Listeria monocytogenes mutants lacking phosphatidylinositol-specific phospholipase C are avirulent.

A number of bacterial species secrete phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we show that the facultative intracellular bacterial pathogen, Listeria monocytogenes, contains a gene, plcA, predicting a polypeptide with 31% amino acid identity to a Bacillus thuringiensis PI-PLC. Accordingly, L. monocytogenes secretes PI-PLC activity, while a mutant with a transposon insertion in plcA lacks detectable PI-PLC activity. In addition, expression of plcA in B. subtilis resulted in secretion of PI-PLC activity. The L. monocytogenes PI-PLC-defective mutant was three logs less virulent for mice and failed to grow in host tissues. The mutant was also defective for in vitro growth in mouse peritoneal macrophages. These results strongly suggest that PI-PLC is an essential determinant of L. monocytogenes pathogenesis. Whether the PI-PLC acts on a bacterial or host substrate remains to be determined.

Novel approaches to monitor bacterial gene expression in infected tissue and host.

Elucidating the complex and dynamic host-microbe interactions during infection requires, among other things, detailed knowledge of microbial gene expression in vivo. Recently, advances in fluorescence and bioluminescence detection techniques, as well as recombinase-based in vivo expression technology, have rendered monitoring virulence gene expression in vivo a feasible task. These techniques have been adapted by several laboratories to study the spatial and temporal patterns of virulence gene expression by organisms such as Salmonella typhimurium, Listeria monocytogenes, Yersinia entercolitica and Vibrio cholerae during infection of tissue culture or animal models of infection.

Effects of passive smoking on theophylline clearance.

Theophylline disposition was examined in seven passive smokers, defined as nonsmokers with long-term exposure to cigarette smoke, and seven age-matched nonsmokers with minimal smoke exposure. Subjects were given an intravenous infusion of aminophylline (6 mg/kg) and blood samples were drawn before and during the 48-hour postinfusion period. Clearance for passive smokers was 6.01 x 10(-2) L/hr.kg and for nonsmokers, clearance was 4.09 x 10(-2) L/hr.kg (p less than 0.025). Terminal elimination half-life for passive smokers was 6.93 hours versus 8.69 hours for nonsmokers (p less than 0.05). The mean residence time for passive smokers was 9.89 hours. For nonsmokers, the mean residence time was 13.11 hours (p less than 0.05). These measurements were statistically different, whereas there was no difference in volume of distribution between the groups, suggesting that passive smokers metabolize theophylline more rapidly than nonsmokers. Plasma and urine cotinine and nicotine concentrations were measured in all subjects. There was a significant difference between the subject groups in plasma (p less than 0.004) and urine (p less than 0.002) cotinine concentrations. Theophylline clearance correlated with both plasma (r = 0.73, p less than 0.01) and urine (r = 0.79, p less than 0.01) cotinine concentrations. Additional studies should be conducted to further define the pharmacokinetic characteristics of passive smokers and to assess the effects of passive smoking on drugs metabolized by the mixed function oxidase system.

IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues.

IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect. The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. The IVET system has several applications in the area of vaccine and antimicrobial drug development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.

Umbilical cord blood: the role of apoptosis in the control of CD34+ cell counts.

BACKGROUND AND OBJECTIVE: Cord blood obtained at delivery can be used for hematopoietic precursor cells (HPC) transplantation. The major limit for its success is represented by the low cellular yield of the stem cell population. The objective of this study was to determine the role played by apoptosis in the numerical control of CD34+ cell counts. DESIGN AND METHODS: Umbilical cord blood samples were collected from 15 women at the time of the delivery and cord blood units processed. Cells, collected following 24h and 48h of incubation, were analysed by flow cytometry using the gating strategy. RESULTS: Remarkable levels of apoptosis were detected in the stem cell population and a significant difference between apoptosis mean values at 24h and 48h within CD34+ cells were found. The difference between the percentage of apoptosis in CD34+ cells and that in the remaining population was significant both at 24h and at 48h. CONCLUSIONS: CD34+ cells have a higher likelihood to undergo apoptosis in comparison to the remaining ones present in umbilical cord blood. This process of cellular death plays a major role in the control of CD34+ cell counts in placental blood and influence, for this reason, the possibility of success of a cord blood transplantation.

