A comparative study of the analysis of human urine headspace using gas chromatography-mass spectrometry.

First-void urine samples were obtained from 24 elderly, asymptomatic men (median age 62.9 years). The headspace above pH adjusted urine samples were extracted using a carboxen/polydimethylsiloxane solid phase micro-extraction fibre and the volatile organic compounds analysed by gas chromatography/mass spectrometry. A total of 147 compounds were identified in the headspace of urine. The acidified samples recorded a total of 92 compounds, 27 of which were ubiquitous, basified samples 70 compounds, with 12 ubiquitous and unmodified pH samples 49, with 6 ubiquitous. Five compounds were ubiquitous irrespective of pH: acetone, methylene chloride, 4-heptanone, 2-pentanone and 2-butanone. A comparative analysis of unfrozen and frozen-thawed urine (stored at room temperature for 0, 1 and 8 h) showed that samples retained the same number of compounds although variations in the peak areas for some compounds were observed.

Prostasomes--their effects on human male reproduction and fertility.

The prostate is a glandular male accessory sex organ vital for normal fertility. It provides the prostatic component of seminal plasma which nourishes and protects sperm following ejaculation. Prostasomes are small (40-500 nm) membrane-bound vesicles produced by epithelial cells lining the prostate acini and are a component of prostatic secretions. Although the existence of these particles has been known for many years, their full function and relevance to reproductive health are largely unknown. Proteomic studies have shown a wide range of proteins (enzymes, structural proteins and novel, unannotated proteins) present in or on the surface of prostasomes providing them with a diverse nature. Interestingly prostasomes are able to fuse with sperm, this event and the associated transfer of proteins lies at the heart of many of their proposed functions. Sperm motility is increased by the presence of prostasomes and their fusion prevents premature acrosome reactions. Prostasomes have been shown to aid protection of sperm within the female reproductive tract because of immunosuppressive, antioxidant and antibacterial properties. Clinically these functions imply a role for prostasomes in male factor infertility. However, the very functions that promote fertility may have negative connotations in later life; recent work has suggested that prostasomes are involved in prostate cancer. Clearly more work is needed to clarify the role of these novel particles and their impact on men's health.

Genetic ablation of interferon-gamma up-regulates interleukin-1beta expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain.

OBJECTIVE: To determine whether the lack of interferon-gamma (IFNgamma) alters resistance to collagen-induced arthritis (CIA) in a nonsusceptible mouse strain, and if so, to identify changes in the antibody, cellular type II collagen (CII)-specific immune responses, and cytokine gene expression that might account for the altered susceptibility. METHODS: CIA-resistant C57BL/6 and C57BL/6 IFNgamma-/- mice were immunized with bovine CII in Freund's complete adjuvant (CFA) or in CFA alone. Animals were monitored for signs of arthritis for up to 80 days; arthritis severity was assessed visually and histologically. Sera were collected at various time points after immunization for measurement of anti-CII antibody levels. T cell responses to bovine CII were assessed in proliferation assays. Cytokine messenger RNA (mRNA) expression in lymph node cells and in synovial cells from arthritic paws was measured by RNase protection assays, and levels of cytokine protein production were determined by enzyme-linked immunosorbent assay. RESULTS: IFNgamma-/- mice developed a severe autoimmune arthritis that was dependent on immunization with CII. IFNgamma-/- mice produced significantly higher amounts of IgG1 and IgG2b antibody to the autoantigen, murine CII, compared with wild-type C57BL/6 mice and had an enhanced T cell proliferative response to bovine CII. Enhanced production of mature interleukin-1/beta (IL-1beta) protein was observed, but no significant changes in Th1 or Th2 cytokines. Although IL-6 and tumor necrosis factor alpha transcripts were clearly evident in the synovial cells from the arthritic paws of IFNgamma-/- mice, neither message was elevated to the levels measured for IL-1beta expression. Treatment of IFNgamma-/- mice with anti-IL-1beta significantly reduced the incidence and severity of the inflammation. CONCLUSION: Endogenous IFNgamma plays a role in the regulation of IL-1beta, in this model of autoimmune arthritis.

Function and localization of oxytocin receptors in the reproductive tissue of rams.

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.

