An epistatic interaction between pre-natal smoke exposure and socioeconomic status has a significant impact on bronchodilator drug response in African American youth with asthma.

BACKGROUND: Asthma is one of the leading chronic illnesses among children in the United States. Asthma prevalence is higher among African Americans (11.2%) compared to European Americans (7.7%). Bronchodilator medications are part of the first-line therapy, and the rescue medication, for acute asthma symptoms. Bronchodilator drug response (BDR) varies substantially among different racial/ethnic groups. Asthma prevalence in African Americans is only 3.5% higher than that of European Americans, however, asthma mortality among African Americans is four times that of European Americans; variation in BDR may play an important role in explaining this health disparity. To improve our understanding of disparate health outcomes in complex phenotypes such as BDR, it is important to consider interactions between environmental and biological variables. RESULTS: We evaluated the impact of pairwise and three-variable interactions between environmental, social, and biological variables on BDR in 233 African American youth with asthma using Visualization of Statistical Epistasis Networks (ViSEN). ViSEN is a non-parametric entropy-based approach able to quantify interaction effects using an information-theory metric known as Information Gain (IG). We performed analyses in the full dataset and in sex-stratified subsets. Our analyses identified several interaction models significantly, and suggestively, associated with BDR. The strongest interaction significantly associated with BDR was a pairwise interaction between pre-natal smoke exposure and socioeconomic status (full dataset IG: 2.78%, p = 0.001; female IG: 7.27%, p = 0.004)). Sex-stratified analyses yielded divergent results for females and males, indicating the presence of sex-specific effects. CONCLUSIONS: Our study identified novel interaction effects significantly, and suggestively, associated with BDR in African American children with asthma. Notably, we found that all of the interactions identified by ViSEN were "pure" interaction effects, in that they were not the result of strong main effects on BDR, highlighting the complexity of the network of biological and environmental factors impacting this phenotype. Several associations uncovered by ViSEN would not have been detected using regression-based methods, thus emphasizing the importance of employing statistical methods optimized to detect both additive and non-additive interaction effects when studying complex phenotypes such as BDR. The information gained in this study increases our understanding and appreciation of the complex nature of the interactions between environmental and health-related factors that influence BDR and will be invaluable to biomedical researchers designing future studies.

Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

Stented ureterovesical anastomosis in renal transplantation: does it influence the rate of urinary tract infections?

OBJECTIVE: Our objective was to evaluate the impact of routine use of double-J stents on the incidence of urinary tract infection after renal transplantation. METHODS: We conducted a retrospective-comparative single-centre study in 310 consecutive adult deceased donor kidney recipients transplanted from 2002 to 2006. Patients were divided in two groups, with or without urinary stent implantation. To evaluate the predictive factors for UTI, donor and recipients pre- and post-transplantation data were analysed. Early urological complications and renal function within 12 months of transplantation were included as well. RESULTS: A total of 157 patients were enrolled to a stent (ST) and 153 patients to a no-stent (NST) group. The rate of urinary tract infection at three months was similar between the two groups (43.3% ST vs. 40.1% NST, p = 0.65). Of the identified pathogens Enterococcus and Escherichia coli were the most common species. In multivariate analysis neither age nor immunosuppressive agents, BMI or diabetes seemed to have influence on the rate of UTI. When compared to males, females had a significantly higher risk for UTI (54.0% vs. 33.5%). CONCLUSION: Prophylactic stenting of the ureterovesical anastomosis does not increase the risk of urinary tract infection in the early postoperative period.

Use of marginal organs in kidney transplantation for marginal recipients: too close to the margins of safety?

OBJECTIVE: Due to organ shortage, average waiting time for a kidney in Germany is about 4 years after start of dialysis. Number of kidney grafts recovered can only be maintained by accepting older and expanded criteria donors. The aim of this study was to analyse the impact of donor and recipient risk on kidney long-term function. METHODS: All deceased kidney transplantations were considered. We retrospectively studied 332 patients between 2002 and 2006; divided in 4 groups reflecting donor and recipient risk. RESULTS: Non-marginal recipients were less likely to receive a marginal organ (69 of 207, 33%) as compared to marginal recipients, of whom two-thirds received a marginal organ (p<0.0001). Graft function significantly differed between the groups, but detrimental effect of marginal recipient status on eGFR after 12 months (-6 ml/min/1.73qm, 95% CI -2 to -9) was clearly smaller than the effect of marginal donor status (-10 ml/min/1.73qm, 95% CI -7 to -14). CONCLUSIONS: As we were able to show expanded criteria donor has a far bigger effect on long-term graft function than the "extra risk" recipient. Although there have been attempts to define groups of recipients who should be offered ECD kidneys primarily the discussion is still ongoing.