Heterotopic ossification complicating critical illness.

Heterotopic ossification (HO), deposition of para-articular ectopic bone, is associated with musculoskeletal trauma and certain congenital and metabolic disorders. Additionally, HO may follow paralysis from diverse traumatic and nontraumatic neurologic insults. We describe three cases of HO associated with catastrophic nontraumatic respiratory illness requiring prolonged chemical paralysis and cardiorespiratory support.

Relationships between pulmonary function and changes in chronic respiratory symptoms. Comparison of Tucson and Cracow longitudinal studies.

Parallel analyses of data from two longitudinal studies, one in Poland and one in the United States, were performed to assess the relationships between pulmonary function and respiratory symptoms. Similar relationships were seen in the both cities using the same methods of analysis. The rate of FEV1 decline and its final level were related to the prior presence of attacks of breathlessness or to a syndrome that also included wheezing and diagnosed asthma. Initial FEV1 level was lower in subjects with dyspnea appearing during the follow-up than in the never-symptom group. These relationships were independent of smoking habits. The consistencies in the parallel analyses strengthen the relationships observed. In Tucson, Ariz, the FEV1 decline in smokers with persistent chronic cough was greater than that due to separate effects of the symptom and smoking. This suggests that chronic cough may be an indicator of an increased effect of tobacco smoke on pulmonary function.

Respiratory effects in relation to estimated tar exposure from current and cumulative cigarette consumption.

Relationships between estimated "tar" exposure from current and past cigarette consumption, were related to respiratory symptoms and function during the seventh survey (1981-1983) of the Tucson epidemiologic study of airways obstructive diseases. Smokers (n = 582; 280 males and 302 females) and exsmokers (n = 621; 345 males and 276 females), answered detailed questions on current and past smoking, and performed flow-volume maneuvers. Cough and phlegm were significantly associated with estimated "tar" exposure from current cigarette consumption. Cough, phlegm, and dyspnea were associated with reported deep inhalation in both sexes, while wheeze was so associated in females. In young smokers (less than 35 years), symptoms were more prevalent in those who had predominantly smoked cigarettes with estimated high "tar." Total exposure was more important in older smokers. Generally, exsmokers showed a lower prevalence of symptoms than smokers. Cross-sectional lung function measurements were significantly related to total estimated "tar" exposure and age. Multiple logistic regressions provided models which helped predict symptoms and lung function impairment in smokers and exsmokers on the basis of estimated "tar" exposure and pack-years of smoking.

Smoking and pregnancy: a comparison of Mexican-American and non-Hispanic white women.

OBJECTIVE: To describe ethnic differences in tobacco use during pregnancy in a clinical population of Mexican-American and non-Hispanic white women. METHODS: Subjects were randomly selected from all patients attending a university-based obstetrics clinic in Tucson, Arizona. Of 555 subjects selected, 367 were interviewed and had their urinary cotinine levels measured. The 37 subjects who refused an interview and the 136 who were selected but not interviewed did not differ from other subjects regarding ethnicity, education, obstetric history, or smoking status. The interviewer gathered standard demographic and obstetric information as well as data on smoking behavior. Subjects were interviewed in Spanish or English by a bilingual interviewer using a standardized questionnaire. Statistical techniques included chi 2 test, t test, and logistic regression. RESULTS: The odds for Mexican-American women to have been smokers were 3.39 times lower than for non-Hispanic white women. The odds of quitting during pregnancy were 4.71 times higher for Mexican-Americans (95% confidence interval 1.66-13.38). Urinary cotinine values verified the latter rates. Mexican-American smokers reported smoking significantly fewer cigarettes than non-Hispanic white women (t = 2.34, P < .05). CONCLUSIONS: In comparing Mexican-American and non-Hispanic white women, we found marked differences in smoking behavior during pregnancy. Mexican-Americans were nearly three times more likely to quit than non-Hispanic whites, and those who continued to smoke showed greater reductions in cigarettes per day. These results show behavioral changes in Mexican-Americans during pregnancy that meaningfully alter the risk profile for the fetus. Understanding the basis for this behavioral change may facilitate the development of preventive measures for other ethnic groups.

