Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Cells of the central nervous system as targets and reservoirs of the human immunodeficiency virus

The availability of highly active antiretroviral therapies (HAART) has not eliminated HIV-1 infection of the central nervous system (CNS) or the occurrence of HIV-associated neurological problems. Thus, the neurobiology of HIV-1 is still an important issue. Here, we review key features of HIV-1-cell interactions in the CNS and their contributions to persistence and pathogenicity of HIV-1 in the CNS. HIV-1 invades the brain very soon after systemic infection. Various mechanisms have been proposed for HIV-1 entry into the CNS. The most favored hypothesis is the migration of infected cells across the blood-brain barrier ("Trojan horse" hypothesis). Virus production in the CNS is not apparent before the onset of AIDS, indicating that HIV-1 replication in the CNS is successfully controlled in pre-AIDS. Brain macrophages and microglia cells are the chief producers of HIV-1 in brains of individuals with AIDS. HIV-1 enters these cells by the CD4 receptor and mainly the CCR5 coreceptor. Various in vivo and cell culture studies indicate that cells of neuroectodermal origin, particularly astrocytes, may also be infected by HIV-1. These cells restrict virus production and serve as reservoirs for HIV-1. A limited number of studies suggest restricted infection of oligodendrocytes and neurons, although infection of these cells is still controversial. Entry of HIV-1 into neuroectodermal cells is independent of the CD4 receptor, and a number of different cell-surface molecules have been implicated as alternate receptors of HIV-1. HIV-1-associated injury of the CNS is believed to be caused by numerous soluble factors released by glial cells as a consequence of HIV-1 infection. These include both viral and cellular factors. Some of these factors can directly induce neuronal injury and death by interacting with receptors on neuronal membranes (neurotoxic factors). Others can activate uninfected cells to produce inflammatory and neurotoxic factors and/or promote infiltration of monocytes and T-lymphocytes, thus amplifying the deleterious effects of HIV-1 infection. CNS responses to HIV-1 infection also include mechanisms that enhance neuronal survival and strengthen crucial neuronal support functions. Future challenges will be to develop strategies to prevent HIV-1 spread in the brain, bolster intrinsic defense mechanisms of the brain and to elucidate the impact of long-term persistence of HIV-1 on CNS functions in individuals without AIDS.     

Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health's experience

In October 2001, a letter containing a large number of anthrax spores was sent through the Brentwood post office in Washington, D.C., to a United States Senate office on Capitol Hill, resulting in contamination in both places. Several thousand people who worked at these sites were screened for spore exposure by collecting nasal swab samples. We describe here a screening protocol which we, as a level A laboratory, used on very short notice to process a large number of specimens (3,936 swabs) in order to report preliminary results as quickly as possible. Six isolates from our screening met preliminary criteria for Bacillus anthracis identification and were referred for definitive testing. Although none of the isolates was later confirmed to be B. anthracis, we studied these isolates further to define their biochemical characteristics and 16S rRNA sequences. Four of the six isolates were identified as Bacillus megaterium, one was identified as Bacillus cereus, and one was an unidentifiable Bacillus sp. Our results suggest that large-scale nasal-swab screening for potential exposure to anthrax spores, particularly if not done immediately postexposure, may not be very effective for detecting B. anthracis but may detect a number of Bacillus spp. that are phenotypically very similar to B. anthracis.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

Receptors for Treponema pallidum attachment to the surface and matrix proteins of cultured human dermal microvascular endothelial cells

Pathogenicity of Treponema pallidum may depend upon the binding of Treponema pallidum to matrix proteins, especially to fibronectin. Infectious organism or cell to matrix interactions are mediated by a family of adhesion molecule receptors known as integrins. Once in the host, the pathogenic Treponema pallidumdum adheres to the vascular endothelium and readily penetrates surrounding tissues. Fibronectin plays an important role in the mediation of the attachment of Treponema pallidum to host cells, including endothelial cells. We found that the binding of Treponema pallidum to human dermal microvascular endothelial cells and to a glass surface coated with fibronectin is inhibited by the presence of arginine-glycine- aspartic acid (RGD), and analysis of the surface receptor revealed an antigenic similarity to an integrin molecule, namely alpha5. This ability to adhere to host endothelium and fibronectin is quite unique to T. pallidum among the treponemes, and may be a key pathogenic factor.     