I-Aq and I-Ap bind and present similar antigenic peptides despite differing in their ability to mediate susceptibility to autoimmune arthritis.

Susceptibility to collagen induced arthritis (CIA) in the murine model is linked to expression of the MHC class II alleles, I-Aq and I-Ar. We have examined the molecular basis for this MHC-linked susceptibility by studying the antigen presentation function of two class II molecules, I-Aq and I-Ap, that are closely related yet differ in mediating susceptibility to CIA. These class II molecules differ by only 4 amino acids, yet only mice expressing I-Aq develop CIA. Although the I-Ap molecule can bind the same immunodominant determinant from type II collagen as I-Aq, H-2p APC have difficulty generating I-Ap:CII peptide complexes when processing of CII is required. Immunization of H-2p mice with type II collagen (CII) generated only a weak T cell response when compared to H-2q mice, whereas immunization with the a CII peptide containing the dominant determinant induced a strong T cell response in both strains. In antigen presentation assays, H-2p APC were very inefficient in stimulating T cells when native CII was used as antigen, however they presented CII synthetic peptides with similar efficiency as H-2q APC. Processing and presentation of other antigens by H-2p APC was not affected. Using soluble class II binding assays, the affinity of I-Ap for the CII dominant peptide was 10 to 50 fold lower than I-Aq, however, this reduced affinity was not a general defect in I-Ap function. I-Aq and I-Ap had virtually identical affinities for binding other antigenic peptides. These data indicate that MHC-based susceptibility to autoimmunity may involve more than simple determinant selection and that the successful generation of an antigenic peptide by processing may be related to the overall affinity of the peptide for the MHC molecule.

A national study of pressure ulcer prevalence and incidence in acute care hospitals.

OBJECTIVE: This study sought to establish national benchmarks for pressure ulcer prevalence and incidence among acute care health organizations served by Novation LLC. SETTING AND SUBJECTS: One hundred sixteen acute care facilities from 34 states participated; the sample consisted of 17,560 patients in hospital-based medical-surgical or intensive care units. INSTRUMENTS: Standardized education kits were provided to each participating site. The kits included an educational video about pressure ulcer staging, a post-test and answer key, and assessment form for patient data collection. METHODS: Pressure ulcer prevalence was measured during a predetermined 24-hour period at each facility. Incidence was measured over the average length of stay determined for each participating facility. Subjects were assessed by teams consisting of a registered nurse and one other health care professional (e.g. licensed practical nurse, physical therapist). Demographic, wound, and other data were collected for these patients. Data collection forms were audited and submitted to a central site for database entry, analysis, and generation of reports. RESULTS: The average length of stay for the participating facilities was 5 days. Pressure ulcers developed in 7% of the subjects (n = 383); 90% were stage I or II pressure ulcers, and 73% occurred in patients older than 65 years. The most sites based on both prevalence and incidence measurements were the sacrum and coccyx at 26% and 31%, respectively. CONCLUSIONS: Prevalence and incidence studies must be routinely conducted to clearly identify the extent of the pressure ulcer problem to provide guidance for efficient and effective corrective action.

Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions.

The contribution of leukocytes and of spermatozoa to reactive oxygen species (ROS) production in prepared sperm suspensions from donors and subfertility patients was compared. In both groups, more leukocytes/10(6) spermatozoa were counted in samples which produced detectable ROS than in those that did not: Donors-645 vs. 170 (medians, n = 7; p < 0.01, Kruskal-Wallis), Subfertile group-1785 (n = 18) vs. 11 (n = 8) (p < 0.005, Kruskal-Wallis), respectively. Leukocyte concentrations were correlated with basal (r = 0.826, p < 0.001) and with ROS production stimulated with 50 mumol N-formyl, met, leu, phe l-1 (N-FMLP) (r = 0.835, p < 0.001) and 100 nmol phorbol 12-myristate 13-acetate l-1 (PMA) (r = 0.835, p < 0.001) measured using a chemiluminescence assay. Leukocytes were removed from the sperm suspensions of 6 donors and from 96 ejaculates from 21 subfertility patients and ROS production was determined. Subsequently, in all 6 donors, N-FMLP did not stimulate ROS production indicating that leukocyte removal was complete, though in one case PMA stimulated low levels of ROS production. In 65 ejaculates from subfertile men the N-FMLP response was completely eliminated but in 7 of these samples PMA continued to stimulate ROS production. We conclude that infiltrating leukocytes are the predominant source of ROS production in unpurified sperm preparations. Some purified sperm suspensions could be stimulated to produce ROS by the addition of PMA indicating that spermatozoa themselves may produce ROS, albeit in much smaller amounts.

