Membrane recycling and calcium dynamics during settlement and adhesion of zoospores of the green alga Ulva linza.

Recruitment of individuals of the marine alga Ulva linza on to a suitable habitat involves the settlement of motile zoospores on to a substratum during which a preformed adhesive is secreted by vesicular exocytosis. The fluorescent styryl dye FM 1-43 and fluorescent Ca(2+) indicators were used to follow membrane cycling and changes in cytosolic Ca(2+) ([Ca(2+)](cyt)) associated with settlement. When swimming zoospores were exposed continuously to FM 1-43, the plasma membrane was preferentially labelled. During settlement, FM 1-43-labelled plasma membrane was rapidly internalized reflecting high membrane turnover. The internalized membrane was focused into a discrete region indicating targeting of membrane to an endosome-like compartment. Acetoxymethyl (AM)-ester derivatives were found to be unsuitable for monitoring [Ca(2+)](cyt) because the dyes were rapidly sequestered from the cytoplasm into sub-cellular compartments. [Ca(2+)](cyt) was, however, reliably measured using dextran-conjugated calcium indicators delivered into cells using a biolistic technique. Cells loaded with Oregon Green BAPTA-1 dextran (Invitrogen, Paisley, UK) showed diffuse cytosolic loading and reliably responded to imposed changes in [Ca(2+)](cyt). During settlement, zoospores exhibited both localized and diffuse increases in [Ca(2+)](cyt) implying a role for [Ca(2+)](cyt) in exocytosis of the adhesive.

Role of the C-terminal actin binding domain in BCR/ABL-mediated survival and drug resistance.

Philadelphia chromosome-positive, chronic myeloid leukaemia (CML) stem and progenitor cells have a survival and growth advantage compared with their normal counterparts. The mechanisms through which the BCR/ABL protein tyrosine kinase (PTK) induces these effects and the important domains within this protein are not fully defined. The F- and G-actin binding region of the BCR/ABL C-terminus may be important in BCR/ABL-mediated events, and we have investigated this by expressing a C-terminus deletion mutant of the temperature-sensitive BCR/ABL PTK, in a haemopoietic progenitor cell line, which models the chronic phase of CML. The truncated BCR/ABL PTK displayed similar levels of PTK activity when compared with wild type and activation of second messenger formation (in the form of sn-1,2-diacylglycerol) remains intact. On fibronectin substrata, localisation of the protein to the periphery of the cell was, however, dependent on the C-terminus of BCR/ABL PTK. Deletion of the C-terminus reversed both BCR/ABL-mediated apoptotic suppression and drug resistance although the progenitor cells did retain a proliferative advantage at low concentrations of growth factor. These results demonstrated that the C-terminal actin-binding domain of BCR/ABL is important for some of BCR/ABL PTK-mediated leukaemogenic effects.

Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385.

A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.

Abdominal helical CT: milk as a low-attenuation oral contrast agent.

One hundred ten consecutive patients were given either whole (4%) milk, 2% milk, water, barium suspension, or no oral contrast agent before abdominal computed tomography (CT). Results with whole milk were superior to those with all other agents for gastrointestinal distention, mural visualization, and pancreas-duodenum discrimination. In bowel loop discrimination, results with 4% milk were equal to those with barium but superior to those with all other agents. Whole (4%) milk is an effective low-attenuation oral contrast agent.

Declining CD4+ T-lymphocyte counts are associated with increased risk of enteric parasitosis and chronic diarrhea: results of a 3-year longitudinal study.

From January 1991 through September 1994, we observed people who were infected with HIV to assess the impact of enteric parasite-associated diarrhea. Respondents answered comprehensive questionnaires covering clinical and epidemiologic information and provided stool specimens monthly, which were examined unstained as well as stained with trichrome, chromotrope 2R, and with Kinyoun carbol-fuchsin, and with indirect immunofluorescence for Cryptosporidium. In all, 602 participants, who were interviewed, provided stool specimens at 3254 monthly visits. Parasites were associated with 50 of 354 (14.1%) acute diarrheal episodes (lasting < or = 28 days) and with 97 of 279 (34.8%) chronic episodes (lasting > 28 days). A parasite was associated with 31 of 222 (14.0%) episodes that occurred when CD4+ counts were > or = 200 cells/microl and with 150 of 566 (26.5%) episodes that occurred when CD4+ counts were < 200 cells/microl. The most commonly identified parasite was C. parvum, which was associated with 18 of 354 (5.1%) acute episodes and 36 (12.9%) of the 279 chronic episodes of diarrhea. In this patient population, enteric protozoan parasites were commonly associated with illness, particularly as immunosuppression worsened, and were more likely to be associated with chronic rather than acute diarrhea.

Antimicrobial resistance in Neisseria gonorrhoeae in the United States, 1988-1994: the emergence of decreased susceptibility to the fluoroquinolones.

