Specific characteristics of peritoneal leucocyte populations during sterile peritonitis associated with icodextrin CAPD fluids.

BACKGROUND: Icodextrin dialysate used for peritoneal dialysis contains an iso-molar glucose polymer solution, which provides sustained ultrafiltration over long dwell times and is considered a valuable approach to reduce intraperitoneal glucose exposure. However, several side effects have been described, including abdominal pain and allergic and hypersensitivity reactions. Also, reactions compatible with chemical peritonitis have been reported. Over the period of a few months (January 2002-May 2002), a remarkable increase in the number of continuous ambulatory peritoneal dialysis (CAPD) patients using icodextrin dialysate diagnosed with sterile peritonitis was observed in our unit. METHODS: Five of the CAPD patients using icodextrin dialysate in our unit and diagnosed with sterile peritonitis were screened for leucocyte count and leucocyte differentiation during a follow-up period of 77 +/- 23 days. In addition, expression of CD14, a receptor for lipopolysaccharide (LPS), on the peripheral and peritoneal monocyte population was analysed. These results were compared to CAPD patients suffering from bacterial peritonitis. RESULTS: The peritoneal leucocyte count of CAPD patients using icodextrin dialysate and diagnosed with sterile peritonitis did not decrease significantly before treatment with icodextrin dialysate was interrupted, whereas it currently disappeared within 2-4 days in proven bacterial peritonitis. The sterile, cloudy icodextrin effluent contained an excess of macrophages on the day of diagnosis, whereas in bacterial peritonitis essentially an increase in the granulocyte population was observed. No elevation in the eosinophil population was observed. In contrast to bacterial peritonitis, we observed no increase in CD14 expression on the peripheral and peritoneal macrophages on the day of presentation and during the follow-up period. CONCLUSIONS: Specific batches of the icodextrin CAPD fluids contain a macrophage chemotactic agent, which causes a sustained inflammatory state in the peritoneal cavity. Because no increase in the expression of the LPS receptor CD14 could be observed, the increased peritoneal leucocyte count is probably not caused by LPS or LPS-like (possibly peptidoglycan-like) contamination.     

Glucose degradation products in peritoneal dialysis fluids: do they harm?

BACKGROUND: Severe limitations in biocompatibility of conventional peritoneal dialysis fluids (PDF) can be partially attributed to the presence of glucose degradation products (GDP), which are generated during autoclaving of PDF. Formation of GDP can be significantly reduced by the use of multi-chamber bag systems. Recent clinical studies have revealed increased dialysate levels of pro-collagen I C-terminal peptide (PICP) in patients dialyzed with these solutions. Here, we briefly review the current knowledge on various aspects of GDP toxicity toward peritoneal cells and analyze the impact of GDP on PICP release by human peritoneal mesothelial cells (HPMC) in vitro. METHODS: HPMC were exposed to a mixture of known GDP added to culture medium at clinically relevant doses. After 12 days, the amount of PICP released was measured using an immunoassay. Furthermore, the protein synthesis was assessed by 3H-proline incorporation in HPMC exposed to peritoneal effluent obtained from patients after three months of CAPD with either conventional PDF or low-GDP solution. RESULTS: Exposure to GDP resulted in a significant decrease in PICP release by HPMC. In addition, the synthesis of new proteins secreted by HPMC was preserved significantly better in HPMC treated with effluent obtained when patients were dialyzed with low-GDP solutions rather than conventional PDF. CONCLUSIONS: Exposure to GDP may impair protein synthesis and secretion by HPMC. Therefore, increased dialysate PICP levels in response to GDP-free PDF may be viewed as evidence of improved mesothelial cell function.     