Molecular modeling of the axial and circumferential elastic moduli of tubulin.

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for alpha- and beta-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for alpha- and 460 MPa for beta-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 +/- 0.14 GPa for alpha-tubulin structures and 1.39 +/- 0.68 GPa for beta-tubulin. Circumferentially the alpha- and beta-moduli were 936 +/- 216 MPa and 658 +/- 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.

High levels of antipeptidoglycan antibodies in psoriatic and other seronegative arthritides.

Bacterial cell wall peptidoglycan, the arthritogenic factor in adjuvant induced arthritis, may also be involved in the etiology of some human rheumatic diseases. Patients with some seronegative rheumatic diseases like ankylosing spondylitis and Reiter's syndrome have elevated antibody titers to peptidoglycan. Using an ELISA with soluble peptidoglycan, we examined the sera of 110 patients with psoriatic arthritis, psoriasis without arthritis and a variety of other joint diseases. Antibody titers were significantly higher (p less than 0.001) among the 22 patients with psoriatic arthritis than the 16 patients with psoriasis without arthritis. Patients with other seronegative arthritides also had higher levels of antipeptidoglycan antibodies than patients with rheumatoid (seropositive) arthritis, osteoarthritis and crystal induced arthritis. Our results furnish additional support for the suggestion for a bacterial role in the pathogenesis of psoriatic and some other seronegative arthritides.

Detection and location of single-base mutations in large DNA fragments by immunomicroscopy.

A technique whereby single-base mutations can be detected by immunomicroscopy of DNA heteroduplexes is described. Four constructs of the filamentous phage M13 were prepared so as to differ by a single base at the same site. Heteroduplexes were prepared and reacted with a water-soluble carbodiimide, with polyclonal antibodies specific for the carbodiimide, and then with a second antibody linked to an electrondense marker. Electron microscopy of the heteroduplexes indicated that the label was located at 4.9 to 5.1 kb in the 7.2-kb phage. The known site of the mismatch was 4.96 kb. Also, plasmids containing inserts of a fragment from the 5' end of hemoglobin A or hemoglobin S were prepared. The median location of the label in heteroduplex molecules was 2.9 kb. The known site of the mismatch was 2.65 kb in the 4.9-kb plasmid. The procedure requires about 10 days to analyze two samples of plasmid or phage DNA.

Murine Gc globulin binds to a synthetic sequential polypeptide that is a murine B lymphocyte mitogen.

A non-immunoglobulin component in mouse serum was found to compete with antibody for the binding of a polyanionic antigen, poly (Tyr-Glu-Ala-Gly). The percent of the polymer (44 ng/ml) bound at physiological pH by the serum component in normal mouse serum was low (18%), but was much higher at pH 6.0 (85%). As a result, the binding of the polyanionic peptide antigen by antibody in immune serum was considerably lower at pH 6.0 than it was at physiological pH. These results should be taken into account when dealing with similar antigens that have been frequently studied in immunology, such as poly (Glu60 Ala40) and poly (Glu60 Ala30 Tyr10). The non-immunoglobulin serum component has been identified as Gc globulin, a vitamin D-binding protein that has been shown to be present on the surface of B lymphocytes. This binding might be responsible for the murine B cell mitogenicity of poly (Tyr-Glu-Ala-Gly) that has been previously reported.

Immunogenicity in mice of the sequential polypeptides (Ala-Tyr-Glu-Gly)n, (Ala-Glu-Tyr-Gly)n, (Glu-Ala-Tyr-Gly)n and (Glu-Tyr-Ala-Gly)n and their linkage with the major histocompatibility complex.

The immunogenicity of the four sequential polymers of alpha-L-amino acids referred to below was studied in inbred strains of mice. The responses were linked to the H-2 haplotype, and the Ir genes controlling the responses were mapped to the IA region. The responses were restricted to two haplotypes as follows: (Ala-Tyr-Glu-Gly)n and (Ala-Glu-Tyr-Gly)n - H-2k; (Glu-Ala-Tyr-Gly)n - H-2b; (Glu-Tyr-Ala-Gly)n - H-2k and b. The absence of reciprocal crossreactions among all of these polymers, plus the sequential polymers (Tyr-Ala-Glu-Gly)n and (Tyr-Glu-Ala-Gly)n at both the antibody and T-cell levels, indicated that each of these six polymers was indeed structurally unique.