Transformation of a type 4 encapsulated strain of Streptococcus pneumoniae.

Streptococcus pneumoniae strain JNR.7/87 is a highly virulent, type 4 encapsulated Gram-positive bacterium whose transformability has not been tested previously, and whose genome is currently being sequenced. The strain was transformed at very low efficiency by addition of exogenous competence-stimulating peptide: However, the efficiency was too low and irreproducible to be useful in many genetic studies. Therefore, the effects on transformation efficiency of changing different components of competence-stimulating peptide-induced transformation have been examined. Screening of growth media was followed by optimization of pre-induction culture acidification, glycine concentration, and induction time. An optimized protocol was developed whereby S. pneumoniae strain JNR.7/87 was transformed reproducibly with a streptomycin resistance (SmR) marker at an efficiency of approximately 10(5) colony forming units per 10(8) cells.

Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae.

CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae.

Parental childhood respiratory illness and respiratory illness in their infants. Group Health Medical Associates.

Parental histories of childhood respiratory trouble (CRT) were examined as risk factors for lower respiratory tract illnesses in healthy infants enrolled in the Children's Respiratory Study, Tucson, Arizona. A parental history of childhood respiratory trouble before age 16 was a risk factor for infantile lower respiratory tract illnesses (LRIs). Early age of onset of the parental CRT and those illnesses described as asthma or bronchiolitis showed the greatest risk: odds ratio = 2.8, P < 0.05. After controlling for known and suspected confounders, a parental history of CRT described as asthma or bronchiolitis with onset before age 3 was associated with wheezing LRIs in their children, with an odds ratio of 2.6, P < 0.05. A parental history of CRT described as bronchitis/croup was associated with nonwheezing LRIs in their children: odds ratio = 2.2, P < 0.05. These findings suggest a familial component to childhood respiratory trouble which may have a hereditary basis.

Two cases of "multicystic peritoneal mesothelioma": description and critical review of the literature.

Two cases of multicystic peritoneal mesothelioma (MPM) are reported. Ultrastructural and immunohistochemical techniques confirmed the mesothelial nature of the lesion. The biologic and clinical behaviour, pathogenesis and differential diagnoses of this rare pathology are discussed. Although regarded as a neoplasm, many analogies seem to link MPM to fibromatoses and other non-neoplastic lesions, suggesting a reactive hyperplastic process. The relationships between mesothelium and the secondary Müllerian system, to date not fully investigated, are stressed and a classification of the coelomatic reactive and neoplastic processes, both metaplastic (müllerian metaplasia) and non-metaplastic, is suggested.

Postchemotherapy late recurrence of non metastatic gestational trophoblastic disease following a partiale mole. A case report.

The authors describe a case of a 35-year-old woman who showed elevation of betahCG 13 months after the complete regression of betahCG values following chemotherapy for an incomplete mole. This case outlines the necessity for careful monitoring of betahCG levels in low risk gestational trophoblastic diseases for a period of time longer than one year after achieving the first clinical remission.

[Hormone replacement therapy (HRT). The effects at 12 months on the risk factors for menopausal osteoporosis]. / Terapia ormonale sostitutiva (HRT). Effetti a dodici mesi sugli indici di rischio per l'osteoporosi menopausale.