Reactive oxygen species (ROS) production and the outcome of diagnostic tests of sperm function.

Washed sperm suspensions from 64 out of 89 (72%) randomly selected infertility patients produced detectable reactive oxygen species (ROS) compared to 17 out of 67 (25%) prospective semen donors (p < 0.01, Chi-square test). Among patients, the median sperm concentration in ejaculates which yielded sperm suspensions that generated detectable levels of ROS was lower than in those which did not: 36.2 (15.63-57.64) vs. 71.5 (22-108) x 10(6)/mL, respectively (median (interquartile range), p < 0.05, Kruskal-Wallis test). In samples that produced ROS, the basal rate of production and the rates after stimulation with 50 mumol N-formyl met leu phe (N-FMLP) l-1 or with 100 nmol phorbol 12-myristate 13-acetate (PMA) l-1 were significantly and inversely correlated with sperm concentration in the ejaculate (r = -0.43, -0.41 and -0.35, respectively, p < 0.01 Spearman's rank correlation). The rate of ROS production showed no relationship to the motility of spermatozoa in semen, whether evaluated visually or via computer assisted semen analysis. However, there was a significant negative correlation (r = -0.370) between the motile, normal sperm concentration (MNSC) and basal ROS production, and when stimulated with N-FMLP (r = -0.311) or with PMA (r = -0.249) (all p < 0.05). In patient samples that generated detectable ROS, the ability of the spermatozoa to retain motility for 24 h after preparation on a 40/80% Percoll gradient was negatively correlated with basal ROS production (r = -0.310, p < 0.05). ROS production was also related to the outcome of in vitro sperm mucus penetration tests. Unstimulated levels of ROS production showed a significant (p < 0.05), negative correlation with the number of progressively motile spermatozoa present in mucus after 15 (r = -0.379) and 60 (r = -0.362) min. These results suggest that sperm samples with increased ROS tend to have poor semen quality and reduced performance in a number of routine, diagnostic sperm function tests.

The clinical and cost effectiveness of externally applied negative pressure wound therapy in the treatment of wounds in home healthcare Medicare patients.

Pressure ulcers, a devastating and costly healthcare problem, often occur in home healthcare settings. We sought to determine if these and other chronic wounds treated at home with negative pressure wound therapy close faster and reduce treatment costs compared to conventional therapies. Records for 1,032 Medicare home healthcare patients with 1,170 wounds that failed to respond to previous interventions--and were subsequently treated with negative pressure wound therapy--were reviewed. Reductions in wound area and volume were compared to rates reported by Ferrell in 1993, and costs were analyzed. Ferrell reported trochanteric and trunk pressure ulcers averaging 4.3 cm2, treated with a low-air-loss surface and saline-soaked gauze closed at an average of 0.090 cm2 per day. For comparison to Ferrell's outcomes, we analyzed our Stage III and IV trochanteric and trunk wounds treated with low-air-loss and negative pressure wound therapy. Ours averaged 22.2 cm2 in area and closed at an average of 0.23 cm2 per day. The average 22.2 cm2 wound in our study, treated as described by Ferrell, would take 247 days to heal and cost $23,465. Using negative pressure wound therapy, the wound would heal in 97 days and cost $14,546. The study concluded that negative pressure wound therapy is an efficacious and economical treatment modality for a variety of chronic wounds.

Managing pressure ulcers: a multisite CQI challenge.

Think preventing and tracking pressure ulcers is expensive? Treatment may cost more. The Nurse Executive Council of VHA Alabama partnered with KCI USA, Inc., to implement a CQI project to manage pressure ulcer incidence.

The effect of incubation periods under 95% oxygen on the stimulated acrosome reaction and motility of human spermatozoa.

