Cyclic AMP inhibits increased collagen production by cyclically stretched smooth muscle cells

Rabbit arterial smooth muscle cells, grown on elastin membranes which were cyclically elongated and relaxed, responded by increasing their rates of synthesis of protein and, in particular, of collagen, compared to stationary controls. Raising intracellular cyclic AMP (cAMP) levels by adding theophylline or dibutyryl cAMP to the culture medium prevented the synthetic response to cyclic stretching, but did not alter the rates of protein or collagen synthesis by stationary controls. Both synthesis and degradation of collagen by cyclically stretched cells increased in parallel such that the proportion of synthesized collagen that was degraded was similar to that found in the stationary cultures. Collagen degradation was not affected by theophylline administration to stationary cell cultures but the drug increased degradation of collagen by cyclically stretched cells. We conclude that the net production of protein, and in particular of a structural protein, collagen, by arterial smooth muscle cells subjected to the mechanical force of stretching was inhibited when intracellular levels of cAMP were raised. The results suggest that cAMP may play a role in the modulation of structural protein content of artery walls in response to changes in tensile stress.     

Problems in the conduct and analysis of randomized clinical trials. Are we getting the right answers to the wrong questions?

To achieve the goal of validity, the randomized clinical trial has emerged as the scientific "gold standard" for evaluating therapies in clinical medicine. Regardless of how well randomized clinical trials are designed, however, problems often occur during the conduct of the trials that give rise to methodologic challenges in the analysis of results. Primarily two types of problems, changes in intended treatment and the failure to ascertain the study outcomes, occur during the conduct of randomized clinical trials. We studied the current analytic strategies that are used to deal with these problems and how the use of these analytic strategies can change the focus of the research so that the trial no longer answers the relevant question. To ensure that the right question is answered, new methods of design and analysis are required that balance the goals of validity and clinical pertinence.     

New epidemiologic evidence confirming that bias does not explain the aspirin/Reye's syndrome association

To determine the validity of the aspirin/Reye's syndrome association, we developed an epidemiologic investigation to assess the effects of five potential sources of bias. A case-control study incorporated procedures to avoid temporal precedence and susceptibility bias. These included classifying cases as having monophasic or biphasic patterns of illness and matching for severity of symptoms at zero-time. To evaluate the effect of a potential recall bias, an "alternate-condition" control group was enrolled. A medical record review study was conducted to assess the potential for diagnostic bias, and a blanket surveillance of all hospitals in a region was conducted to evaluate reporting bias. Twenty-four case subjects and 48 matched controls were enrolled. Eight-eight percent of case subjects and only 17% of controls had received aspirin prior to the onset of Reye's syndrome (matched odds ratio, 35; 95% confidence interval, 4.2 to 288). Further analyses demonstrated that the association could not be attributed to the five potential sources of bias.     

Hemolytic uremic syndrome after bone marrow transplantation: clinical characteristics and outcome in children.

Hemolytic uremic syndrome (HUS) is an uncommon but potentially life-threatening complication of hematopoietic stem cell transplantation. We retrospectively studied the medical records of 293 children who underwent allogeneic bone marrow transplantation at St. Jude Children's Research Hospital between 1992 and 1999 to describe the clinical course of and to identify risk factors for transplant-associated HUS. Conditioning regimens included cyclophosphamide, cytarabine, and total body irradiation for patients with hematologic malignancies (n = 244); patients with nonmalignant diseases (n = 49) received disease-specific regimens. Grafts from unrelated or mismatched related donors were depleted of T lymphocytes, whereas matched sibling grafts were unmanipulated. All patients received cyclosporine as prophylaxis for graft-versus-host disease. Recipients of grafts from matched siblings also received pentoxifylline or short-course methotrexate. HUS developed in 28 (9.6%) patients at a median of 171 days after transplantation. We identified older donor age (P = .029), use of antithymocyte globulin in the conditioning regimen (P = .008), and recipient CMV seronegativity (P = .011) as being associated with an increased risk of HUS. With a multiple regression analysis, the use of antithymocyte globulin (beta = .86; P = .04) and recipient cytomegalovirus seronegativity (beta = .93; P = .035) remained significant risk factors for the development of HUS.     

Serum concentrations of oestradiol-17beta, progesterone, relaxin and chorionic gonadotrophin during blastocyst implantation in natural pregnancy cycle and in embryo transfer cycle in the rhesus monkey

The present study was undertaken to assess the temporal association between the profiles of serum concentrations of oestradiol-17beta, progesterone, chorionic gonadotrophin (CG) and relaxin in pregnancies established naturally, and after embryo transfer, as well as in failed pregnancies in rhesus monkeys. In naturally mated cycles (group 1) a conception rate of 75% was obtained. In group 1, the mean day of CG detection in serum was 11.5 +/- 1.9 day post-ovulation, and for relaxin, 9.0 +/- 2.5 day post-ovulation. In group 2, embryo transfer to synchronous, non-mated surrogate recipients was performed; seven embryo transfer cycles yielded three pregnancies which were allowed to continue to term and normal infants were delivered. In embryo transfer cycles the mean day of CG detection was 14.8 +/- 1.8 day post- ovulation, and for relaxin, 11.4 +/- 2.6 day post-ovulation. A delay of about 3 days was observed in the appearance in circulation of CG (P < 0.05) and also of relaxin (P < 0.05) between natural mated and embryo transfer conception cycles. Significant differences (P < 0.05 for progesterone and P < 0.03 for oestradiol) were obtained for the areas under the curves for progesterone and oestradiol between days 12 and 16 in conception cycles compared with failed pregnancies. These data provide the first observation of the normal hormonal signals associated with maternal recognition of transferred embryos during the peri- implantation period, and suggest that the use of such an experimental primate embryo transfer model may help to elucidate components of maternal and embryonic signal-response mechanisms during embryo implantation.      

A rapid microagglutination test for the diagnosis of Legionella pneumophila (serogroup 1) infection

A rapid microagglutination test has been developed which can be performed in 30 minutes. Ninety-seven percent of 96 patients diagnosed as having Legionella pneumophila (serogroup 1) infection by indirect immunofluorescence were also detected by the rapid microagglutination test.     

Identification of a novel mycobacterial histone H1 homologue (HupB) as an antigenic target of pANCA monoclonal antibody and serum immunoglobulin A from patients with Crohn's disease

pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved approximately 32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.     

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.