Partial deletion of chromosome 8 ß-defensin cluster confers sperm dysfunction and infertility in male mice.

ß-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine ß-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that ß-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

Differential alveolar epithelial injury and protein expression in pneumococcal pneumonia.

The integrity of the alveolar epithelium is a key factor in the outcome of acute lung injury. Here, we investigate alveolar epithelial injury and the expression of epithelial-selective markers in Streptococcus pneumoniae-induced acute lung injury. S. pneumoniae was instilled into rat lungs and alveolar type I (RTI(40)/podoplanin, MMC6 antigen) and alveolar type II (MMC4 antigen, surfactant protein D, pro-surfactant protein C, RTII(70)) cell markers were quantified in lavage fluid and lung tissue at 24 and 72 hours. The alveolar epithelium was also examined using electron, confocal, and light microscopy. S. pneumoniae induced an acute inflammatory response as assessed by increased total protein, SP-D, and neutrophils in lavage fluid. Biochemical and morphological studies demonstrated morphologic injury to type II cells but not type I cells. In particular, the expression of RTI(40)/podoplanin was dramatically reduced, on the surface of type I cells, in the absence of morphologic injury. These data demonstrate that type II cell damage can occur in the absence of type I cell injury without affecting the ability of the lung to return to a normal morphology. These data also demonstrate that RTI(40)/podoplanin is not a type I cell phenotypic marker in experimental acute lung injury caused by S. pneumoniae. Given that RTI(40)/podoplanin is an endogenous ligand for the C-type lectin receptor and this receptor plays a role in platelet aggregation and neutrophil activation, we hypothesize that the reduction of RTI(40)/podoplanin on type I cells might be important for the regulation of platelet and/or neutrophil function in experimental acute lung injury.

Human ß-defensin 3 affects the activity of pro-inflammatory pathways associated with MyD88 and TRIF.

ß-Defensins are cationic host defense peptides that form an amphipathic structure stabilized by three intramolecular disulfide bonds. They are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in TLR4-stimulated macrophages. In the present study, we investigate the mechanism underlying the anti-inflammatory effect of human ß-defensin 3 (hBD3). We show that the canonical structure of hBD3 is required for this immunosuppressive effect and that hBD3 rapidly associates with and enters macrophages. Examination of the global effect of hBD3 on transcription in TLR4-stimulated macrophages shows that hBD3 inhibits the transcription of pro-inflammatory genes. Among the altered genes there is significant enrichment of groups involved in the positive regulation of NF-κB including components of Toll-like receptor signaling pathways. We confirm these observations by showing corresponding decreases in protein levels of pro-inflammatory cytokines and cell surface molecules. In addition, we show that hBD3 reduces NF-κB signaling in cells transfected with MyD88 or TRIF and that hBD3 inhibits the TLR4 response in both MyD88- and TRIF-deficient macrophages. Taken together these findings suggest that the mechanism of hBD3 anti-inflammatory activity involves specific targeting of TLR signaling pathways resulting in transcriptional repression of pro-inflammatory genes.

Integrating Physician Services in the Home: evaluation of an innovative program.

OBJECTIVE: To evaluate a new program, Integrating Physician Services in the Home (IPSITH), to integrate family practice and home care for acutely ill patients. DESIGN: Causal model, mixed-method, multi-measures design including comparison of IPSITH and non-IPSITH patients. Data were collected through chart reviews and through surveys of IPSITH and non-IPSITH patients, caregivers, family physicians, and community nurses. SETTING: London, Ont, and surrounding communities, where home care is coordinated through the Community Care Access Centre. PARTICIPANTS: A total of 82 patients receiving the new IPSITH program of care (including 29 family physicians and 1 nurse practitioner), 82 non-randomized matched patients receiving usual care (and their physicians), community nurses, and caregivers. MAIN OUTCOME MEASURES: Emergency department (ED) visits and satisfaction with care. Analysis included a process evaluation of the IPSITH program and an outcomes evaluation comparing IPSITH and non-IPSITH patients. RESULTS: Patients and family physicians were very satisfied with the addition of a nurse practitioner to the IPSITH team. Controlling for symptom severity, a significantly smaller proportion of IPSITH patients had ED visits (3.7% versus 20.7%; P = .002), and IPSITH patients and their caregivers, family physicians, and community nurses had significantly higher levels of satisfaction (P < .05). There was no difference in caregiver burden between groups. CONCLUSION: Family physicians can be integrated into acute home care when appropriately supported by a team including a nurse practitioner. This integrated team was associated with better patient and system outcomes. The gains for the health system are reduced strain on hospital EDs and more satisfied patients.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.

