An inducible bundle-forming pilus of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC), a cause of childhood diarrhea, grow on the surface of the small intestine and on cultured epithelial cells as colonies of adherent bacteria. When propagated on solid medium containing blood or attached to HEp-2 cells, EPEC express ropelike bundles of filaments, termed bundle-forming pili (BFP), that create a network of fibers that bind together the individual organisms. BFP were found to be expressed by five EPEC serogroups, each harboring a approximately 92-kilobase plasmid previously known to be important for virulence in humans. When two of these strains were cured of this plasmid, they neither expressed BFP nor grew as adherent colonies. An antiserum to BFP reduced the capacity of EPEC to infect cultured epithelial cells. BFP are composed of a repeating subunit of 19,500 daltons, the amino-terminal amino acid sequence of this subunit is homologous to that of the toxin-coregulated pilin of Vibrio cholerae.

Hepatitis C virus core protein up-regulates anergy-related genes and a new set of genes, which affects T cell homeostasis.

Hepatitis C virus (HCV) infection is the main cause for chronic hepatitis, leading to cirrhosis and hepatic carcinoma. Virally induced immune dysfunction has been called as the cause for viral persistence. Previous results demonstrate that CD4 Jurkat cells stably expressing the HCV core protein show an increased activation of NFAT transcription factor and an impaired IL-2 promoter activity, affecting intracellular signaling pathways in a manner that mimics clonal anergy. We had shown previously that NFAT activates a transcriptional program, ensuing in immunological tolerance. In the present work, we have engineered lentiviral vectors expressing the HCV core to analyze the events, which unfold in the initial phase of HCV core-induced anergy. We show that genes initially described to be up-regulated by ionomycin-induced anergy in mice are also up-regulated in humans, not only by ionomycin but also by HCV core expression. We also show that HCV core is sufficient to cause NFAT nuclear translocation and a slow-down in cell-cycle progression, and using whole genome microarrays, we identify novel genes up-regulated in Jurkat cells expressing HCV core. The relevance of our results is highlighted by the presence of HCV in CD4 T cells from HCV chronically infected patients.

Streptococcus mitis endocarditis. Report of 17 cases.

Seventeen patients with Streptococcus mitis endocarditis were treated at a municipal hospital over a three-year period. Thirteen patients were intravenous drug addicts. Streptococcus mitis has a predilection for right-sided endocarditis in intravenous drug addicts and left-sided endocarditis in non-drug addicts. Streptococcus mitis is highly susceptible to therapy with penicillin G potassium (minimal inhibitory concentration less than or equal to 0.1 mg/L of penicillin in all of these 17 cases), and four to six weeks of therapy is safe and effective.

Longus pilus of enterotoxigenic Escherichia coli and its relatedness to other type-4 pili--a minireview.

Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili. These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E. coli and both longus and the colonization factor antigen III (CFA/III) of ETEC. These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements. While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids. Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them. This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC. Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively. This suggests that lngB may be an accessory gene involved in biogenesis of longus.

Serial analysis of the effects of methimazole or radical therapy on circulating CD16/56 subpopulations in Graves' disease.

The distribution of peripheral blood CD16/56 cytotoxic T and natural killer (NK) cells in Graves' disease patients is analyzed in order to correlate them with disease activity and with prognosis. Eighteen patients with Graves' disease, twenty-four patients with Hashimoto's thyroiditis and thirty-two sex- and age-matched healthy control subjects were studied. Peripheral blood CD16/56 (cytotoxic T and NK) cells were analyzed by cytofluorometry. A decreased proportion of CD16/56+ and CD16/ 56+CD3+ cells were detected in Graves' disease patients when compared with thyroiditis patients and healthy control groups. No correlation was detected with serum free thyroxine. On diagnosis, patients who would require a radical treatment for thyrotoxicosis control showed a significant decrease of cytotoxic CD56+ T (CD3+) and NK (CD3-) cells compared with those who would maintain the euthyroid state after methimazole. These results suggest that the cytotoxic compartment, both T and NK cells, of the immune system is altered in patients with Graves' disease, independently of the functional thyroid status. Changes in peripheral blood lymphocytes in Graves' disease patients could be useful as predictive markers of an unfavorable outcome.

The expression of the beta 1 integrin CD29 and the beta 2 integrin CD11b is decreased in peripheral blood lymphocytes from Graves' disease patients.