Host Defence in Continuous Ambulatory Peritoneal Dialysis: The Effect of the Dialysate on Phagocyte Function

Peritoneal dialysis fluid was examined after dwell periods offrom 30 mm to 18 h. Macrophages formed more than 70% of allcells recovered, irrespective of dwell time. The viability ofthese cells was 95% or greater even in 30 mm effluent. Peritoneal macrophages and polymorphonuclear leucocytes wereincubated in unused peritoneal dialysis fluid. By 30 mm theviability of polymorphonuclear leucocytes had fallen to 50%but that of peritoneal macrophages was still 84%. However, phagocytosisof unopsonised zymosan by both cell types was depressed afteronly 10 mm exposure. Peritoneal dialysis effluents obtained after dwell times of30–180 min were examined for their effect on phagocytosisin vitro. These fluids suppressed peritoneal macrophages functionas compared to RPMI 1640. Effluent after an overnight dwelldid not affect phagocytosis. The suppressant effect decreasedwith increasing dwell time. Polymorphonuclear leucocytes wereaffected to a greater degree than peritoneal macrophages. Tests showed that this decreased phagocytosis was not due tocell death nor was it due to the osmolality or lactate contentof the dialysate. Adjusting pH only improved cell function slightly. Phagocyte function appears to be depressed for clinically significantperiods of the CAPD cycle.     

Contribution of mononuclear leucocytes to the progression of experimental focal glomerular sclerosis

Uninephrectomized Sprague-Dawley rats repeatedly administered with aminonucleoside of puromycin and protamine sulfate developed progressive focal glomerular sclerosis (FGS). The contribution to disease progression of both glomerular and interstitial infiltrating leucocytes was studied throughout the disease evolution. Leucocyte subsets were quantitated with an immunoperoxidase technique using monoclonal antibodies for rat leucocyte surface antigens: OXI (total leucocytes) OX6 (Ia positive cells), OX8 (suppressor/cytotoxic T cells), OX19 (total T cells), OX22 (B cells and subsets of T cells), and ED1 (macrophages/monocytes). In the glomeruli, macrophages and Ia positive cells were significantly increased when sclerotic lesions appeared, but T lymphocytes and subsets of T lymphocytes were not found. However, in the interstitium, all leucocytes were identified and increased in number throughout the disease evolution. Early in the disease, monocytes and lymphocytes were both present in large numbers, but at the end stage of the process, the predominant infiltrating leucocytes were CD4+ve T cells. In FGS rats treated throughout the disease with oral prednisolone (begun after disease induction), renal function was significantly better than in the untreated group, whereas the sclerosis and leucocyte accumulation in the glomeruli were unchanged. However, prednisolone treatment resulted in significantly fewer interstitial leucocytes and especially reduced the numbers of CD4+vc cells. These results suggest that the glomerular sclerotic lesions are related to the participation of macrophages independent of T cells, and that immune mechanisms mediated by T cells in the interstitium have an important role in the progression of this disease to end-stage renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal interstitial tenascin immunostaining and immune cell infiltration in IgA nephropathy

SUMMARY: Interstitial expression of tenascin and interstitial leucocyte infiltration were examined by an indirect immunoperoxidase method using monoclonal antibodies against tenascin, CD45 (all leucocytes), CD45RO (T cells) and CD68 (monocytes/macrophages) on renal biopsy specimens from 25 patients with mesangial proliferative IgA-positive glomerulonephritis (IgAN). Ten biopsy kidney specimens, which were removed because of renal trauma, were used as the control group. In patients with IgAN, the mean interstitial expression of tenascin was significantly higher than in the control group. Strong tenascin staining was detected in areas with interstitial damage. In patients with IgAN there were positive correlations between the interstitial expression of tenascin and the relative interstitial cortical volume, as well as serum creatinine. In the IgAN patents, a significant increase in the total number of interstitial CD45-immunopositive cells, CD45RO-positive and CD68-positive cells was seen compared with the control group. In patients with IgAN, immunostaining of tenascin did not correlate with the number of T-cells, monocytes/macrophages or all leucocytes in the renal interstitium. These results suggest that in patients with IgAN the interstitial accumulation of tenascin did not depend on the type or the density of interstitial inflammatory infiltrates.     

Genotyping: a new application for the spent dialysate in peritoneal dialysis.

BACKGROUND: The dialysate of patients on peritoneal dialysis (PD) is used to determine the concentration of growth factors and cytokines, and as a source of resident peritoneal cells for subsequent culture experiments. We hypothesized that the cells contained in spent dialysate samples obtained at the time of the peritoneal equilibration test (PET) and subsequently stored may represent a source of DNA from a given PD patient. METHODS: We characterized a protocol of DNA extraction from dialysates obtained in PD patients after a long dwell during the initial PET and kept frozen up to several years. After amplification of the source DNA by strand displacement using the bacteriophage phi 29 DNA polymerase, we performed polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for the Glu298Asp polymorphism of ENOS to demonstrate the suitability of the extracted DNA for genotyping. RESULTS: A significant amount of DNA (mean yield 12 microg/ml dialysate) was extracted from frozen dialysate samples. The extraction yield was not influenced by the duration of storage at -20 degrees C. Following amplification, the DNA extracted from the dialysate was used successfully for genotyping the Glu298Asp polymorphism of ENOS, as demonstrated by parallel analyses using DNA extracted from the peripheral blood and sequencing. CONCLUSIONS: These results demonstrate that the dialysis effluent obtained at the time of the initial PET and stored at -20 degrees C is a reliable source of DNA that can be used subsequently for PCR amplification, RFLP analysis and sequencing.     

Effect of immunosuppression on damage,leukocyte infiltration,and regeneration after severe warm ischemia/reperfusion renal injury

BACKGROUND: Post-ischemia/reperfusion (I/R) damage, accompanied by leukocyte infiltration, is unavoidable in renal transplantation, as is the need for immunosuppressive treatment. Influence of immunosuppressive treatment on post-I/R renal damage, nonalloimmune cellular infiltration, and regeneration is not well studied. METHODS: Uninephrectomized inbred LEW rats were submitted to warm renal ischemia of 45 minutes/60 minutes, and received different immunosuppressive regimens: cyclosporine (CsA) 10 mg/kg/day subcutaneously in the neck daily, or mycophenolate mofetil (MMF) 20 mg/kg/day by daily oral gavage. Control animals underwent sham operation (unilateral nephrectomy) with immunosuppressive treatment or ischemia with vehicle administration. In addition the effect of MMF/mycophenolic acid (MPA) on renal tubule cell proliferation in culture was studied with bromodeoxyuridine incorporation. RESULTS: The post-I/R interstitial cellular infiltration/proliferation consisted mainly of mononuclear leukocytes [first monocytes/macrophages (Mo/MPhi) followed by CD4+ cells]. This mononuclear cell infiltration became apparent 24 hours after injury at the time of acute tubular necrosis, and was most prominent during the phase of regeneration. Severe I/R combined with CsA aggravated morphologic damage and dysfunction, without effect on tubular cell proliferation and tubular regeneration. Early leukocyte infiltration was qualitatively and quantitatively comparable to control animals, yet decreased moderately later in time. In contrast, MMF in combination with severe I/R did not influence initial morphologic damage and dysfunction. Although the initial leukocyte infiltration was comparable to control animals, the subsequent mononuclear cell accumulation, especially CD4 T cells decreased dramatically during MMF treatment. This was concomitant with a decrease of tubular cell proliferation and hence tubular regeneration. Increasing MPA concentrations in renal tubular cell culture caused a significant decrease in total cell number, and an almost arrest of bromodeoxyuridine incorporation, as measurement of cell proliferation. CONCLUSION: Immunosuppressive treatment with CsA or MMF affected significantly and in a different manner post-I/R renal morphologic damage, interstitial leukocyte, accumulation and regeneration.     

Generation of functional islet-like clusters after monolayer culture and intracapsular aggregation of adult human pancreatic islet tissue

BACKGROUND: Cellular replacement therapy represents a promising strategy for treating type I diabetes; however, such an approach is limited due to the inadequate availability of human donor tissue. Here we investigated the extent to which human islet tissue can be expanded in monolayer culture and brought back to islet function. METHODS: Adult human pancreatic cells were proliferated with a serum-free media in monolayer cultures through multiple passages. Expanded cells were dispersed and encapsulated in alginate-poly-l-lysine microcapsules wherein the cells spontaneously coalesced into islet-like clusters. Encapsulated cell clusters were subsequently transplanted into the peritoneal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: The cultured monolayer cells secreted insulin in response to glucose stimulation and maintained endocrine gene expression. Encapsulated islet-like clusters displayed cellular architecture similar to freshly isolated and encapsulated adult human islets maintained in culture, exhibiting an immunoreactive core of insulin, glucagon, and somatostatin, as well as peripheral cytokeratin-19 staining. Encapsulated aggregates significantly reduced hyperglycemia in transplanted mice within 1 week and normoglycemia was achieved after 5 weeks. Human C-peptide was detected in transplanted mice concomitant with the reduction in hyperglycemia. Capsules recovered 8 weeks posttransplantation exhibited insulin immunoreactivity. CONCLUSIONS: Collectively, these data indicate that adult human pancreatic islet cells can be expanded by three serial passages while maintaining their endocrine properties and can yield functional islet-like cell clusters through intracapsular aggregation that reverse hyperglycemia in diabetic mice. This culture and aggregation process could serve as a platform for proliferation and differentiation studies of endocrine lineage cells.     

Leucocyte subpopulations in the seminal plasma and their effects on fertilisation rates in an IVF cycle

Controversy exists on the role of leucocytospermia on fertilisation rates and IVF outcomes. The aim of our study was to identify the effect of leucocytes and leucocyte subpopulations on fertilisation rates in an IVF cycle. A prospective comparative study of the leucocyte subpopulations of seminal fluid of partners of women attending an IVF cycle was conducted. The samples underwent immunocytochemical staining. The monoclonal antibodies used in this study include CD3, CD4, CD8 (T Cells), CD14 (monocytes/macrophages), CD16 (granulocytes), CD20 (B Cells), CD45 (Pan Leucocytes), CD56 (natural killer cells) and CD69 (activated T and B Cells). Of 21 patients who were recruited into the study, seven were identified as poor fertilisers (<35%) and 14 were identified as good fertilisers (>60%). Data were analysed with SPSS version 14. The total leucocyte counts (CD45) between the poor and good fertilisers were not statistically significant. The macrophages and the monocytes (CD14) were significantly elevated in the good fertilisers group in comparison with the poor fertilisers (P < 0.05). We also found that T cells (CD2, CD4, CD8) and CD14 (macrophages) correlated significantly (r = 0.47, P value < 0.01) with the fertilisation rate. Our study confirms that the presence of leucocytes does not adversely affect the fertilisation rates and the outcome of an IVF cycle. However, macrophages and the monocytes (CD14) were significantly elevated in the good fertilisers group. The increased phagocytic activity in these individuals might increase their fertilising potential by removing spermatozoa with abnormal morphology.     

Intercellular adhesion molecule-1 mediated interactions and leucocyte infiltration in IgA nephropathy

