Effects of diadenosine polyphosphates,ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) on membrane voltage (V _m) and membrane conductance (g _m) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure V _m and g _m. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the V _m values in MC of WKY (–42±1 mV, n=70) and SHR rats (–45±2 mV, n=99) as well as in the agonist-induced changes of V _m, all data were pooled. The V _m of all the cells was –44±1 mV (n=169) and g _m was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K⁺ and a non-selective cation conductance in resting MC whereas a Cl^– conductance or a Na⁺selective conductance could not be observed. Ap₃A, Ap₄A, Ap₅A, AP₆A and ATP each at a concentration of 5 mol/l, led to a significant depolarization of V _m by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap₄A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC ₅₀) as 6 mol/l (n=5–25), for ATP as 2 mol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap₄A 100 mol/l increased g _m significantly by 55±20% (n=16), 100 mol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize V _m (Ang II >ATP> Ap₄A>Ap₃A>Ap₅A>Ap₆A) by activation of a Cl^– conductance and a non-selective cation conductance, as do ATP or Ang II.     

Heterogeneous malignant non Hodgkin's lymphomas as a causative disorder in lethal midline granuloma

Summary The present report describes the results of a combined morphological, enzyme- and immunohistochemical analysis of nine cases of malignant non Hodgkin's lymphomas (NHL) clinically presenting as lethal midline granuloma. In a previous report written before antibodies directed against B and T lymphocytes were available, a histiocytic origin of such neoplasms had been suggested. A panel of antibodies reactive with most B cells (L26, MB1, KiB3) and a majority of T cells (MT1, UCHL1) was applied on paraffin sections of formalin fixed tissues as well as antibodies directed against leukocyte common antigen (LCA), myeloid/histiocyte antigen (MAC 387), lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, S-100 protein, prekeratin and immunoglobulin light chains. Enzyme histochemistry included tests for non-specific acid esterase, acid phosphatase, betaglucuronidase and chloroacetate esterase. As a result, five T, two B and two unclassified (malignant histiocytosis probable) NHL were identified, indicating distinct heterogeneity of NHL as causative disorders in lethal midline granuloma.     

Chemical Characterization of Macrophage Cytotoxicity Factor,Macrophage Migration Inhibitory Factor,T-Helper Cell-Replacing Factor and Colony-Stimulating Factor from Culture Supernatants of Concanavalin A-Stimulated Murine Spleen Cells

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatography of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF at pH 8.2 and 7.2, whereas MIF activity focused from pH 4.5 to 5.5. TRF activity was found in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.     

Protection against colibacillosis in neonatal piglets by immunization of dams with procholeragenoid.

Protection against colibacillosis in neonatal piglets was obtained by immunization of pregnant dams with procholeragenoid. Procholeragenoid is a stable high-molecular-weight aggregate of cholera toxin formed during the heating of cholera toxin. Procholeragenoid retained approximately 1% of the toxicity of native toxin as determined in the rabbit ileal loop and Y-1 adrenal cell assays and 5% of the activity in the rabbit skin assay. Immunization of pregnant dams with 50 micrograms of procholeragenoid 5 and 2 weeks before the expected delivery date elicited high titers of antitoxic immunoglobulin G and toxin-neutralizing antibody in both the colostrum and serum. In three independent field trials, immunization with procholeragenoid resulted in a substantial decrease in diarrhea (73% in controls versus 11% in immunized) and death (4.7% in controls versus 0.77% in immunized) in neonatal piglets. The protection rate in the immunized population was approximately 85% for both diarrhea and death. In the following gestation period, reimmunization of dams with a single dose of procholeragenoid (50 micrograms) 2 weeks before delivery elicited titers of antitoxic immunoglobulin G and toxin-neutralizing antibody comparable to those obtained during the primary immunization. The death rate in neonatal piglets (0.86%) was comparable to that seen after immunization during the first gestation period (0.77%). These results indicate that substantial protection of neonatal piglets against colibacillosis can be obtained by immunization of dams with procholeragenoid. Protection was found to be based solely on antitoxic immunity.     

Detection of methicillin-resistant Staphylococcus aureus and simultaneous confirmation by automated nucleic acid extraction and real-time PCR

A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.     