We have prospectively examined the percentage of peripheral blood lymphocytes which expressed adhesion molecules in untreated Graves' disease patients. Eighteen patients with Graves' disease, twenty-four patients with Hashimoto's thyroiditis and thirty-two sex- and age-matched healthy control subjects were studied. The expression of the lymphocyte adhesion molecules beta-1 integrin CD29, beta-2 integrin CD11b and L-selectin Leu8 (CD62L) was analyzed by cytofluorometry. A decreased percentage of CD29+ and CD11b+ lymphocytes was observed in hyperthyroid patients in comparison with Hashimoto's thyroiditis patients and healthy controls. However, there was no difference in the percentage of CD62L+ lymphocytes in the three groups. Percentages of lymphocyte activation markers, hyperthyroid status, presence or absence of ophthalmopathy or serum levels of antithyroid antibodies were not related to the proportions of CD29+ or CD11b+ lymphocytes. Four Graves' patients required radical therapy but after the treatment, there was no modification in the percentages of CD29+ and CD11b+ lymphocytes compared with those determined at diagnosis. Our findings suggest that the decrease in beta-1 and beta-2 integrins could be a predisposing marker of development of Graves' disease.

Virulence properties of atypical EPEC strains.

Virulence properties of 31 atypical enteropathogenic Escherichia coli (EPEC) strains isolated from cases of diarrhoea were examined. All except two strains adhered to HEp-2 cells in a localised adherence-like (LAL) pattern. With the exception of two strains, all were fluorescent actin staining (FAS) positive. Gentamicin HEp-2 invasion assay studies showed that all strains were invasive. Transmission electron microscopy of infected HEp-2 cells showed the characteristic attaching and effacing lesion and invasion of the cultured cells. Of the nine strains that hybridised with a DNA probe for alpha-haemolysin, five were haemolytic within 3 h of incubation, while the remaining strains were haemolytic only after incubation for 24 h. Three strains produced enterohaemolysin on blood agar. None of the 31 strains of E. coli induced fluid accumulation in the rabbit intestinal loop assay or displayed cytotoxic effects in HeLa and Vero cells. All the strains belonging to serotypes O26:H11, O26:H- and 0119:H2 expressed intimin beta, whereas all the strains from serotype O55:H7 expressed intimin gamma. The strains belonging to serogroup O111 expressed a non-typable intimin. The participation of intimin in LAL was supported by adhesion inhibition experiments in which antibodies to intimin significantly reduced the level of LAL.

Infections in parenteral drug abusers. Further immunologic studies.

In our previous study of soft tissue infections in parenteral drug abusers, two thirds of the infections were polymicrobial. Oral and enteric organisms were frequently recovered. These patients and a group of uninfected addicts showed frequent cutaneous anergy, lymphopenia, and hypergammaglobulinemia. An additional group of uninfected addicts was studied. The mean levels of IgA, IgG, and IgM were higher in the uninfected addicts. In the addict and control groups, elevations in IgA (17 percent of total), IgG (65 percent), and IgM (19 percent) levels were found. Zinc levels were within normal limits. T-cell populations below 70 percent were seen in five of the seven addicts and two of the four control subjects. Reversed helper to suppressor cell ratios were found in three of the seven addicts and control subjects. No consistent pattern of immunologic abnormalities emerged. The interrelationship of the abnormalities in the addict and their relationship to AIDS is unclear.

Male pseudohermaphroditism with 5-alpha-reductase deficiency: report of two new familial cases. The importance of early diagnosis.

We report two new familial cases of male pseudohermaphroditism due to 5-alpha-reductase deficiency, from the south of Spain. They were born with ambiguous genitalia and were reared as females. At the time of puberty, both brothers virilized partially and underwent a change of gender role from female to male with a stormy psychic readjustment period. We stress the value of the prolonged chorionic gonadotropin test for an early diagnosis.

Modulation of hepatic and intestinal glutathione S-transferases and other antioxidant enzymes by dietary lipids in streptozotocin diabetic rats.

Antioxidant enzymes in liver and small intestine were investigated using control and streptozotocin diabetic rats fed diets with 5% olive, sunflower or fish oil for five weeks. In liver, Glutathione Peroxidase and Superoxide Dismutase decreased and in intestine Glutathione-S-transferase (GST) increased by diabetes. In isolated jejunum and ileum, this increase in GST activity was due to an increase in GST-alpha and -mu isoenzymes in jejunum and GST-alpha, mu and -pi in ileum. Since GST plays an important role in protecting tissues from oxidative damage, our results highlight the role of the intestine against free radicals in physiological or pathological situations.

Thyroid nodules. Role of fine needle aspiration and intraoperative frozen section examination.