Murine responses to poly (Phe-Glu-Ala-Gly) and poly (Phe-Ala-Glu-Gly): comparisons between the specificities of the T-cell and B-cell responses.

The murine immune response patterns to (Phe-Glu-Ala-Gly)n and (Phe-Ala-Glu-Gly)n differ from those of the tyrosine analogues (Tyr-Glu-Ala-Gly)n and (Tyr-Ala-Glu-Gly)n. (Phe-Glu-Ala-Gly)n was not immunogenetic in inbred, congenic or recombinant strains of mice. (Phe-Ala-Glu-Gly)n was immunogenic only in mice having f alleles in the IA subregion of the H-2 complex. Reciprocal in vitro cross reactions were noted with T cells from mice of H-2f that respond to either (Phe-Ala-Glu-Gly)n or (Tyr-Ala-Glu-Gly)n. T cells from mice of H-2b, which are responders to (Tyr-Glu-Ala-Gly)n, could not be stimulated with the nonimmunogenic (Phe-Glu-Ala-Gly)n. These results support other studies showing that for any polymer to elicit cross T-cell proliferative responses it must be (a) structurally related to the homologous polymer and (b) immunogenic in the mouse strain whose T cells are being challenged in vitro. Explanations are offered for these statements.

Use of a synthetic peptidoglycan-precursor immunogen and its antibodies as a probe of infectious diseases in man.

Antibodies to a peptide sequence found in peptidoglycan (PG)- precursors (Lys-D-ala-D-Ala) have been found in man and their titers are elevated in several human diseases. Antibodies in rabbits were elicited by a synthetic immunogen containing these PG-precursor sequences, affinity purified with the precursor linked to Sepharose and shown to cross-react with bacterial by-products, such as the high molecular weight soluble peptidoglycan (SPG) from Staphylococcus aureus, which contain these sequences. SPG are secreted by some gram-positive bacteria when grown in the presence of penicillin. Serological studies have indicated that SPG may be the natural immunogen in man. The affinity purified antibodies cross-reactive with Lys-containing SPG plus vancomycin which has a similar specificity have been used to develop an ELISA capable of detecting as little as 50 pg/ml of SPG. Using this ELISA, about half of the human volunteers that took an oral 250 mg dose of penicillin V had detectable levels of SPG in their urine six hours later. There are structural differences among SPG which may be useful in identifying the bacteria responsible for the SPG. The SPG from Bacillus subtilis has diaminopimelic acid (A2pm) instead of lysine. Using the antibody to the synthetic immunogen, the A2pm-containing SPG gave a strong color reaction with the ELISA. However, the sensitivity was about an order of magnitude less than that for the Lys-containing SPG from S. aureus.

Antibodies to peptidoglycan in patients with spondylarthritis: a clue to disease aetiology?

Although the aetiology of the spondylarthritic diseases, ankylosing spondylitis and Reiter's syndrome, is obscure, a clue to the pathogenesis might be an animal model, adjuvant arthritis. Rats with this disease develop a spectrum of pathology with marked similarity to the spondylarthritides. Since peptidoglycan, a major cell wall component of most bacteria is causally implicated in adjuvant arthritis, we sought evidence that peptidoglycan exposure accompanies both Reiter's syndrome and ankylosing spondylitis. Antibodies to the D-Ala-D-Ala moiety of peptidoglycan were measured by a sensitive and specific ELISA. Antibodies were elevated significantly in patients with ankylosing spondylitis or Reiter's syndrome, but not in patients with rheumatoid arthritis or degenerative joint disease in comparison with normal controls. The findings should be considered preliminary, since only a minority of patients had increased antibody titres. However, the findings are compatible with the hypothesis that peptidoglycan is causally related to spondylarthritis. Antibodies to other moieties in the peptidoglycan molecule might be a more sensitive test for significant exposure.

Detection of soluble peptidoglycan in urine after penicillin administration.

A unique enzyme-linked immunosorbent assay was developed to detect soluble peptidoglycans in biological fluids. It makes use of the similar affinities of vancomycin and purified rabbit antibodies to peptidoglycan precursor sequences found in soluble peptidoglycans. This assay has been used to detect as little as 500 pg of soluble peptidoglycan per ml of serum and 5 pg/ml of urine. Studies of normal individuals and Staphylococcus aureus-infected patients revealed only a few sera with detectable levels of soluble peptidoglycans. Studies of normal volunteers who were given a single oral dose of 250 mg of penicillin VK showed that about half had detectable levels of soluble peptidoglycans in their urine up to 6 h after ingestion. This suggests that soluble peptidoglycans can be released by indigenous bacteria in detectable amounts. In one volunteer, a detectable level of soluble peptidoglycan in the urine at 6 h decreased to an undetectable level at 12 h. Such an ephemeral appearance of soluble peptidoglycan in the urine could account for the small number of human sera that had detectable levels of soluble peptidoglycan.