Small-scale field trial of a sensing device for detecting peridomestic populations of Triatoma infestans (Hemiptera: Reduviidae) in northwestern Argentina

Two prototypes of sensing devices for detecting peridomestic populations of Triatoma infestans Klug were tested in paired trials with bamboo canes in Amamá and nearby rural villages under triatomine surveillance. In infested peridomestic structures housing domestic animals, 1-2 pairs of numbered devices were placed per test site, left for 3-9 nights, and inspected for evidence of infestation. Prototype A was a black plastic cylinder 19 cm high and 10 cm diameter, with a screw cap on the top, 2 openings in the bottom, and a removable central structure made of resistant plastic coated with leather. Prototype B had square leather pieces rolled into cylinders instead of the central structure. Prototype A was significantly more sensitive than the bamboo cane with pleated paper inside in 13 test sites in which 20 pairs were tried. In a smaller series involving 7 pairs, prototype B also detected infestations more frequently than the cane. Triatomine feces were the signs most frequently recorded by both prototypes, whereas the bamboo canes recorded no feces. Ten T. infestans and 1 Triatoma guasayana Wygodzinsky & Avalos were collected from the prototypes placed on the ground or walls, not beneath the thatched roofs of the animal shelters, whereas only 3 T. infestans were collected from the canes. This study describes an effective sensing device for detecting T. infestans populations in outdoor animal shelters and provides quantitative field data on its performance.     

Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells

Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (~10- fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, am ongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were ~10-fold and ~15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immuno-reactive PAI-1 was considerably elevated (. 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only ~2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (~10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associ-ated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer. © Rapid Science 1998     

Comparison of screening methods to identify methicillin-resistantStaphylococcus aureus

Screening methods to identify methicillin-resistantStaphylococcus aureus (MRSA) were compared using 96 isolates representing 17 distinct clones. The sensitivity of four commercial agglutination tests was determined in comparison to the tube coagulation test, and the results related to the presence of the coagulase gene. The broth screening test, agar dilution test and disc diffusion test were carried out, and the results related to the presence of themecA gene. Mannitol salt agar and Iso-Sensitest agar with varying salt supplements were used. All agglutination tests had high rates of detection ofStaphylococcus aureus (95.8–99.0%). Resistance in mecA gene-positiveStaphylococcus aureus isolates was correctly detected by the oxacillin broth test, the agar dilution test and the disc diffusion test on mannitol salt agar, whereas on Iso-Sensitest agar detection rates were lower (between 68.5% and 94.4%, depending on the salt supplement). Incubation of the Iso-Sensitest plates for 48 hours significantly improved the rate of detection of resistance, but increased the major error rate up to 71.4%.MecA genepositiveStaphylococcus aureus isolates not detected by the disc diffusion test on Iso-Sensitest agar had significantly lower oxacillin minimal inhibitory concentration values and were significantly less resistant to a variety of antibiotics. Thus, mannitol salt agar might be a suitable medium for use in the disc diffusion and agar dilution test to detect resistance to oxacillin inStaphylococcus aureus.     

The role of accessory cells in polyclonal T cell activation II. Induction of interleukin 2 responsiveness requires cell-cell contact

It has previously been reported (Hünig, T. et al., Eur. J. Immunol. 1983. 13:1) that highly purified peripheral T cells do not respond to concanavalin A (Con A) even in the presence of Con A-induced spleen cell supernatant as a source of interleukin 2 (IL 2). In the present report, the hypothesis was tested whether this unresponsiveness correlates with the observed inability of Con A to mediate cell-cell contact between highly purified T cells. It was found that T cells, pretreated with neuraminidase to reduce their net negative charge, were both aggregated and rendered IL 2-reactive by Con A. In addition, leukoagglutinin (LA) was found to be able to both agglutinate untreated T cells and to make them IL 2-reactive. Neuraminidase treatment reduced the concentration of LA required to mediate both effects. Neuraminidase treatment did not alter the overall Con A-binding capacity of T cells, nor did it induce reactivity to IL 2 in the absence of lectin. Furthermore, irradiated, neuraminidase-treated T cells served as accessory cells (AC) in the induction of responsiveness to IL 2, but not for production of IL2, which depends on Ia+ AC. Finally, Lyt-2- neuraminidase-treated peripheral T cells responded in the same fashion as whole T cell populations, indicating that at least some T cells of the helper phenotype need not interact with Ia+AC for the induction of IL 2 responsiveness.