Human sperm samples were prepared on a 30% Percoll gradient and reactive oxygen species (ROS) production was measured. In samples that generated ROS incubation under 95%O2:5%CO2 for 30 min decreased the proportion of spermatozoa capable of the stimulated acrosome reaction by 40% in comparison to samples incubated under 95%N2:5%CO2 (P< 0.001, repeated measures analysis of variance), but the degree of inhibition did not increase after more prolonged incubation periods (up to 6 h). The addition of the antioxidants catalase and superoxide dismutase prevented the inhibitory effect of 95%O2:5%CO2. Leukocyte removal from samples prior to 95%O2:5%CO2 incubation preserved the ability of the spermatozoa to acrosome react. Sperm motility parameters were less affected by 95%O2:5%CO2 but track velocity was 64.1 microm/s+/-1.96 after 2 h incubation under 95%N2:5%CO2 compared with 54.7 microm/s+/-1.41 after 2 h incubation under 95%O2:5%CO2 (P < 0.05, repeated measures analysis of variance). Sperm samples that did not generate detectable ROS were not affected by 95%O2:5%CO2. The toxic effects of incubation under 95%O2:5%CO2 on human spermatozoa result from increased endogenous ROS production, mostly from leukocytes. High ROS levels inhibit sperm function, with the stimulated acrosome reaction being more susceptible than motility parameters.

Characterization of signal transduction through the TCR-zeta chain following T cell stimulation with analogue peptides of type II collagen 260-267.

The immunodominant T cell determinant of type II collagen (CII) recognized by DBA/1 mice (I-Aq) is CII 260-267. The aims of this study were to determine the role of the amino acid residues within CII 245-270 in T cell signal transduction. To that end, we utilized I-Aq-restricted, CII-specific T cell hybridomas and examined tyrosine phosphorylation of TCR-zeta following stimulation with either wild-type CII 245-270 or a panel of analogue peptides. A variety of patterns occurred, ranging from increased phosphorylation of TCR-zeta to either partial or a complete abrogation of phosphorylation. Critical substitutions also completely abrogated the phosphorylation of ZAP70, a downstream molecule in TCR-zeta signaling. Evaluation of the supernatants of the T cell hybridomas for cytokine production in response to the peptides revealed a close correlation between the induction of phosphorylation of TCR-zeta and the amount of cytokine induced. Selected analogue peptides were tested as tolerogens in neonatal mice. Analogues that did not induce the phosphorylation of zeta chain, such as B3 (CII 251-270s263F-->N), were completely unable to induce tolerance, while analogues that caused a partial phosphorylation, such as B6 (CII 251-270s267Q-->T) and A3 (CII 245-270s269P-->A), induced partial tolerance judged by intermediate degrees of suppression of arthritis. We conclude that discrete alterations in specific amino acid residues of antigenic peptides had profound effects on T cell signaling and that the signaling correlated with T cell cytokine secretion and T cell function in the induction of tolerance and suppression of arthritis.

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen.

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.

An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Reactive oxygen species in human sperm suspensions: production by leukocytes and the generation of NADPH to protect sperm against their effects.

In men from couples consulting for infertility, 72% of washed sperm preparations produced detectable amounts of reactive oxygen species (ROS) compared to only 25% of preparations from a population of fertile donors. High ROS production was associated with oligozoospermia and poor sperm function. The rate of ROS production was closely correlated with the concentration of leukocytes (r = 0.826) and the degrees of stimulation by N-formyl-methionyl-leucyl-phenyl alanine (NFMLP) and by phorbol 12-myristate 13 acetate (PMA) were similar (r = 0.923). The removal of leukocytes with 'Dynabeads' either abolished or substantially decreased ROS production but in a few cases ROS production that could be stimulated by PMA but not NFMLP was observed in leukocyte-free preparations. When sperm preparations which produced ROS were incubated under 95% O2 there was a rapid 40% decrease in the number of sperm that could be stimulated to acrosome react although the acrosome reaction was unaffected by incubation under 95% N2 for up to 6 h. The harmful effect of oxygen was not seen in preparations that produced no ROS and could be prevented by removing leukocytes from the suspension or by adding superoxide dismutase and catalase. We conclude that leukocytes are the predominant source of ROS in human sperm preparations and that the ROS they produce are harmful to sperm. On the other hand these data confirm that highly purified sperm can produce ROS albeit in smaller amounts. We have demonstrated that flux through the pentose phosphate pathway (PPP) in purified sperm preparations increases in response to oxidative stress. This is required to make reduced glutathione available for glutathione peroxidase and we suggest that measurement of PPP flux provides an index of the capacity of glutathione peroxidase to protect sperm against oxidation.

