Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages.

IL-10 is a key anti-inflammatory cytokine secreted by activated macrophages as a feedback control mechanism to prevent excessive inflammatory responses. Here, we define multiple intracellular trafficking pathways involved in the secretion of newly synthesized IL-10 from macrophages following TLR4 activation with LPS, and show how this relates to the previously defined trafficking pathways for IL-6 and TNF in macrophages simultaneously producing these proinflammatory cytokines. IL-10 exits the Golgi in multiple tubular carriers, including those dependent on p230GRIP. Some of the IL-10 is then delivered to recycling endosomes, where cytokine sorting may occur prior to its release. Another portion of the IL-10 is delivered to the cell surface in distinct vesicles colabeled for apoE. Thus, we show at least two post-Golgi pathways via which IL-10 is trafficked, ensuring its secretion from activated macrophages under different physiological conditions.

Intranasal immunisation with Toxoplasma gondii tachyzoite antigen encapsulated into PLG microspheres induces humoral and cell-mediated immunity in sheep.

Proteins from a crude extract of Toxoplasma gondii tachyzoites were encapsulated into poly(D,L-lactide-co-glycolide) (PLG) micro- and nano-particles with a mean encapsulation efficiency of 80%. An intranasal immunisation and infection experiment using 24 sheep was conducted to compare the immune responses elicited by intranasal administration of soluble and particulate T. gondii antigen (with and without cholera toxin). Sheep immunised with particulate toxoplasma antigen produced enhanced levels of both local and systemic antigen-specific IgA antibody, and showed increased cellular immune responses with a corresponding increase in IFNgamma production. After challenge with toxoplasma oocysts larger quantities of both nasal and systemic IgG were measured more rapidly in all animals immunised with toxoplasma antigen than animals infected with oocysts, suggesting a secondary-type IgG response. A slight modification of the febrile response to toxoplasma infection could be observed in animals immunised with particulate toxoplasma antigen and cholera toxin, although none of the immunised animals were protected against the challenge infection. These studies show that intra-nasal delivery has the potential to be an effective route for mucosal immunisation in sheep.

Characterization of ovine nasal-associated lymphoid tissue and identification of M cells in the overlying follicle-associated epithelium.

The distribution of mucosal lymphoid nodules in the ovine nasopharyngeal tract was studied by an acetic acid fixation technique. Nodules, which were concentrated just posterior to the opening of the Eustachian tube, were excised and examined by light microscopy, and scanning and transmission electron microscopy. Immunohistochemical examination revealed that each lymphoid structure consisted of follicles containing discrete B- and T-cell areas, characteristic of a mucosal inductive site of the mucosa-associated lymphoid tissue (MALT). Electron microscopy revealed that specialized epithelial cells, displaying features characteristic of M cells, were present in the follicle-associated epithelium (FAE) that covered the lymphoid nodules. These cells had sparse irregular microvilli and were closely associated with lymphocytes in the underlying tissue. These findings suggest that targeting the nasopharyngeal region may provide a practical and effective route for the stimulation of protective mucosal immune responses.

The use of delayed computerized tomography in the evaluation of blunt abdominal trauma: a preliminary report.

This purpose of this study was to analyze the use of abdominal computed tomography (CT) imaging in patients with possible blunt abdominal trauma. A retrospective analysis of all trauma patients over a 1-year period (1993-1994) was conducted, with prospective study protocol in 52 patients using serial abdominal exam and hematocrits (Hcts) instead of abdominal CT for evaluation of blunt abdominal trauma. Urgent abdominal CT was used as the initial diagnostic test for evaluation of blunt abdominal trauma in 813 patients over this 1-year period. CT was obtained in 379 (46.6%) of these patients who arrived hemodynamically stable (admission systolic blood pressure > or = 90), had a Glasgow Coma Scale > 13, and had admission Hct > or = 35 because of distracting injuries, possible traumatic brain injury, or alcohol/drug use, which might render the abdominal physical exam unreliable. Only 47 CT scans (12.4%) were positive, and three patients (0.8%) required laparotomy. In an effort to more efficiently use abdominal CT, we performed a prospective study in 52 patients with possible blunt abdominal trauma, admission systolic blood pressure > or = 90, Hct > or = 35, Glasgow Coma Scale > 13, and a normal abdominal exam on admission. These patients were followed with serial abdominal examinations and Hcts every 6 hours for 24 hours, and delayed CT, when applicable. CT was obtained in seven patients (13.5%) for evaluation of fall in Hct or abnormal abdominal examination; all were negative for abdominal injury. A protocol using serial abdominal exams, Hcts, and delayed abdominal CT imaging may be useful in select patients to decrease the high number of negative routine abdominal CTs that are obtained in the evaluation of blunt abdominal trauma.

Impact of a critical pathway on postoperative length of stay and outcomes after infrainguinal bypass.

PURPOSE: To determine the effect of a critical pathway on postoperative length of stay and outcomes after infrainguinal bypass. METHODS: A critical pathway for care of patients after infrainguinal bypass was introduced in December 1995 to coordinate postoperative care at our institution. We compared care of 67 consecutively treated patients before institution of the pathway with care of 69 consecutively treated patients with the critical pathway in place. Data collection was done by means of chart review. Univariate analyses were used to identify differences between prepathway and postpathway patients and to identify factors influencing postoperative length of stay. Multivariate analysis was used to identify factors that influenced length of stay and to examine the effect of use of the pathway after adjusting for other factors. RESULTS: Patients on the pathway were similar to prepathway controls with respect to comorbid illnesses, vascular risk factors, indications for surgical treatment, type of conduit, and type of operation. Factors associated with longer postoperative stays included distal anastomoses to tibial rather than popliteal vessels (p = 0.02), preexisting cardiac disease (p = 0.005), postoperative complications (p = 0.0003), lower preoperative hematocrit (p = 0.01), and elevated preoperative creatinine level (p = 0.006). Overall, pathway patients had somewhat shorter postoperative lengths of stay (median value 7 days; range 2 to 29 days) than prepathway patients (median value 6 days; range 2 to 35; p = 0.01), and the two groups had similar frequencies of postoperative complications, readmission, and 6-month mortality. However, patients on the pathway were more likely to be discharged to an intermediate-care facility rather than directly home. After 12 patients with extraordinarily prolonged postoperative stays were excluded, multivariate analysis indicated that pathway patients had significantly shorter postoperative stays (p = 0.001). However, the difference was not significant if patients with extraordinarily long postoperative stays were included in the analysis (p = 0.28). CONCLUSION: Use of a critical pathway was associated with a modest decrease in postoperative length of stay for most patients. This was accomplished without an adverse effect on readmission, complication, or mortality rates. However, the decrease in stay may have been achieved primarily by discharging more patients to intermediate-care facilities. The pathway did not appear to have any effect when the subset of patients with extraordinarily long stays because of complex medical problems was included.

Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors.

PURPOSE: Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood. The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb). METHODS: Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient. Dermal fibroblasts were isolated from explant cultures in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated. In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth. RESULTS: Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different. Normal-fb were compact and tapered, with well-defined nuclear morphologic features. Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features. Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb. Supplementation of media with growth factors stimulated the growth of wound-fb. Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008). Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant. CONCLUSIONS: Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb. These characteristics are typical of aged or senescent cells. This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta. Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers. Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.