OBJECTIVE: To determine the accuracy of fine needle aspiration (FNA) and intraoperative frozen section examination (IFSE) on thyroid nodules. STUDY DESIGN: The study group consisted of 470 patients who underwent thyroidectomy. FNA was performed on 289 patients and IFSE on 326. The FNA and IFSE results were compared with the final histologic diagnosis obtained after examination of permanent sections. RESULTS: The overall FNA sensitivity was 65%, specificity 88% and positive predictive value 61%. The IFSE sensitivity was 50%, and the specificity and positive predictive value were 100%. When both procedures were used together, FNA identified 16 of 45 (36%) carcinomas as malignant and an additional 13 (29%) as follicular proliferative lesions; IFSE correctly identified only 23 of 45 (51%) carcinomas. CONCLUSION: FNA provides enough information for determining the extent of thyroid surgery when a diagnosis of cancer is made. However, IFSE should be considered a supplementary procedure when FNA is not positive for cancer.

Natural killer activity in patients with breast cancer.

The clinical significance of the natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) was analyzed in 83 breast cancer patients and 24 healthy control women. Similar levels of NK cytotoxic activity against K-562 target cells were found in PBMC from either untreated or surgically treated patients with local or disseminated breast cancer and from normal controls. However, a transitory and significant decrease (p less than 0.05) of the NK activity of PBMC from breast cancer patients was found during chemotherapy. But, according to quantitative flow cytometry analysis, similar percentages of phenotypically defined NK cells (CD16+, CD11b+, HNK-1+) were found in PBMC from patients, whether prior to or during chemotherapy, and healthy controls. Our results demonstrate that in breast cancer patients, the percentage of NK cells present in PBMC and their lytic activity are independent of the clinical and pathological stage of the disease.

[Determination of insulin, leptin and neuropeptide y by radioimmunoanalysis in patients with morbid obesity and anorexia nervosa after therapeutic intervention]. / Determinación por radioinmunoanálisis de insulina, leptina y neuropéptido Y en pacientes con obesidad mórbida y anorexia nerviosa después de intervención terapéutica.

The present study was conducted in order to analyze the relationship existing between leptin, insulin and neuropeptide Y (NPY) levels in massive weight loss and weight recovery. Twenty-three patients with severe obesity, 23 patients with anorexia nervosa and 28 healthy control subjects were studied. Patients with severe obesity underwent a vertical banded gastroplasty followed by an 800 kcal/day diet during 16 weeks, with evaluation taking place before (Body mass index, BMI, 52,1 8 Kg/m2) and after the drastic weight loss (BMI 39,2 6,2 Kg/m2). Patients with anorexia nervosa were treated with nutritional therapy exclusively during 16 weeks, and they were evaluated in the low weight situation (BMI 15,3 1,7 Kg/m2) and after weight recovery (BMI 18,9 2,8 Kg/m2). Normal subjects had a normal BMI from 20 to 27 (average 21,8 2 Kg/m2). BMI, percentage of body fat, and serum levels of leptin, insulin, and NPY, were determined in each patient and normal subjects. In severe obese patients serum leptin and insulin decreased significantly after drastic weight reduction (leptin: from 48,8 19,2 to 24,3 9,8 ng/ml; insulin: from 26,2 10,8 to 18 6 U/ml). In patients with anorexia nervosa serum leptin mean levels were significantly higher after weight recovery (3,7 1,9 vs 9,2 5,1 ng/ml). In subjects with morbid obesity NPY levels decreased after weight loss below those of control group (43,5 16,1 vs 57,3 12,8 pmol/l). On the other hand, patients with anorexia nervosa had NPY levels superior to those of control group. In subjects with anorexia, NPY levels decreased after weight recovery (69,1 16,7 a 59,1 20,3 pmol/l). In the whole population, Leptin and NPY plasma levels were correlated with body fat percentage. Leptin was positively correlated with BMI and body fat percentage in obese and anorectic subjects after weight loss or recovery, respectively. NPY was inversely correlated with body fat percentage in controls and obese subjects before treatment. These data reveal that the concentration of serum leptin and NPY correlates significantly with the total adiposity in subjects with a wide weight range and caloric intake. Leptin plasma levels are proportional to fat stores in patients with severe obesity and anorexia nervosa after drastic weight loss or recovery, respectively. NPY serum levels are negatively correlated with de total body fat in normal weight subjects and obese patients in their initial weight.

[Plasma levels of insulin and leptin in patients with morbid obesity and anorexia nervosa after weight loss or gain, respectively]. / Niveles plasmáticos de insulina y leptina en pacientes con obesidad mórbida y anorexia nerviosa después de pérdida o ganancia de peso, respectivamente.