Antagonistic effects upon cutaneous circulation of muscular exercise and exposure to a high ambient temperature

Measurements were made in man of heart rate (Fc), arterial blood pressure (Pa), cutaneous blood flow (Z) (by plethysmography of the hand) as well as variation sin venous volume (deltaV) in the course of muscular exercise of 70 Watts intensity corresponding from 48% to 60% of the maximal oxygen consumtion of the subjects. These exercises were carried out at ambient temperatures of 22 degrees and 29 degrees C. for 8 min. In every case, an initial lowering of the Q and deltaV followed by a progressive climb was found. The average maximal reduction in output was virtually identical at 22 degrees and 29 degrees C (4.10 +/- 2.05 and 4.00 +/- 2.31 ml/min. 100 cm3). But the average maximal fall in volume turned out to be less at 29 degrees C (0.30 +/- 0.40 ml/100 cm3) than at 22 degrees C (0.60 +/- 0.60 ml/100 cm3). The resistive sector of cutaneous circulation conserves the same vasomotor capacities at the two ambient temperatures studied; the cutaneous capacitive sector shows lower reactivity at the higher ambient temperature. This would suggest that diminished venous return may be partially responsible for poor tolerance to exercise in warm climates.     

Evaluation of a one-year Masters of Public Health program for medical students between their third and fourth years.

For physicians to better treat and advise their patients on the roles of behavioral and social factors in health and disease, greater levels of competency in social and behavioral sciences are needed. Physicians should also understand the structure, financing, and administration of the health care delivery system, so that they will be able to practice medicine effectively and participate in planning and managing the delivery of care. And, improving overall public health requires that physicians understand the basic tenets of population-based medicine. One way to achieve these goals is to develop education and training programs for integrating formal public health training with formal medical training.There are many models by which a medical student or practitioner can obtain a master of public health (MPH) degree. In this article, the authors describe an accelerated one-year MPH program for competitively selected New York City medical students who have completed their third year of training and enroll at the Mailman School of Public Health, Columbia University. The Macy Scholars Program, offered between 1999 and 2007 to 12 students per year, is completed between the third and fourth years of medical school. Under full-tuition scholarships, students complete a practicum experience, attend seminars, and write a master-level paper or thesis, among other requirements. Data from an evaluation of this program demonstrate participant satisfaction and support of the program, outstanding academic performance, and the effect of public health training on their residency and career choices.     

Activation of immunological memory cells in vitro

Peritoneal cell populations from NMRI mice injected three times intraperitoneally with sheep red blood cells do not contain a significant number of plaque-forming cells to sheep red blood cells. However, plaque-forming cells do arise in such populations 3–5 days after transfer into tissue culture. The in vitro activation of these cells into antibody-producing cells occurs without further exposure to the antigen. The culture system contained, in addition to the peritoneal cells, irradiated spleen cells from non-immunized animals which were shown not to respond when cultured alone. The peritoneal cells as well as non-producing spleen cells could be activated in vitro at varying times after the last stimulation of the donor animal up to a period of several weeks. These observations suggest that there is a compartment of resting memory cells whose activation is not dependent on the addition of antigen.     

Functional differences between human cutaneous mast cells and basophils: a comparison of morphine-induced histamine release

Intravenous administration of morphine sulfate often produces urticarial and hypotensive reactions associated with elevations in plasma histamine. The source of this histamine and mechanisms controlling its release are poorly understood. Previous studies of morphine-induced histamine release compared human leukocytes to rat peritoneal mast cells. The effects of morphine on human cutaneous mast cells has not been examined. We studiedin vitro histamine release from human basophils and human skin preparations containing cutaneous mast cells to evaluate their relative, contribution to the pharmacologic effects of morphine.Human skin mast cell preparations showed dosedependent histamine release over a morphine concentration range of 1.5×10^–5 to 4.5×10^–3 M, with peak release occurring at 5×10^–4 M, with peak release occurring at 5×10^–4 M. Clinically, morphine sulfate is usually injected as a 1.5×10^–2 M solution. Histamine release was calcium dependent and equivalent to that obtained with 3 and 10 mM strontium. Morphologic examination revealed degranulation and exocytosis occurring in morphine-stimulated tissue but not in specimens exposed to buffer alone. Lactate dehydrogenase levels did not increase following morphine incubation, thus supporting a noncytolytic mechanism of histamine release.Basophils, in contrast, showed no significant histamine release from exposure to morphine up to 10^–2 M. Concanavalin A, as a positive control in these same preparations, produced a mean histamine release of 21.0%.Our studies indicate distinct functional differences between human skin mast cell and human blood basophil responses to morphine sulfate. We conclude that the cutaneous and systemic reactions to morphine sulfate probably result from the release of histamine from mast cells rather than from basophils.This study was supported by: National Institutes of Health Grants 2-T32 AMO7153, 5-RO1 AI18615, AI 15557, HL 25831, American Society of Anesthesiologists Starter Grant 1283, and a grant from the Bramble Foundation.