Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Surgery for severe rhinosinusitis

Sinusitis is one of the most common reasons patients visit their primary care physician. The etiology of sinusitis is multifactorial in most cases. However, the final common pathway of disruption is usually made with a thorough history. The physical examination is often unremarkable. Complaints of nasal obstruction, mucopuluent nasal drainage, and facial pain/pressure are most diagnostic chronic sinusitis. Isolated headache is an uncommon presenting symptom. Computed tomography scans are the gold standard for diagnostic imaging. They can be used both for diagnosis and surgical treatment. All chronic sinusitis patients, being considered for endoscopic sinus surgery, should have failed a trial of maximal medical therapy. This includes a 4–6 wk course of oral antibiotics, nasal steroids, topical nasal decongestants, and oral prednisone if possible. Patients who fail maximal medical therapy have persistent symptoms that significantly effect their daily activities, have chronic abnormalities on computet tomography scan, and are candidates for endoscopic sinus surgery. Appropriate patient selection and preoperative counseling are key factors in patient satisfaction. Most patients with symptoms that significantly impact their daily activities will receive marked improvement in symptoms after sinus surgery. Endoscopic sinus surgery has undergone radical changes in the last 15 yr. Minimally invasive techniques, combined with advances in instrumentation and computers have reduced postoperative discomfort and improved patient satisfaction.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.     

Site-specific homologous recombination mutagenesis in group B streptococci

We describe the use of suicide vectors to generate site-specific mutations in group B streptococcus. This is accomplished by cloning gene-specific sequences into a temperature sensitive plasmid and selecting for clones which have undergone homologous recombination. The recombinan clones can be easily isolated by selecting for clones which have retained the antibiotic resistance markers that are present on the vector or cloned into the gene-specific sequences. To confirm the fidelity of the recombination events, PCR analysis is performed on chromosomal DNA isolated from the recombinant clones. Using this strategy, we have generated site- specific insertions in several capsule genes and have found that these insertions occurred as expected and that the mutations result in loss of capsule expression. In this report, we specifically detail the construction of cpsB mutants by single and double cross-over recombination and demonstrate that the resulting strains are acapsular.     

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.     

Sociotropic cognition moderates stress-induced cardiovascular responsiveness in women through effects on total peripheral resistance, but not cardiac output.

Although cardiovascular disease (CVD) remains the leading cause of mortality in women, few studies have examined the role of psychosocial factors in its development. This study examined the moderating effects of sociotropic cognition (SC), a need for social acceptance and approval, on psychosocial stress-induced cardiovascular responsiveness (CVR) and affect reactivity in women. Sixty-eight normotensive, college-aged females were randomly assigned to a low or high social threat condition. Measures of systolic, diastolic and mean arterial blood pressures (SBP, DBP and MAP, respectively), heart rate (HR), cardiac output (CO), total peripheral resistance (TPR) and negative affect were collected during rest, and under conditions of high vs. low interpersonal threat. A two-step hierarchical regression analysis was performed to predict all response variables (BPs, HR, CO, TPR and affect). Increases in SBP, DBP, MAP, TPR and negative affect were greater in the high threat than low threat condition. Changes in SBP, MAP and TPR positively covaried with SC under conditions of high interpersonal threat, but showed no significant covariation in the low threat condition. The data suggest that an excessive need for social acceptance may contribute to rises in BP through an increase in TPR, but not CO under conditions of high social threat.     

Obesity and upper body fat distribution in Mexican American children from families with a diabetic proband

Upper and centralized body fat distribution is associated with non-insulin dependent diabetes mellitus (NIDDM). Few studies have focused on anthropometric characteristics of preadults from families in which there is a diabetic (NIDDM) proband. This study explores the prevalence of upper and centralized body fatness in Mexican American children from the Diabetes Alert study (1981–1983) in Starr County, Texas. Anthropometric data on 165 males and 224 females 9–19 years include measures of adiposity such as skinfold thicknesses and the body mass index (BMI), a measure of overweight. They show rates of obesity two to three times that of White children of comparable age and sex from National Health Surveys. In comparison with U.S. White subjects, Mexican American adults are shorter, have more adiposity and arm muscle mass and have sitting heights and body breadths at the mean of these dimensions for the U.S. population. Children from Diabetes Alert families show only marginal excess of severe obesity (> 95th percentile of BMI) when compared to the general population of children surveyed in Starr County schools. Girls from these families, but not boys, have excess fatness in the BMI compared to Mexican American children from the Hispanic Health and Nutrition Examination Survey (HHANES); suprailiac skinfold thicknesses are also greater in children of the Diabetes Alert study than in HHANES children. From 1972 through 1982, Mexican American children in South Texas showed an increase in average stature, weight, and the BMI. These data together suggest that excessive obesity exists and may be increasing in children in populations at risk for NIDDM. The prevention of NIDDM in the Mexican American population may be more effective if educational and promotional interventions include the school aged population. © 1993 Wiley-Liss, Inc.     

Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779–784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171–177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.