The present study was conducted in order to analyze the relationship existing between leptin and insulin levels in massive weight loss and weight recovery. Thirteen patients with severe obesity, 14 patients with anorexia nervosa and 13 healthy control subjects were studied. The patients with severe obesity underwent a vertical banded gastroplasty followed by an 800 kcal/day diet for 12 weeks. They were evaluated prior to (body mass index [BMI] 51.2 +/- 8.8 Kg/m2) and after drastic weight loss (BMI 40.6 +/- 6.7 Kg/m2). Patients with anorexia nervosa were treated exclusively with nutritional therapy during 12 weeks, and they were evaluated at their lowest weight status (BMI 16.2 +/- 2.2 Kg/m2) and after weight recovery (BMI 17.9 +/- 2.3 Kg/m2). The BMI of the normal subjects was in the normal range of 20 to 27 Kg/m2 (average 22.8 +/- 2.6 Kg/m2). BMI, percentage of body fat, waist circumference, and serum levels of leptin, insulin, and C-peptide were determined in each patient and normal subject. In severely obese patients, serum leptin and insulin decreased significantly after drastic weight reduction (leptin: from 51.8 +/- 22.3 to 23.7 +/- 10.2 ng/ml; insulin: from 27.1 +/- 13.3 to 17.2 +/- 7.2 mU/ml). In patients with anorexia nervosa, the mean serum leptin levels were significantly higher after weight recovery (5.5 +/- 3.2 vs 7.6 +/- 6 ng/ml). Serum leptin in the severe obesity group correlated positively with BMI, percentage body fat and waist circumference before and after weight loss. In those patients suffering from anorexia nervosa, serum leptin correlated positively with the BMI, percentage of body fat, and waist circumference in the low weight state and after weight recovery. In addition, their serum insulin correlated with BMI and waist circumference after weight recovery. These data reveal that serum leptin concentration correlates significantly with the BMI and body fat content 1) in subjects with a range of weight and caloric intake, 2) in obese patients after drastic weight loss; 3) in anorexic patients after weight gain; and that hyper- or normoinsulinemia do not seem to have any influence on the leptin changes caused by weight loss or gain.

Expression of flagella and motility by Shigella.

Since the discovery of Shigella as the aetiologic agent of acute dysentery almost 100 years ago, this organism has been described as a non-motile and nonflagellated organism that invades the human colonic mucosa. In this study, the production of flagella by prototypic strains of all four Shigella species and, moreover, by fresh clinical isolates was demonstrated by electron microscopy. The flagellum of Shigella (flash) is approximately 10 microns long and 12-14 nm in diameter and is typically seen emanating from one pole of the bacterium. Flash is composed of a putative structural polypeptide subunit of 33-38 kDa that shares immunological similarities with Escherichia coli, Salmonella spp., and Proteus mirabilis flagellins, and with the recently described recombinant Shigella flagellins (FliCSS and FliCSF) expressed in E. coli K-12. A fliCSS-specific oligo probe hybridized with all four Shigella species, while a fliCSF probe hybridized with all Shigella flexneri and Shigella dysenteriae strains, but not with all Shigella sonnei or Shigella boydii strains, indicating genetic divergence among their flagellin genes. Shigella exhibits motility in low-concentration motility agar under physiological growth conditions. The expression of flash and motility appears to be strictly regulated by unidentified genetic and environmental factors. These heretofore undescribed features may allow the bacteria to circumvent the natural intestinal mucosal defences leading to bacterial colonization and disease. The motility of shigellae may represent an evolutionary adaptation important for bacterial survival.

Bacteroides fragilis pelvic abscess: resolution with metronidazole therapy alone.

An 81-year-old woman had Bacteroides fragilis sepsis with a sonographically demonstrated, multiply echogenic collection in the pelvis. She failed to respond to clindamycin therapy. The patient and her family refused surgical intervention. On intravenous metronidazole alone, the patient's fever decreased, she improved, and the pelvic collection disappeared in a period of between six and 30 weeks.

Biliary concentrations of piperacillin in patients undergoing cholecystectomy.

Piperacillin is a new semisynthetic, expanded-spectrum penicillin with marked activity against Pseudomonas aeruginosa. The biliary excretion of piperacillin was studied in patients undergoing cholecystectomy. Concentrations of piperacillin in common duct bile at 35 to 90 min postinfusion of 1-g doses ranged from 31 to 920 micrograms/ml, with a mean (+/- standard deviation) of 467 +/- 363 micrograms/ml. Gallbladder piperacillin levels at 30 to 75 min postinfusion ranged from 2.2 to 80 micrograms/ml, with a mean of 27 +/- 31 micrograms/ml. No correlation occurred with peak serum level of antibiotic, creatinine, bilirubin, or alkaline phosphatase. Significant amounts of piperacillin were excreted via the biliary system.

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.

Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera-like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH2-terminal amino acid sequence revealed homology with the toxin-coregulated pilus of Vibrio cholerae, the bundle-forming pilus of enteropathogenic E. coli and type IV pilins of some Gram-negative bacterial pathogens. The longus structural gene (lngA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K-12. The presence of lngA was restricted to human ETEC strains. In contrast to other ETEC pili, lngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immunoprophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat-stable toxin only.