Antimicrobial susceptibilities of Neisseria gonorrhoeae have been prospectively determined in the Gonococcal Isolate Surveillance Project of the Centers for Disease Control and Prevention. From 1988 through 1994, susceptibilities were determined for 35,263 isolates from 27 clinics. Patients were demographically similar to those in nationally reported gonorrhea cases. In 1994, 30.5% of isolates had chromosomally or plasmid-mediated resistance to penicillin or tetracycline. Penicillin resistance increased from 1988 (8.4%) to 1991 (19.5%) and then decreased in 1994 (15.6%). Tetracycline resistance decreased from 1988 (23.4%) to 1989 (17.3%) and then increased in 1994 (21.7%). Most isolates (99.9%) were highly susceptible to broad-spectrum cephalosporins. Isolates with decreased susceptibility to ciprofloxacin increased from 1991 (0.4%) to 1994 (1.3%); 4 isolates were ciprofloxacin-resistant. Ciprofloxacin-resistant strains may not respond to therapy with recommended doses of fluoroquinolones, and the clinical importance of strains with decreased susceptibility is unknown. The emergence of fluoroquinolone resistance in N. gonorrhoeae in the United States threatens the future utility of this class of antimicrobials for gonorrhea therapy.

Protection from Mycobacterium avium complex disease in human immunodeficiency virus-infected persons with a history of tuberculosis.

Risk of Mycobacterium avium complex disease was examined in human immunodeficiency virus (HIV)-infected patients with and without a history of tuberculosis. Information was obtained by retrospective review of charts of patients in HIV clinics in 10 US cities. Among 1363 patients with <200 CD4 cells/mm3 seen at Grady Memorial Hospital (GMH), 11 (17%) of 66 with a history of a positive purified protein derivative (PPD) skin test acquired M. avium infection, while 29 (16%) of 185 who were PPD-negative (but not anergic) did not (P = .85). Only 4 (8%) of 49 GMH patients with a history of tuberculosis acquired M. avium infection compared with 252 (19%) of 1314 GMH patients without a history of tuberculosis (P = .05). Proportional hazards analysis of risk factors for M. avium infection among 441 persons with and 8702 persons without a history of tuberculosis in 9 other cities confirmed protection from M. avium infection in persons with a history of tuberculosis (relative risk, 0.52; 95% confidence interval, 0.36-0.76; P < .001). Prior tuberculosis provides protection against M. avium infection in HIV-infected persons, possibly by stimulation of antimycobacterial immunity.

Talc pleurodesis: talc slurry versus thoracoscopic talc insufflation in a porcine model.

BACKGROUND: Pleurodesis using both talc slurry and thoracoscopic talc insufflation has been shown to be clinically effective. This study compares these two modalities of pleural talc instillation in an animal model. METHODS: Eleven immature pigs underwent general endotracheal anesthesia. On one side, a slurry of 5 g sterile United States Pharmacopeia talc in 50 mL of saline solution was instilled through a thoracostomy tube. On the other side, the lung was deflated and 5 g of dry talc was insufflated under thoracoscopic visualization. The animals were sacrificed 30 days later, and the quality of pleural adhesions was graded from 0 to 2 (0 = absent; 1 = light; 2 = dense) in each of six regions of each hemithorax. The distribution of adhesions on each side was graded from 0 to 6, according to the number of areas that contained adhesions. RESULTS: One animal died of anesthetic complications. Among the survivors, adhesions produced by both methods were dense and diffuse in 8 of 10 animals, and light and diffuse in 1 animal. One animal had light or absent adhesions on the talc slurry side, and dense and diffuse adhesions on the thoracoscopic talc insufflation side. There was no difference between the techniques for density of adhesion scores (talc slurry, 9.9 +/- 2.2; thoracoscopic talc insufflation, 10.0 +/- 2.5) or distribution of adhesion scores (talc slurry, 5.5 +/- 1.0; thoracoscopic talc insufflation, 5.8 +/- 0.4) (p > 0.1). CONCLUSIONS: Effective pleurodesis in a porcine model can be obtained with either talc slurry or thoracoscopic talc insufflation.

Radiological features of a symptomatic splenic hamartoma.

Symptomatic splenic hamartomas are rare in the pediatric age group, with only four previous reports in the literature. Splenic hamartoma has been reported as a solid homogeneous mass without calcification on CT and ultrasound (US), and only one previous report of the findings on MRI has been published. We report a case of a large symptomatic splenic hamartoma in a 14-year-old girl who presented with splenomegaly, pancytopenia and growth retardation. A solid mass with multiple punctate foci resembling calcifications was seen on US. The mass was heterogeneous and better demarcated on enhanced CT. Radiocolloid scintigraphy demonstrated uptake within the lesion, but less than that of normal spleen. The mass was isointense relative to normal splenic tissue on T1-weighted MRI (0.5 T) and of increased intensity with T2 weighting. At splenectomy, a red pulp hamartoma was identified, which contained nodules of hyalinization and necrosis thought to account for the punctate foci seen on US.