Background: Mononuclear leucocytes have a role in IgA nephropathy (IgAN). Renal leucocyte recruitment is mediated by adhesive interactions between leucocytes and their ligands on renal cells. Methods: We have assessed interstitial and glomerular leucocytes by avidin-biotin-peroxidase with monoclonal antibodies (MA) against leucocytes (CD45), {beta}2-integrin (CD18), monocyte-macrophages (CD14), T (CD3) and T-cell subsets (CD4, CD8), and intercellular adhesion molecule 1 (ICAM-1) (CD54), and analysed their relation with the abnormal expression of ICAM-1 on proximal tubule epithelium in sequential renal sections from 48 patients with IgAN stratified according to the severity of the interstitial cellular infiltration observed by light microscopy. Results: In IgAN without (n=15) and with (n=7) interstitial cellular infiltration of 1+, ICAM-1 expression on vascular endothelium was unchanged with respect to that observed in the normal kidney; the proximal tubule epithelium was negative for this stain. In IgAN with interstitial cellular infiltration of 2+ (n=10), 3+ (n=11), and 4+ (n=5), ICAM-1⁺ stain was observed on the proximal tubule epithelium, the median value of its quantitative expression being 0.3, 0.1, and 0.2 (P=0.0008), respectively. The tubular ICAM-1⁺ stain was significantly associated with the interstitial leucocytes identified by MA, and correlated with CD45⁺ (r=0.59, P=0.02), CD14⁺ (r=0.54, P<0.02), and CD3⁺ (r=0.51, P=0.02) interstitial leucocytes in IgAN with interstitial cellular infiltration. Interstitial ICAM-1⁺ and CD18⁺ leucocytes were correlated (r=0.56, P<0.001). Correlation was found between the quantitative tubular expression of ICAM-1⁺ and the number of CD45⁺ (r=0.98, P<0.0001), CD3⁺ (r=0.48, P=0.02), and CD8⁺ (r=0.76, P<0.02) glomerular leucocytes. Conclusion: Our results suggest that tubular and interstitial ICAM-1⁺ cells may participate in adhesive interactions with interstitial leucocytes. Interstitial T-cells and macrophages as well as glomerular T-cells bearing predominantly CD8⁺ phenotype could play a role in the induction of the tubular expression of ICAM-1 in IgAN.     

Peritoneal dialysis with solutions low in glucose degradation products is associated with improved biocompatibility profile towards peritoneal mesothelial cells.

BACKGROUND: In vitro experiments point to a better biocompatibility profile of new pH-neutral peritoneal dialysis fluids (PDFs) containing low levels of glucose degradation products (GDPs). The present study examines the impact on human peritoneal mesothelial cells (HPMCs) of equilibrated dialysates obtained during dialysis with either conventional or new PDFs. METHODS: Peritoneal dialysate was collected from 17 patients participating in a randomized, controlled, cross-over trial comparing a pH-neutral low-GDP solution (Balance) to a conventional solution (S-PDF). All patients were treated sequentially for 3 months with both PDFs. At the end of each treatment phase, peritoneal effluent was drained after a timed 10 h dwell. Samples of dialysate were then mixed with standard culture medium and added to in vitro cultures of HPMCs from healthy donors. Cells were assessed for proliferation, viability and cytokine release. RESULTS: Proliferation and viability of HPMCs were better preserved in the presence of effluent obtained during dialysis with Balance (P<0.046 and P<0.035, respectively). The proliferative response of HPMCs correlated with the concentration of fibronectin in dialysates (P = 0.0024). Effluent drained following a 3 month dialysis with Balance contained significantly increased levels of fibronectin (P = 0.004) and CA125 antigen (P = 0.0004) compared with S-PDF. There was no significant difference in constitutive and stimulated cytokine (IL-6, MCP-1, VEGF) synthesis by HPMCs treated with either Balance- or S-PDF-derived effluents. CONCLUSIONS: These results suggest that therapy with new pH-neutral low-GDP solutions contribute to an intraperitoneal milieu that improves mesothelial cell proliferation and viability. It may positively impact on the preservation of the peritoneal membrane integrity during long-term dialysis.     