Soluble peptidoglycan from Staphylococcus aureus is a murine B-lymphocyte mitogen.

Soluble peptidoglycan from Staphylococcus aureus has been shown to be capable of causing murine B lymphocytes from the spleen to proliferate and to secrete immunoglobulins in both an in vitro and an in vivo assay. The optimal concentration in vitro was between 33 and 100 micrograms/ml. A 3-day incubation with soluble peptidoglycan was more stimulatory than was a 1- or 2-day incubation. Removal of most of the T lymphocytes with anti-theta serum did not result in any significant change in the mitogenic activity of soluble peptidoglycan on the remaining B cells.

Immunogenicity of four sequential polypeptides in inbred guinea-pigs and their recognition at the T lymphocyte and antibody levels.

Four sequential polypeptides containing equimolar amounts of tyrosine, glutamic acid, alanine and glycine were shown to be T lymphocyte-dependent immunogens in inbred guinea-pigs. Poly (Glu-Ala-Tyr-Gly) was immunogenic only in strain 2 guinea-pigs; poly (Glu-Tyr-Ala-Gly) and poly (Ala-Tyr-Glu-Gly) were immunogenic only in strain 13 guinea-pigs; and poly (Ala-Glu-Tyr-Gly) was immunogenic in both inbred strains. The specificity of immune recognition was probed at the T lymphocyte and humoral levels with the heterologous polypeptides. Only a few cases of heterologous cross-stimulation or cross-reaction were observed, indicating the great selectivity of immune recognition. The results showed considerable variability in immune recognition from animal to animal. Nevertheless, at the T-cell level, cross-stimulation appears to necessitate that the antigen is itself immunogenic in that strain, whereas at the antibody level, cross-reaction is not similarly restricted. The structural basis for mutual recognition of immunogen and antigen at these levels is discussed.

Evidence for the secretion of soluble peptidoglycans by clinical isolates of Staphylococcus aureus.

Four isolates of Staphylococcus aureus from patients with endocarditis and bacteremia were capable of secreting high-molecular-weight soluble peptidoglycans when grown in a minimal cell wall medium containing penicillin G. Vancomycin was not able to substitute for penicillin G in triggering this secretion. Secretion reflected de novo synthesis of soluble peptidoglycan and was strongly dependent on time of incubation (30 to 60 min), and number of bacteria (2 X 10(8) to 5 X 10(8) colony-forming units per ml), but not on penicillin G concentration (10 to 250 micrograms/ml). The incorporation of alanine into the peptidoglycans secreted in vitro by these isolates incubated in the presence of penicillin G under optimal conditions was variable. The least incorporation of alanine into peptidoglycan occurred with an isolate from a patient treated with nafcillin who had no detectable antipeptidoglycan titer.

Antibody levels to bacterial peptidoglycan in human sera during the time course of endocarditis and bacteremic infections caused by Staphylococcus aureus.

Sera from patients with endocarditis and bacteremia due to Staphylococcus aureus were compared for peptidoglycan-binding capacity with those from normal blood donors. Those patients treated with beta-lactam antibiotics had higher antigen-binding levels than normal donors and patients treated exclusively with vancomycin (P less than 0.01). The factor responsible for this activity was purified by affinity chromatography from a normal donor and shown to be an immunoglobulin. Specificity studies indicated that the immunodominant determinant was a peptide sequence found in peptidoglycan precursors. Since soluble peptidoglycan molecules having the precursor peptide sequence are known to be secreted by some gram-positive bacteria like Micrococcus luteus when grown in the presence of beta-lactam antibiotics, these soluble molecules may constitute the "natural" immunogen. Such a hypothesis is consistent with the study of the peptidoglycan-binding capacities in the sera of these patients during the course of treatment. For most of the responding patients studied (four of four with bacteremia and seven of nine with endocarditis), a significant increase in peptidoglycan-binding capacity was observed in sera taken 1 to 5 weeks after the initiation of beta-lactam antibiotic therapy (compared with the initial serum studied). No such increase in the peptidoglycan-binding capacity over a similar time span was noted in the sera of people not receiving beta-lactam antibiotics (none of seven).