Fluconazole compared with endoscopy for human immunodeficiency virus-infected patients with esophageal symptoms.

BACKGROUND & AIMS: The best initial treatment of human immunodeficiency virus (HIV)-infected patients with esophageal symptoms is unknown. The outcome, including safety and cost-effectiveness, of fluconazole compared with endoscopy as a treatment strategy for HIV-infected patients with new-onset esophageal symptoms was evaluated. METHODS: During a 53-month period, 134 HIV-infected patients with esophageal symptoms were randomized prospectively to groups receiving either standard doses of fluconazole or endoscopy. RESULTS: Among the 68 patients in the fluconazole group, a complete symptomatic response was observed in 56 patients (82%), usually within 1 week. The most common endoscopic findings in the 66 patients in the endoscopy group included Candida esophagitis alone in 42 patients (64%) and ulcerative esophagitis in 10 patients (15%). Patients responding to empirical antifungal therapy or who had Candida esophagitis alone at endoscopy were less like to have severe symptoms (P = 0.027) or odynophagia as the only symptom (P < 0.001) but more frequently had odynophagia and dysphagia (P = 0.007) and thrush (P = 0.002). Empirical fluconazole was cost-effective, saving $738.16 per patient. CONCLUSIONS: Empirical oral antifungal therapy with fluconazole is highly efficacious, safe, and cost-effective for HIV-infected patients with new-onset esophageal symptoms.

Chronic unexplained diarrhea in human immunodeficiency virus infection: determination of the best diagnostic approach.

BACKGROUND & AIMS: Chronic unexplained diarrhea is a common complication of human immunodeficiency virus infection, although the best diagnostic approach is unknown. The purpose of our study was to evaluate the clinical use of endoscopy for the evaluation of this problem. METHODS: Patients infected with human immunodeficiency virus with chronic unexplained diarrhea underwent upper endoscopy to the jejunum followed by colonoscopy. RESULTS: Forty-eight patients were evaluated. A potential cause of diarrhea was found in 21 patients (44%; 95% confidence interval, 30%-58%). Colonoscopy with biopsy identified an etiology in 13 patients, including cytomegalovirus colitis alone in 9. In all but 1 patient with colonic disease, the diagnosis was made by biopsy of the rectosigmoid colon. Upper endoscopy with biopsy identified microsporidiosis in 7 patients and cryptosporidiosis in 2 patients. Logistic regression analysis identified weight loss and duration of diarrhea (P < 0.001) as the only independent predictors for diagnosis. No patient without weight loss and a CD4 lymphocyte count of > 100/mm3 had a diagnosis established. Of the 25 patients without a diagnosis in whom long-term follow-up was available, improvement or spontaneous resolution of diarrhea occurred in 9 (38%). CONCLUSIONS: Clinical parameters are helpful in predicting which patients may benefit from endoscopic examination.

The presence of Enterocytozoon bieneusi spores in the lamina propria of small bowel biopsies with no evidence of disseminated microsporidiosis. Enteric Opportunistic Infections Working Group.

Enterocytozoon bieneusi is the most frequently reported microsporidial infection of humans. In patients with the acquired immunodeficiency syndrome, Enterocytozoon infects the lining epithelial cells of the small intestine, hepatobiliary tract, and gallbladder. Because Enterocytozoon has been thought to be limited to infecting lining epithelial cells, the mechanism of spread of E bieneusi within the intestine, to the biliary tract, and, in two case reports, to distant organs remains unknown. This report describes a patient with acquired immunodeficiency syndrome and intestinal microsporidiosis due to E bieneusi. Histopathologic examination of well-oriented biopsies from the duodenum and jejunum revealed both intra- and extracellular spores of Enterocytozoon extending deeply into the lamina propria, where they were located adjacent to capillaries. The patient has not developed disseminated disease 20 months after the initial diagnosis. In this patient, the demonstration of E bieneusi spores in extraepithelial tissues does not appear to be associated with development of subsequent systemic infection.

Leucine enkephalin effects on paracellular and transcellular permeation pathways across brain microvessel endothelial cell monolayers.

Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size-dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular-weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances.

Predictors of survival in patients with AIDS and disseminated Mycobacterium avium complex disease.

