Short-circuit current and ionic fluxes in the isolated colonic mucosa of Bufo arenarum

1. The unidirectional fluxes of ²²Na, ³⁶Cl and [¹⁴C]bicarbonate ions were measured in paired portions of the isolated and short-circuited colonic mucosa of Bufo arenarum, separated from its muscular layer. Pharmacological effects as well as effects of changes in the composition of the nutrient solutions on the electrical parameters of membrane activity (potential difference, short-circuit current and total membrane resistance) are described.2. The net fluxes of both Cl and bicarbonate ions were not significantly different from zero in the absence of electrochemical gradients across the membrane.3. The net Na flux from mucosa to serosa represented a variable proportion of the short-circuit current ranging from 62 to 100%.4. The proportion of membranes with high discrepancies between net Na flux and short-circuit current decreased with the duration of captivity of the toads.5. When Na was entirely replaced by choline in the mucosal bathing solution, the short-circuit current dropped by a variable amount within the range of 64 to 98% of its control values in different membranes. This effect was completely reversible. Similar changes in the serosal solution had no effect.6. The short-circuit current and potential difference were very sensitive to the serosal concentration of bicarbonate ions. In different membranes, 60-100% of the short-circuit current was reversibly abolished by bathing the serosal surface with a bicarbonate-free solution. The mucosal bicarbonate level had no effect on either the potential difference or the short-circuit current. 5 mM bicarbonate in the serosal solution restored at least 50% of the short-circuit control value and full recovery was attained by concentrations near 30 mM bicarbonate.7. Anoxia brought the potential difference and short-circuit current reversibly down to zero in about 50 min.8. Ouabain reduced the short-circuit current up to 80% in about 40 min when present in the serosal solution at a concentration of 10^-4 M. At this or lower concentrations the ouabain effect was reversible. Above this level ouabain produced 100% inhibition in 3-4 hr, but this was no longer reversible. Ouabain had no effect on the short-circuit current either when applied to the mucosal surface or in the absence of Na from the mucosal solution.9. Diamox produced a variable inhibition of the short-circuit current of up to 30% only at concentrations above 10 mM.10. Possible mechanisms are discussed for the appearance of the non-Na component of the short-circuit current. A theory concerning its nature is proposed.     

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.     

Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.     

Glomerulonephritis induced by pre-formed immune complexes containing monoclonal antibodies of defined affinity and isotype.

The work presented here represents the first report of the induction of experimental immune complex (IC) disease in mice using monoclonal antibodies (MoAb) derived from somatic cell hybridization. IC were formed using two antigens of either high (DNP19BSA) or low (DNP4BSA) epitope density and five MoAb (four IgGl with varying affinities for the dinitrophenol hapten and one IgM with a similar affinity to that of the lowest affinity IgGl). Circulating levels and sizes of IC were dependent on the affinity of the antibody component of the complex. When antigen of high epitope density was used, the glomerular localization of injected IC was diffuse mesangial for the IgM antibody, focal mesangial for the highest affinity IgG and diffuse, and predominantly capillary for the low affinity IgG antibodies. Subepithelial electron dense deposits were observed only with IC made with the low affinity IgG antibodies. When IC containing antigen of a lower epitope density were injected, localization was only observed with IC made near equivalence. Deposition of these IC was less prominent than that found when IC containing antigen of higher epitope density were injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.     

Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.     

Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

Responses against complex antigens in various models of CD4 T-cell deficiency

The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4^−/−) and major histocompatibility complex (MHC) class II knockout (class II^−/−) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II^−/− and CD4^−/− mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II^−/− and CD4^−/− mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4^−/− mice produce IgG against both simple model and complex antigens, but class II^−/− and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.