to parasitaemia

Fibrin(ogen) degradation products, platelet counts, antithrombin III, and the components of the Factor VIII complex were studied in a total of 80 patients with Plasmodium falciparum, Plasmodium vivax or Plasmodium ovale infections. The haemostatic findings were correlated to the numbers of parasitized erythrocytes and to each other. The results indicate that haemostatic changes in malaria correlate with the degree of parasitaemia. Evidence for moderate hyperfibrinolysis was found in patients with high P. falciparum parasitaemias only. Thrombocytopenia closely corresponded to parasitaemia and to von Willebrand factor levels, but appeared not to be linked to a consumption of coagulation factors. It was concluded that thrombocytopenia in malaria is not indicative of disseminated intravascular coagulation (DIC) but may relate to endothelial damage.     

Isolation of rabbit C3, Factor B, and Factor H and comparison of their properties with those of the human analog

The rabbit complement components C3, Factor B, and Factor H were isolated and characterized and were compared to the corresponding proteins of human serum. Chromatographic behavior, chemical properties, and functional interactions show great similarities between the components in both species. By SDS polyacrylamide gel electrophoresis, the m.w. were estimated to be 195,000 for C3, 86,000 for Factor B, and 155,000 for Factor H. The amino acid compositions of the rabbit proteins resembled those of the human analog. The total carbohydrate content of rabbit C3 and Factor H was approximately one-half that of the human proteins. In addition, a qualitative difference in the carbohydrate moieties of the C3 proteins was observed. The serum concentration of the rabbit proteins was markedly lower than that of the human proteins. The rabbit C3b,Bb enzyme resembled the human analog with respect to half-life, control by Factor H, and stabilization by nickel ions.     

Mucopolysaccharidosis-like alterations in cardiac valves of rats treated with tilorone

The purpose of this study was to examine whether the aortic and mitral valves of rats are involved in the mucopolysaccharidosis-like disorder induced by tilorone. Rats were treated with large doses of the drug for periods of 1-21 weeks. After chronic drug treatment the leaflets of both heart valves were thickened and opaque. In all treated animals the spongiosa layer of the stroma was crowded with vacuolated cells; the fibrosa layer was altered only after prolonged treatment. Ultrastructurally, the vacuolated cells of the spongiosa could be identified as histiocytes and fibroblasts, the former being the most susceptible cell type. The fibroblasts of the fibrosa represented the least sensitive cell type. The histochemical results showed that the clear cytoplasmic vacuoles in the spongiosa cells were due to lysosomal storage of polyanionic material with staining characteristics similar to cartilage matrix. After discontinuation of drug treatment the alterations persisted for several weeks. The present study shows that heart valves are involved in the mucopolysaccharidosis-like disorder induced by tilorone. The molecular pathomechanism of the disorder and the exact identification of the storage material must await further analysis.