Patients with AIDS and disseminated Mycobacterium avium complex disease (DMAC), as defined by the presence of a positive blood culture for MAC, were studied retrospectively to define the natural history of DMAC. All patients had fevers, severe anemia (hematocrit < 26%), or both. Eighty-seven (76%) had signs, symptoms, or laboratory findings related to the gastrointestinal tract, but no distinct syndrome was identified. Sixty-nine patients received antimycobacterial therapy; assignment to therapy was not randomized. In a proportional hazards analysis, shorter survival was associated with higher initial level of mycobacteremia (relative risk [RR], 1.86; 95% confidence interval [CI], 1.49-2.31; P < .001), while administration of antimycobacterial chemotherapy (RR, 0.42; 95% CI, 0.26-0.70; P < .001) and antiretroviral therapy (RR, 0.40; 95% CI, 0.22-0.73; P < .01) had protective effects. Thus, the initial level of mycobacteremia of patients with DMAC may have prognostic value, and administration of antimycobacterial and antiretroviral agents may be associated with prolonged survival.

Leucine-enkephalin metabolism in brain microvessel endothelial cells.

The metabolism of leucine-enkephalin was investigated in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Leucine-enkephalin was hydrolyzed slowly with less than 40% of the peptide metabolized following a 5-h incubation with intact BMEC monolayers at 37 degrees C. Following separation and extraction of BMEC enzymes into cytosolic and membrane-bound fractions, leucine-enkephalin was observed to be labile in the presence of both cytosolic and membrane-associated enzymes. A much greater concentration of enkephalin-hydrolyzing enzyme was associated with the cytosolic fraction. Resulting metabolite profiles for leucine-enkephalin appeared to be the result of interactions of the peptide with primarily aminopeptidases and angiotensin-converting enzyme. Our results suggested that extensive metabolism of leucine-enkephalin solely by BMECs in the cerebro-vasculature would require internalization by the cells and presentation to cytosolic enzymes.

Low serum levels of oral antimycobacterial agents in patients with disseminated Mycobacterium avium complex disease.

Twenty-seven human immunodeficiency virus-infected patients with disseminated Mycobacterium avium complex disease who were treated with oral antimycobacterial agents (clofazimine, ciprofloxacin, ethambutol, and rifampin) were studied to evaluate the usefulness of monitoring serum drug concentrations and testing in vitro susceptibility of M. avium complex (MAC) isolates. Twenty patients tolerated treatment with three or four antimycobacterial agents for at least 8 weeks; mycobacteremia was eradicated in 7 (35%). The in vitro susceptibilities of MAC isolates to antimycobacterial agents were similar for these 7 and for the 13 who did not respond to antimycobacterial treatment. Serum drug levels were below the expected range in 6 of the 7 whose mycobacteremia was cleared and in 9 of the 13 nonresponders (P = .41). These low serum concentrations of antimycobacterial drugs may be due to impaired drug absorption in patients with AIDS and disseminated MAC disease.

A prospective evaluation of Mycobacterium avium complex colonization of the respiratory and gastrointestinal tracts of persons with human immunodeficiency virus infection.

To describe the natural history of Mycobacterium avium complex (MAC) in the respiratory or gastrointestinal tract of persons with human immunodeficiency virus (HIV) infection, 67 HIV-infected patients with CD4+ cell counts < 200/mm3 and initial negative MAC blood cultures were followed prospectively. Patients were screened every 3 months with cultures and smears of sputum, rectal swab, and blood for mycobacteria. Fourteen patients (20.9%) developed positive blood cultures for MAC (23.4%/year). Sputum cultures revealed MAC in 3 (21%) of the 14 patients at 1, 2, and 8 months before dissemination; no smears were positive. No rectal swab cultures or smears were positive before dissemination. Colonization of the respiratory and gastrointestinal tracts in persons with HIV infection and < 200/mm3 CD4+ cells is infrequently detected with currently available techniques. Screening cultures and smears of sputum and stool do not appear to be sensitive methods for detection of early MAC infection.

Survival prognosis of HIV-infected patients.

Previous studies of survival after a diagnosis of acquired immunodeficiency syndrome (AIDS) have reported variation in temporal trends in association with age, gender, race, mode of transmission, lymphadenopathy, antiretroviral therapy, and presence of specific opportunistic infections at diagnosis. We used a logistic regression model to assess the effect of these factors while controlling for other potential predictors of time from initial CD4 cell count to death in 839 HIV-infected patients at a public hospital in Atlanta, Georgia. Our study found that a CD4 level of < 200 cells/microliters [odds ratio (OR) = 1.71; 95% confidence interval (CI) of 1.58, 1.85] and the presence of an AIDS-indicating condition (initial diagnosis OR = 2.50 and CI of 1.93, 3.24; diagnosis of a second AIDS condition OR = 3.02 and CI of 2.08, 4.40) are independently predictive of survival in HIV-infected persons. Furthermore, specific clinical manifestations of AIDS vary in their contribution to progression from diagnosis of AIDS to death. Therefore, changes in survival of AIDS patients must take into account changes over time in the relative frequency of specific AIDS-indicating diagnoses.