A codon 891 exon 15 RET proto-oncogene mutation in familial medullary thyroid carcinoma: a detection strategy

A ser891ala RET proto-oncogene mutation has been previously discovered in a single kindred with familial medullary thyroid carcinoma (FMTC). An additional two probands with this mutation and with medullary thyroid carcinoma (MTC) without any other manifestations of MEN 2 have been identified. In one of thse families, two other individuals also had the mutant sequence and FMTC. Analysis of both cases showed cosegregation of the mutation and MTC. To facilitate detection of this mutation, a primer was engineered which creates a Hha I recognition site in the presence of the mutant sequence. As a result, this codon 891 exon 15 mutation can be identified with a restriction enzyme digestion.     

Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts

Migration of pericytes such as hepatic stellate cells is fundamentally important for diverse biological and pathological processes including tumor invasion and fibrosis. In prototypical migratory cells such as fibroblasts, the small GTPases Rac1 and RhoA govern the assembly of lamellipodia and stress fibers, respectively, cytoskeletal structures that are integral to the cell migration process. The gaseous signaling molecule nitric oxide (NO) influences growth factor chemotactic responses, although this occurs primarily in cell-type-specific ways and through cell biological effects that are poorly characterized. In this study, we use complementary molecular and cell biological approaches to delineate important roles for Rac1, RhoA, and NO in migration of the human hepatic stellate cell line LX2 and primary rat hepatic stellate cells. Both platelet-derived growth factor (PDGF) and Rac1 overexpression drove migration through formation of actin-positive filopodia spikes in LX2 as compared to the formation of lamellipodia in fibroblasts. NO inhibited PDGF- and Rac1-driven migration in LX2 by abrogating filopodia formation and inhibited migration of fibroblasts by attenuating lamellipodial protrusions. Additionally, RhoA conferred resistance to NO inhibition of migration and restored chemotactic responses to PDGF in the absence of functional Rac1 in LX2. In conclusion, these studies identify novel crosstalk between small GTPases, cytoskeletal structures, and NO in pericyte-specific pathways, providing counterbalances in the chemotactic responses to growth factors.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the United States

Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of > or =94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection.     

Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins

"Centralized" (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.     

Comparative genomic hybridization of ductal carcinoma in situ of the breast—evidence of multiple genetic pathways

There is strong evidence that ductal carcinoma in situ (DCIS) represents a precursor lesion of invasive breast cancer. In order to analyse specific chromosomal alterations of DCIS, 38 paraffin-embedded specimens of DCIS and six associated invasive carcinomas were examined by means of comparative genomic hybridization (CGH). Losses of 16q material were seen almost exclusively in well- and intermediately-differentiated DCIS. These two subgroups differed in the average number of genetic imbalances, 2·5 and 5·5 respectively. Additionally, a higher frequency of gains of 1q and losses of 11q material was seen in intermediately-differentiated in contrast to well-differentiated DCIS. Poorly-differentiated DCIS displayed a higher frequency of amplifications (17q12, 11q13) and a higher average rate of genetic imbalances (7·1). Analysis of adjacent invasive breast carcinoma revealed a genetic pattern almost identical to the one seen in the DCIS counterpart. These data characterize DCIS as a genetically far-advanced, heterogeneous lesion and as a direct precursor of invasive breast cancer. Copyright © 1999 John Wiley & Sons, Ltd.     

Association between dementia and reduced walking ability and 30-day mortality in patients with extended-spectrum beta-lactamase-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> bacteremia

Previous studies have shown controversial results of factors associated with short-term mortality in patients with extended-spectrum beta-lactamase (ESBL)-producing E. coli bacteremia and no research has investigated the impact of the geriatric assessment criteria on short-term mortality. Our objective was to determine whether dementia and walking ability are associated with 30-day mortality in patients with ESBL-producing E. coli bacteremia. All blood bottle cultures, analyzed from January 2008 to April 2015, in the Bacteriology Department of a 2,600-bed, university-affiliated center, Nantes, France, were retrospectively extracted. Factors associated with short-term mortality in patients with ESBL-producing E. coli bacteremia: 140 patients with an ESBL-producing E. coli bloodstream infection were included; 22 (15.7%) patients died within 30 days following the first positive blood bottle culture of ESBL-producing E.coli. In multivariate analysis, a reduced ability to walk (OR = 0.30; p = 0.021), presence of dementia (OR = 54.51; p = 0.040), a high Sepsis-related Organ Failure Assessment (SOFA) score (OR = 1.69; p < 0.001), presence of neutropenia (OR = 12.94; p = 0.049), and presence of a urinary tract infection (OR = 0.07; p = 0.036), were associated with 30-day mortality. Our findings provide new data showing an independent association between 30-day mortality with dementia and reduced walking ability, in patients with ESBL-producing E. coli bacteremia. These criteria should be considered in the therapeutic management of patients with ESBL-producing E. coli bacteremia.