Peptide thioester synthesis through N-->S acyl-transfer: application to the synthesis of a beta-defensin.

Peptide thioesters readily prepared through N-->S acyl transfer of a specific C-terminal motif provide access to biologically active mini-proteins using native chemical ligation.

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types.

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.

Re-utilization outcomes and costs of minor acute illness treated at family physician offices, walk-in clinics, and emergency departments.

OBJECTIVE: To examine factors associated with re-utilization of health services and to estimate and compare costs of treatment for minor acute illnesses in family physicians' offices (FPOs), walk-in clinics (WICs), and emergency departments (EDs). DESIGN: Prospective cohort study using questionnaires, telephone follow up, medical chart data, and costs according to Ontario Health Insurance Plan (OHIP) schedules. SETTING: 16 FPOs, 12 WICs, and 13 EDs in three Ontario cities. PARTICIPANTS: Consecutive patients with one of eight predefined minor acute illnesses found in all three settings (upper respiratory infection, pharyngitis, acute bronchitis, acute otitis media, serous otitis media, low back pain, gastroenteritis, and urinary tract infection). MAIN OUTCOME MEASURES: "Early" (< 3 days) versus "later" (3 days to 2 weeks) re-utilization of health services after initial encounter and direct cost to OHIP. RESULTS: The overall rate of re-utilization of health services for the same episode of illness was 11.3% for early and 20.6% for later re-utilization. Factors associated with early re-utilization were initial evaluation in ED setting (odds ratio [OR] = 6.5, confidence interval [CI] = 2.2-19.2) and, regardless of setting, less satisfaction with patient-centred care (OR = 1.7 for each one-point decrease on a four-point scale; CI = 1.1-2.7). Factors associated with later re-utilization were ED setting (OR = 4.9; CI = 2.4-9.9) and diagnosis of urinary tract infection (OR = 2.4; CI = 1.1-5.2). Factors tested and found not signifcantly associated with rate of re-utilization were patients' age, sex, responses to a variety of questions assessing psychosocial factors (stress, social support, independence), and opinions on health care. Cost of care was similar for FPOs and WICs and higher for EDs for all diagnoses. The initial visit was the largest component of cost in all settings, and this component (as well as total cost) was consistently higher in EDs. CONCLUSION: Both re-utilization rates and costs are higher for those seeking care in EDs for minor acute illness. Patient-centred care, an important feature of health care encounters regardless of setting, can reduce re-utilization rates.

Patient satisfaction and quality of care in walk-in clinics, family practices and emergency departments: the Ontario Walk-In Clinic Study.

BACKGROUND: Although walk-in clinics are an increasingly common feature of Ontario's health care system, the quality of care they provide is the subject of continuing debate. In this study we examined differences in patient satisfaction and quality of care for common acute conditions in walk-in clinics, family practices and emergency departments. METHODS: For this prospective cohort study, we recruited 12 walk-in clinics, 16 family practices and 13 emergency departments from 11 geographic areas in greater Toronto, Hamilton-Burlington and London, Ont. An expert review panel selected and established quality-of-care criteria for 8 common acute conditions. Patients who sought initial care for 1 of the 8 conditions were recruited by an on-site data collector. We used a questionnaire to assess the satisfaction of 433 patients with patient-centred communication, the physician's attitude and any delay in the waiting room during the study visit. Abstractors reviewed 600 charts for the study patients to assess whether the quality-of-care criteria had been met. A quality score for each case was computed as the percentage of applicable criteria that were met. Mean quality scores for the 3 settings were computed, with adjustment for potentially confounding variables (sex, age, city and diagnosis). RESULTS: After adjustment for 12 patient characteristics, walk-in clinic patients were significantly more satisfied than emergency department patients on all 3 satisfaction scales. Family practice patients were more satisfied than walk-in clinic patients on all 3 satisfaction scales, but the difference was statistically significant only for satisfaction with waiting time. Adjusted mean quality-of-care scores were 73.1% for emergency departments, 69.9% for walk-in clinics and 64.1% for family practices. The scores for walk-in clinics and emergency departments were significantly higher than that for family practices. INTERPRETATION: Satisfaction with waiting time was highest among family practice patients. Both family practices and walk-in clinics were perceived more positively than emergency departments on all 3 dimensions of satisfaction. Overall quality-of-care scores were higher in walk-in clinics and emergency departments than in family practices.

Increased virulence of a fibronectin-binding protein mutant of Staphylococcus aureus in a rat model of pneumonia.

Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia.