Evaluation of a western blot system (DAVIH-BLOT) for the confirmation of HIV-1 antibodies]

Matrix metalloproteinases (MMPs), a sort of important enzymes involved in extracellular matrix metabolism, play critical roles in the process of tissues remodeling, wound healing and metastasis of tumors. Dot blot and in situ hybridization were used in this study to detect the expression and localization of MMP-9, an important proteolytic enzyme implicated in bone resorption in bone tissues. The results showed that the level of MMP-9 mRNA expression in osteoporotic bone tissues was significantly higher than that in normal control group and the cell types that expressed MMP-9 mRNA included mono- and multi-nuclear osteoclasts and some lining cells on the surface of bone matrix. It was suggested that MMP-9 play a key role in the development of bone loss in osteoporosis.     

Staphylococcal enterotoxin A-induced fever is associated with increased circulating levels of cytokines in rabbits

Rabbits were injected intravenously with 10 to 100 ng of staphylococcal enterotoxin A (SEA) per kg, and colonic temperatures were monitored. The febrile responses were compared with circulating levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2, and IL-6 just before the injection of SEA. Both colonic temperatures and circulating levels of IFN, TNF, and IL-2 started to rise at 1 to 2 h and reached their peak levels at 3 to 5 h after SEA injection. Both the fever and the increased circulating levels of IFN, TNF, and IL-2 produced by SEA were decreased by pretreatment with indomethacin (a cyclo-oxygenase inhibitor) (15 mg/kg, intraperitoneally), anisomycin (a protein synthesis inhibitor) (15 mg/kg, subcutaneously), or dexamethasone (an effective anti-inflammatory and immunosuppressive agent) (4 mg/kg, intravenously) in rabbits. Rabbits were injected intravenously with 30 ng of SEA per kg on four consecutive days, and colonic temperatures were monitored. Compared to rabbits that received the single injection of SEA, rabbits that received four consecutive injections of SEA showed a lesser increase in circulating levels of IFN, TNF, and IL-2 as well as colonic temperatures in response to an intravenous dose of SEA (30 ng/kg). The data suggest that the prevention of the febrile response elicited by SEA by indomethacin, anisomycin, or dexamethasone is due to prevention by these compounds of the increase in the circulating levels of IFN, TNF, and IL-2. The pyrogenic hyporesponsiveness to repeated injection of SEA is associated with decreased production of these circulating cytokines.     

Tamoxifen downregulates TGF-beta production in keloid fibroblasts

BACKGROUND: It has recently been suggested that primary lactase deficiency might have been selected for by malaria, as occurred for beta-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. However, recently we have found that the prevalence of primary lactase deficiency in the area of Sassari (Northern Sardinia), where, in the past, there was intermediate malarial endemicity, is comparable to that observed in the adult population from other areas of Southern Italy where malaria was less endemic. AIMS: To address the problem further, we have determined the prevalence of primary lactase deficiency, glucose 6-phosphate dehydrogenase deficiency deficiency and beta-thalassaemia trait in the populations of three Sardinian villages which differ in altitude above sea-level, socioeconomic features, history of endemic malaria and prevalence of b-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. SUBJECTS: We tested 138 adult males: 53 were from Fonni (a non-malarial mountain village, with a strong pastoral tradition), 38 from Lodé (a village with a similar pastoral tradition, but high malarial endemicity in the past) and 47 from Terralba (a lowland fishing village with an agricultural tradition and heavy malarial morbidity and mortality). METHODS: A blood sample was obtained in all subjects for determination of HbA2 and glucose 6-phosphate dehydrogenase activity. Lactase deficiency was assessed by measuring breath hydrogen production after oral administration of lactose (50 g), by gas-chromatography. RESULTS: The frequencies of glucose 6-phosphate dehydrogenase deficiency and of beta-thalassaemia trait in the non-malarial village of Fonni were strikingly low, compared to frequencies found in the two villages (Terralba and Lodé) with a very high past malarial morbidity. In contrast, there was no significant difference in the prevalence of lactase deficiency in the three groups of subjects from the three villages. CONCLUSIONS: These data obtained in Northern Sardinia do not support the hypothesis of a selection of primary lactase deficiency by malaria. For definitive conclusions, however, the malaria hypothesis should be tested in other parts of the world.     

Biosynthesis of growth hormone-releasing factor by fetal rat cerebrocortical and hypothalamic cells

The biosynthesis of growth hormone-releasing factor (GRF) by cerebrocortical tissue is controversial. Although several reports have indicated its presence in certain rat cortical areas and in cultured rat hypothalamic cells, no data exist demonstrating its biosynthesis in these areas. In this study, we have investigated the capacity of fetal rat cerebrocortical and hypothalamic cells in culture for synthesizing GRF. Fetal cerebrocortical and hypothalamic cells were exposed to [3H]Arg for 48 h. Medium and cell extracts were processed and [3H]Arg-IR-rGRF was isolated by affinity chromatography and characterized by HPLC. Intracellular [3H]Arg-IR-rGRF from both hypothalamic and cerebrocortical cells exhibited four major peaks, one of them coeluting with synthetic rGRF. In cerebrocortical cultures, newly synthesized and released [3H]Arg-IR-rGRF showed a similar pattern to the cell content. However, in media from hypothalamic cells, higher hydrophobicity molecular forms were absent. The data demonstrated that fetal cerebrocortical and hypothalamic cells in primary culture synthesize GRF with similar posttranslational processing, but with different molecular patterns of secretion.     

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.     

Modulation of the p27kip1 cyclin-dependent kinase inhibitor expression during IL-4-mediated human B cell activation

IL-4 activates resting B cells and, in conjunction with cosignals such as anti-IgM (anti-mu) Ab or CD40 ligand, modulates progression of B cells through the cell cycle, leading to proliferation. In this study, we show that the mitogenic combination of IL-4 and anti-mu Ab triggered induction of cyclin D3 and up-regulated cyclin-dependent kinase (cdk) 6 expression, whereas such regulation was not observed in B cells activated by IL-4 or anti-mu Ab alone. Furthermore, cyclin D3 immunoprecipitated fron as associated with cdk6, and the cyclin D3/cdk6 complex was able to phosphorylate recombinant retinoblastoma protein in vitro. In addition, B cells activated with either IL-4 or 1L-13 alone expressed a higher amount of p27kip1 (p27) cdk inhibitor than nonstimulated cells. In contrast, p27 expression was decreased when cells were activated with mitogenic combinations of IL-4 and anti-mu Ab or anti-CD40 mAb. We also observed that the IL-4-mediated inhibition of the proliferation of anti-mu/IL-2- or anti-mu/phorbol 12,13-dibutyrate-activated human leukemic B cells was associated with the maintenance of large amounts of p27 in these cells. These data suggest that IL-4 controls B cell proliferation by action during at least two steps of the regulation of the cell cycle, cyclin D3/cdk6 complex regulation and p27 inhibitor expression.