Identification of MHC class II and TCR binding residues in the type II collagen immunodominant determinant mediating collagen-induced arthritis.

Collagen-induced arthritis (CIA), an autoimmune arthritis model, is elicited by the immunization of genetically susceptible strains of mice with type II collagen (CII). We have analyzed the molecular interactions that occur during the presentation of the immunodominant determinant within CII(257-270) by the murine class II susceptibility allele, I.Aq. Utilizing a soluble I-A binding assay and clonally distinct CII-specific T cells, we have identified the residues that control the ability of the CII(257-270) peptide to bind to I-Aq and those that interact with the TCR. In competitive binding assays with a panel of analog peptides, only two residues within CII(257-270) were found to participate in the binding of this peptide to I-Aq, residues 260 (Ile) and 263 (Phe). When these substitutions were combined into a single peptide, no binding of the peptide to I-Aq could be detected. Although no other substitutions decreased the binding affinity of the peptides, substitution of several amino acid residues lying outside of the determinant core increased the peptide's affinity for I-Aq and in some instances greatly enhanced the potency of the peptide in stimulating T cells. In antigen presentation assays, clonotypic variation in the recognition of several analog peptides indicated that residues 261, 262, 264, 266, and 267 are likely TCR contact sites. Since residue 266 interacts with the TCR and is the only residue in this determinant that differs between chick/bovine CII and mouse CII, these data indicate that immunity to the autoantigen may play a role in this model.

Vaccination with a recombinant V alpha domain of a TCR prevents the development of collagen-induced arthritis.

A recombinant TCR domain, derived from a T cell hybridoma that recognizes an immunodominant type II collagen epitope, was used to vaccinate against collagen-induced arthritis in DBA/1 (H-2q) mice. The recombinant TCR domain comprises VA11.1-JA17 gene segments and is representative of the V alpha domains expressed by oligoclonal T cells in this disease model. Vaccination of mice 28 days before type II collagen (CII) immunization with this V alpha 11.1 domain resulted in a significantly decreased incidence of arthritis in DBA/1 mice, in contrast to vaccination with a V alpha 4-J alpha 40 domain derived from an encephalitogenic T cell hybridoma specific for MBP. Disease blockade is accompanied by a reduction in T and B cell responses to both the immunogen bovine CII and the autoantigen murine CII. V alpha 4 and V alpha 11.1 domains were found to be highly immunogenic in DBA/1 mice, inducing both T cell proliferation and the production of V alpha specific Abs, indicating that the vaccination effect of V alpha 11.1 is specific. This is the first report of V alpha-directed immunotherapy in an autoimmune disease model and demonstrates the potential use of recombinant TCR vaccines in the treatment of autoimmune diseases that involve oligoclonal autoreactive T cells.

Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine alpha 1(II)-CB11 in H-2q mice.

Collagen-induced arthritis (CIA) is an experimental autoimmune disease elicited in genetically susceptible strains of mice by immunization with heterologous type II collagen. This experimental disease is mediated by the immune response of both T and B cells, and susceptibility is restricted by the class II molecules of the MHC. To study the T cell determinants of bovine type II collagen (CII) that mediate the autoimmune response in H-2q mice, we have identified a cyanogen bromide fragment of bovine CII, CII(124-402), that induces arthritis in DBA/1 mice. Using an overlapping set of peptides to map the T cell response to CII(124-402), we have determined that the I-Aq-restricted T cell response to this collagen fragment is mediated by a single immunodominant antigenic determinant. Consequently, this determinant plays a central role in promoting the production of the collagen-specific Abs and the induction of CIA in H-2q mice. Characterization of this immunodominant determinant revealed that the core residues required for T cell stimulation consists of only eight amino acids and is located at amino acids 260 through 267 of bovine CII. The systematic analysis of the contribution of each of these amino acids, in conjunction with sequences of other peptides known to bind to I-Aq, have allowed us to propose a peptide binding motif for the collagen arthritis susceptibility allele, I-Aq.