Cessation of ovarian activity is accompanied by a more or less marked and more or less accelerated reduction in bone mass; the degree and speed of the process--which occurs in all women--depends on individual (genetic factors influencing peak bone mass, duration of child-bearing period), iatrogenic (treatment with corticosteroids or thyroid hormones) or accidental factors (post-traumatic immobilization). Whatever the factor that triggers off the process, the end result is the destruction of bone tissue. This process may be documented by hematochemical (Nordin's test) and instrumental parameters (MOC and similar techniques). Oestroprogestin (and to a lesser extent calcitonin) hormone replacement therapy has been demonstrated to be highly efficacious in countering this involutive process. The authors report data obtained following the evaluation of 35 women in menopause undergoing. Nordin's test and measurement of the plasma level of estradiol using RIA, before and after twelve months after the start of osteoprotective treatment. Of the 35 patients 9 received only progestin, 22 an oestroprogestin combination (of these 18 patients received estrogen transdermally and 4 orally), and 4 calcitonin administered parenterally. A statistically significant positive correlation with Nordin's test was only found in the group receiving oestrogen therapy. In conclusion, it may be affirmed that in the absence of contraindications oestrogens represent the elective form of treatment for menopausal osteoporosis. Acceptable results have been reported in the literature also using calcitonin, but this treatment could not be evaluated in this study owing to the reduced number of the sample treated.

[The effects of hormone replacement therapy (HRT) on lipid metabolism in the menopause]. / Effetti della terapia ormonale sostitutiva (HRT) sul metabolismo lipidico in menopausa.

INTRODUCTION AND AIMS: During menopause the number of cardiovascular attacks increases parallel to the elimination of estradiol production. The administration of the latter reverses this tendency owing to a compound mechanism (improved HDL) cholesterol/total cholesterol ratio, diminished vasal resistance). MATERIALS AND METHODS: The authors present a study performed in 34 patients in menopause receiving oestroprogestinic replacement therapy using an oral or transdermal route. Metabolic status (serum concentrations of total cholesterol (TC) and HDL cholesterol (C.HDL) was evaluated in all patients before treatment and after 12 months. RESULTS: A statistically significant positive correlation (p < 0.0001) was found between the use of oestrogen and serum levels of HDL. This correlation appeared to be more evident in patients using transdermal treatment compared to the oral form. CONCLUSIONS: The authors conclude that in the absence of contraindications, hormone replacement therapy in menopause exercises a beneficial effect on the lipid status, contributing to diminishing the risk of cardiovascular attacks. The possibility of an increased incidence of breast cancer is now being evaluated, whereas effective protection of the endometrium against the risk of hyperplasia and cancer was shown using the doses of progestin used in this study, which coincide with those currently prescribed in the literature.

[Intestinal endometriosis. Three new cases and review of the literature]. / Endometriosi intestinale. Tre nuovi casi e revisione della Letteratura.

OBJECTIVE: The study was undertaken to identify some features of the intestinal endometriosis such as symptoms, helpful investigations, pattern of distribution and surgical management. PATIENTS: Three consecutive cases, observed during a sixteen month period, are reported. The most frequent symptoms were chronic pelvic and abdominal pain, dysmenorrhea, alterated bowel habit and menorrhagia. The diagnosis of intestinal endometriosis was incidental in all but one case admitted for an intestinal subocclusive syndrome in patient with a past history of pelvic endometriosis previously documented by laparoscopy. RESULTS: All patients presented a sigmoid localization of endometriosis with different degree of stenosis and underwent sigmoid resection, followed by a resolution of abdominal symptoms. DISCUSSION: Although the exact frequency of intestinal endometriosis is difficult to know because of the lack of specific symptoms and reliable investigations, it has been estimated that implants to the bowel may occur in 3%-37% of women affected by endometriosis. The sigmoid colon is the most common site of localization. The main symptoms are pelvic pain, dysmenorrhea, infertility and diarrhoea or constipation; rarely patients present bowel occlusion due to stenosis (less than 15% of the cases) or cyclic rectal bleeding. CONCLUSION: Generally, intestinal endometriosis is not suspected preoperatively in those patients without a past history of this condition; however an accurate diagnosis can be provided throughout laparoscopy, before open surgery. The hormonal therapy is not successful in alleviating moderate to severe obstructive symptoms. Thus surgery still remains the most effective treatment for advanced intestinal endometriosis.