Response of human peritoneal mesothelial cells to inflammatory injury is regulated by interleukin-1b and tumor necrosis factor-a

Peritoneal injury is often associated with alterations of the mesothelium, resulting in peritoneal healing and adhesion formation. We analyzed the effects of pro-inflammatory cytokines on cell morphology and proliferation of human peritoneal mesothelial cells (HPMC). After 48 hours, HPMC formed a confluent layer with cell volumes of 2,662+/-111 fL. Treatment of HPMC with interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) induced mesothelial disintegration and alterations in mesothelial cell morphology, which were associated with an interleukin-1beta-triggered increase in cell volume (3,028+/-118 fL; p<0.05) and exfoliation of cells into the supernatants of cell cultures (p<0.05). Whereas TNF-alpha arrested HPMC in the G0/G1 phase (p<0.05), interleukin-1beta caused an increase of cells into the S phase of the cell cycle. In addition, interleukin-1beta and interferon-gamma exerted a proliferative effect on HPMC. These changes were independent from mesothelial Na+/H+ antiporter-1 expression. Our data indicate that the response of HPMC to inflammatory injury is regulated by interleukin-1beta and TNF-alpha reflecting their putative role in peritoneal wound healing and adhesion formation.     

Inhibition of cytokine synthesis by peritoneal dialysate persists throughout the CAPD cycle.

The current study focused on the effect of continuous ambulatory peritoneal dialysis (CAPD) dialysate obtained following different intraperitoneal dwell periods on the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha) from mononuclear leukocytes (PBMC). Aliquots of 5 x 10(6)/ml healthy peripheral PBMC were exposed to fresh or spent CAPD dialysate (10-240 min of intra-peritoneal dwell) and stimulated with Escherichia coli endotoxin (10 micrograms/ml, 2h). IL-6 and TNF alpha in cell supernatants were determined by specific enzyme immunoassays. Control PBMC in physiological buffer released 361 +/- 70 pg/ml IL-6 and 717 +/- 147 pg/ml TNF alpha (mean +/- SEM, n = 8), whereas exposure to fresh dialysis fluids severely suppressed cytokine release from PBMC (less than 30 pg/ml IL-6 and less than 15 pg/ml TNF alpha). A significant inhibition of IL-6 and TNF alpha release was also observed in PBMC exposed to spent dialysate. The inhibitory capacity of the spent fluids was pronounced with increasing intra-peritoneal dwell time (10 min: 183 +/- 45 pg/ml IL-6 and 538 +/- 109 pg/ml TNF alpha; 240 min: 26 +/- 5 pg/ml IL-6 and 105 +/- 30 pg/ml TNF alpha; mean +/- SEM, n = 16). These data indicate that the impairment of cell responsiveness following exposure of PBMC to peritoneal dialysate is not restricted to the unused fluids, but is also observed following intra-peritoneal equilibration. Moreover, our findings suggest the presence of cytokine inhibitory factors in the peritoneal dialysate of CAPD patients which appear to accumulate in the peritoneal effluent during the CAPD cycle.     

Correction of anemia in uremic mice by genetically modified peritoneal mesothelial cells

BACKGROUND: During peritoneal dialysis, mesothelial cells become detached from the peritoneum and accumulate in the dialysate. Our aim was to evaluate the potential of peritoneal effluent (PF)-derived human peritoneal mesothelial cells (HPMC) as target for gene therapy. We used erythropoietin (EPO) as our target gene. METHODS: Various extracellular matrixes (ECM) were tested for optimal adhesion and growth of HPMC. The EPO gene was introduced to mouse peritoneal mesothelial cells (MPMC) and HPMC by transfection or retroviral transduction. EPO secretion from PMC was measured by enzyme-linked immunosorbent assay (ELISA) and by the TF-1 cell proliferation assay. We performed intraperitoneal or intramuscular transplantations of the genetically modified cells into regular or 5/6 nephrectomized Balb/c mice and nude mice. Finally, we measured serum EPO and hematocrit levels. RESULTS: ECM-coated plates provided up to sixfold increase in the efficiency of PMC isolation from PF. Gelatin coated dishes (20 microg/cm2) were found optimal for isolation of PF-HPMC. RPR-120535 liposome was found to be best for PMC transduction. In vitro studies showed EPO secretion from modified HPMC over 6 months. Intraperitoneal transplantation aided with collagen matrix was the most effective. EPO, in MPMC transplanted mice, was detected up to 3 weeks (peak at 13 +/- 1 mIU/mL), and anemia of uremic mice was corrected (35.3 +/- 0.9 mIU/mL to 41.9 +/- 1.1 mIU/mL). CONCLUSION: PF-HPMC can be considered as an appropriate target for gene therapy since these cells can be efficiently isolated, modified, and transplanted. Nevertheless, implantation techniques in the peritoneum should be directed at obtaining longer duration of transgene expression in vivo, and means should be developed for enabling regulated expression of the gene.     

