Blast exposure causes dynamic microglial/macrophage responses and microdomains of brain microvessel dysfunction.

Exposure to blast overpressure (BOP) is associated with behavioral, cognitive, and neuroimaging abnormalities. We investigated the dynamic responses of cortical vasculature and its relation to microglia/macrophage activation in mice using intravital two-photon microscopy following mild blast exposure. We found that blast caused vascular dysfunction evidenced by microdomains of aberrant vascular permeability. Microglial/macrophage activation was specifically associated with these restricted microdomains, as evidenced by rapid microglial process retraction, increased ameboid morphology, and escape of blood-borne Q-dot tracers that were internalized in microglial/macrophage cell bodies and phagosome-like compartments. Microdomains of cortical vascular disruption and microglial/macrophage activation were also associated with aberrant tight junction morphology that was more prominent after repetitive (3×) blast exposure. Repetitive, but not single, BOPs also caused TNFα elevation two weeks post-blast. In addition, following a single BOP we found that aberrantly phosphorylated tau rapidly accumulated in perivascular domains, but cleared within four hours, suggesting it was removed from the perivascular area, degraded, and/or dephosphorylated. Taken together these findings argue that mild blast exposure causes an evolving CNS insult that is initiated by discrete disturbances of vascular function, thereby setting the stage for more protracted and more widespread neuroinflammatory responses.

Prevalence of enterohemorrhagic Escherichia coli in raw and treated municipal sewage.

Municipal sewage was screened for DNA encoding Shiga-like Toxin (SLT) II, a key protein involved in the virulence of enterohemorrhagic Escherichia coli. PCR analysis of sewage concentrates showed that DNA encoding SLT II was present in a single sample of untreated sewage and absent in all other samples tested (n = 6). Thermotolerant E. coli cultured from the sewage (n = 1,520) also tested negative for SLT II by colony hybridization.

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.

Cell extracts of Candida albicans block adherence of the organisms to endothelial cells.

Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.

Candida albicans adherence to endothelial cells.

Mechanisms of adherence to vascular endothelial cells by microorganisms on a molecular level can be elucidated by using monoclonal antibodies, purified cell wall constituents, and receptor analogues. Since these agents are expensive and available in limited quantities, a microsystem for probing adherence mechanisms to these cells has become essential. We studied techniques to accurately quantify the adherence of L-[35S]methionine-labeled Candida albicans to human umbilical vein endothelial cells in a 96-well microtiter plate system while avoiding specific problems related to Candida coadherence and avid binding to plastic. The endothelial cells were grown on a collagen matrix in individually detachable microwells enabling the determination of the number of adherent organisms from radioactive counts of the entire well. This procedure has the critically important advantage of obviating the need to remove adherent Candida from the wells. Expressing adherence to endothelial cell monolayers as the percentage of total organisms added to each well significantly decreases the variability of the assay.

Comparison of fluconazole and amphotericin B for treatment of disseminated candidiasis and endophthalmitis in rabbits.

We compared the efficacy of intravenous fluconazole (80 mg/kg of body weight per day) with that of amphotericin B (1 mg/kg/day) for the long-term treatment of endophthalmitis in rabbits with disseminated candidiasis. After 17 days of therapy, fluconazole decreased the fungal colony counts of the choroid-retinas significantly more than did the saline control (P less than 0.05); however, after 24 days of fluconazole therapy, this treatment effect was lost and fluconazole was no more effective than saline. In contrast, treatment for 24 days with amphotericin B reduced the vitreous and choroid-retina fungal colony counts significantly more than either fluconazole or saline (P less than 0.05 for both treatment groups). After 17 days of therapy, indirect ophthalmoscopy revealed less severe eye involvement in both antifungal treatment groups than in saline controls; however, this difference reached statistical significance only for the amphotericin B-treated rabbits (P less than 0.05). Also, there was a trend towards worsening eye lesions, as seen by indirect ophthalmoscopy, in the fluconazole-treated rabbits after 24 days of therapy, which roughly paralleled the quantitative culture results. Despite the presence of negative choroid-retina cultures, some rabbits in all treatment groups had persistently visible eye lesions, indicating that ophthalmoscopic resolution of Candida endophthalmitis may lag behind lesion sterilization. Amphotericin B was superior to fluconazole in the treatment of Candida endophthalmitis in this model.

Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens.

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.

An enzyme-linked immunosorbent assay for quantifying adherence of Candida to human vascular endothelium.

Success in elucidating the pathogenesis of certain bacterial infections through studies of bacterial adherence to host cells has stimulated interest in parallel investigations of fungal adherence. Fungal adherence differs from bacterial adherence, especially when fungal coadherence (adherence of fungal cells to each other) is a factor. Using human umbilical vein endothelial cells cultured in a living monolayer in microtiter plates, we developed an ELISA to study adherence of Candida albicans to endothelial cells in the absence of yeast coadherence. A rabbit antibody to Candida detected the adherent Candida, and an alkaline phosphatase-conjugated antibody to rabbit IgG was the developing antibody. A linear relationship between the log of the optical density and the log of the number of adherent organisms was seen for wells containing 3 X 10(4)-1 X 10(6) organisms (r = .923- .965). In addition to measuring adherence of living Candida to living target cells and avoiding Candida coadherence, this assay makes it possible to investigate adherence limited to lumenal surfaces, conserves reagents, and facilitates the testing of large numbers of potential adherence modifiers.