Comparative analysis of immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay using virus-like particles or virus-infected mouse brain antigens to detect IgM antibody in sera from patients with evident flaviviral infections

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 - specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.     

The Effect of Accuracy of Perceptions of Dietary-Fat Intake on Perceived Risk and Intentions to Change

Consumption of excess fat increases risk for many health problems and diseases. In the present study, 188 undergraduate students were studied to understand self-perceptions of dietary-fat intake and the impact of those perceptions. Findings indicated that the majority of participants had inaccurate perceptions about the amount of fat in their diets. Further, compared to people who overestimated dietary-fat intake, people who underestimated fat intake had lower perceived risk of cancer, had lower intentions to change, and demonstrated less knowledge about the dietary-fat content of many foods. Findings suggest that this unrealistic underestimation of fat intake is a cognitive barrier to dietary change and people who underestimate dietary fat intake may require more intensive intervention to change their diets.     

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.     

BRCA1 and pancreatic cancer: pedigree findings and their causal relationships

Anecdotal reports and series studies indicate that 5-10% of pancreatic cancer (PC) cases are familial. In addition, PC is associated with a variety of hereditary cancer syndromes. PC appears to be an integral cancer in the hereditary breast-ovarian cancer (HBOC) syndrome, with most notice given to the role of BRCA2. Our purpose is to call attention to BRCA1, which also predisposes to PC. Using data from our familial breast cancer registry, we identified 19 BRCA1/2 families that contain PC affecteds in the pedigrees, 15 with BRCA1 mutations and 4 with BRCA2 mutations. The association between BRCA2 and pancreatic cancer is well established; however, a definite link with pancreatic cancer in families carrying a BRCA1 mutation has been far less studied. Thus, the focus of this report is on 9 of the 15 BRCA1 families, in which PC affecteds were either confirmed carriers of the BRCA1 mutation or were inferred as probable obligate BRCA1 mutation carriers. The numbers are small, but nevertheless illustrate the finding of others of an apparent association between PC and BRCA1-mutation-bearing families. Given the dismal prognosis of PC, with the only current hope for survival being through surgical extirpation of the pancreas prior to metastasis, it is prudent that we realize the potential predisposition toward PC via BRCA1, in the hope of early diagnosis and prevention.     

Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

Importance of anesthesia for the genesis of neurogenic pulmonary edema in spinal cord injury

There are reports describing both provocation and inhibition of neurogenic pulmonary edema by anesthetic drugs. Therefore, we compared the effect of two types of anesthesia on the formation of neurogenic pulmonary edema in rats with balloon-induced acute spinal cord injury. Animals with sham procedure (group 1) were anesthesized by intraperitoneal sodium pentobarbital. In the experimental groups, rats were submitted to acute spinal cord lesion by insufflations of a balloon in the epidural space at T8 for 1 min (group 3 under i.p. sodium pentobarbital and group 2 under i.p. xylazine-ketamine anesthesia). In rats with pentobarbital anesthesia, systolic blood pressure doubled the baseline value during compression, whereas this effect was less pronounced in the ketamine-xylazine group. The pulmonary index (100 x wet lung weight/body weight) was 0.395 (+/-0.018) in sham-operated rats, rose to 0.499 (+/-0.060) in group 2, and was maximum under pentobarbital anesthesia (0.639+/-0.14; p=0.0018). Histologic examination of the spinal cord showed parenchymal ruptures and acute hemorrhage. Comparison of the pulmonary index with histologic slides of lung parenchyma revealed that relevant intra-alveolar edema occurred only for index values above 0.55. On electron microscopy, endothelial alterations, and damage of the alveolar lining cells were found. Our study indicates that neurogenic pulmonary edema caused by spinal cord injury is less pronounced in rats under xylazine-ketamine anesthesia, when compared with pentobarbital.     

Congenital clubfoot in Europe: A population‐based study

We aimed to assess prevalence, birth outcome, associated anomalies and prenatal diagnosis of congenital clubfoot in Europe using data from the EUROCAT network, and to validate the recording of congenital clubfoot as a major congenital anomaly by EUROCAT registries. Cases of congenital clubfoot were included from 18 EUROCAT registries covering more than 4.8 million births in 1995–2011. Cases without chromosomal anomalies born during 2005–2009, were randomly selected for validation using a questionnaire on diagnostic details and treatment. There was 5,458 congenital clubfoot cases of which 5,056 (93%) were liveborn infants. Total prevalence of congenital clubfoot was 1.13 per 1,000 births (95% CI 1.10–1.16). Prevalence of congenital clubfoot without chromosomal anomaly was 1.08 per 1,000 births (95% CI 1.05–1.11) and prevalence of isolated congenital clubfoot was 0.92 per 1,000 births (95% CI 0.90–0.95), both with decreasing trends over time and large variations in prevalence by registry. The majority of cases were isolated congenital clubfoot (82%) and 11% had associated major congenital anomalies. Prenatal detection rate of isolated congenital clubfoot was 22% and increased over time. Among 301 validated congenital clubfoot cases, diagnosis was confirmed for 286 (95%). In conclusion, this large population‐based study found a decreasing trend of congenital clubfoot in Europe after 1999–2002, an increasing prenatal detection rate, and a high standard of coding of congenital clubfoot in EUROCAT.     

Potential involvement of gelatinases and their inhibitors in Mannheimia haemolytica pneumonia in cattle

Mannheimia haemolytica infection of the lower respiratory tract of cattle results in a bronchofibrinous pneumonia characterized by massive cellular influx and lung tissue remodeling and scarring. Since altered levels of gelatinases and their inhibitors have been detected in a variety of inflammatory conditions and are associated with tissue remodeling, we examined the presence of gelatinases in lesional and nonlesional lung tissue obtained from calves experimentally infected with M. haemolytica. Lesional tissue had elevated levels of progelatinase A and B and active gelatinase A and B when compared with nonlesional tissue obtained from the same lung lobe. In vitro, M. haemolytica products stimulated production of gelatinase B, but not its activation, by bovine monocytes. Alveolar macrophages showed constitutive production of gelatinase B but no change in response to M. haemolytica products. Bovine neutrophils exposed to M. haemolytica products also released gelatinase B, and there was a significant increase in the activated form of this enzyme. These effects were virtually identical when recombinant O-sialoglycoprotease was used to stimulate these cells. M. haemolytica products also enhanced the expression by bovine monocytes and alveolar macrophages of the tissue inhibitor of metalloproteinase 1. Our results provide evidence that matrix metalloproteinases are activated in lung lesions from cattle with shipping fever and that M. haemolytica virulence products induce production, release, and especially activation of gelatinase B by bovine inflammatory cells in vitro.