Modulation of postsurgical macrophage function by early postsurgical polymorphonuclear leukocytes.

Surgical trauma to the peritoneum, in the absence of infection, elicits a rapid and transient influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity prior to the accumulation of macrophages. The aim of this study was to characterize the effects of these PMNs on macrophage function in the early postsurgical period. Rabbits underwent intestinal reanastomosis and peritoneal exudate cells were collected at various times after surgery. Macrophage-enriched preparations were incubated with spent media from cultures of PMNs obtained at the corresponding times after surgery. Superoxide anion (O2-) release by macrophages in response to phorbol myristate acetate was determined by cytochrome c reduction. Fibrinolytic and protease inhibitory activities in macrophage-spent media were also evaluated. The release of O2- had already increased at 2 hr, reached peak levels at 6 hr, and decreased by 24 hr after surgery. Spent media from PMNs harvested 6 hr after surgery suppressed, whereas spent media from postsurgical 12- or 24-hr PMNs increased O2- release from macrophages harvested at 6 and 12 hr after surgery. PMN-spent media had no effect on the secretion of plasminogen activator (PA) from macrophages harvested within 12 hr after surgery. In contrast, PA activity in the spent media from macrophages harvested 24 hr after surgery was elevated after exposure to PMN-spent media. PA inhibitory activity was reduced in macrophage-spent media at 2 hr after surgery and increased by 24 hr, while PMN-spent media had no effect on the level of PA inhibitory activity. Thus, soluble factors secreted into the culture medium by PMNs modulate macrophage function as soon as 6-12 hr after surgery.     

Epidermal growth factor modifies the expression and function of extracellular matrix adhesion receptors expressed by peritoneal mesothelial cells from patients on CAPD.

BACKGROUND: Efficient peritoneal dialysis depends on an intact layer of mesothelial cells that line the peritoneal membrane. This layer is disrupted in patents on continuous ambulatory peritoneal dialysis during episodes of peritonitis (acute injury) and replaced by fibrous tissue during extended dialysis (chronic injury). Little is understood of human peritoneal mesothelial cell (HPMC) responses to wounding and episodes of peritonitis. METHODS: HPMC were harvested from spent peritoneal dialysis effluent and maintained under defined in vitro conditions. Adhesive interactions with extracellular matrix (ECM) molecules and chemotactic and wound-healing responses were measured in vitro using purified ECM molecules. RESULTS: HPMC express multiple functional cell receptors recognizing and binding to ECM molecules, including several members of the integrin family. HPMC exhibit directed migration in wound healing and chemotaxis assays with ECM molecules. Epidermal growth factor (EGF) stimulates a reversible change to a fibroblastic phenotype, accompanied by increased expression of beta1 integrins, particularly alpha2beta1, increased adhesion to type I collagen, and significantly greater HPMC migration on type I collagen in wound healing and chemotaxis assays. CONCLUSIONS: HPMC possess the migratory capacity to contribute to the efficient repair of damaged peritoneal membrane after acute injury, and growth factors, such as EGF, facilitate peritoneal membrane healing by augmenting cell adhesion and migration.     

Rapid T-cell-based immunodiagnosis of tuberculous peritonitis in a peritoneal dialysis patient

Tuberculous peritonitis is a rare complication during peritoneal dialysis (PD). This report presents the case of a patient with clinical signs and symptoms indicative of bacterial peritonitis, but without culture growth of conventional bacteria or fungi. Cytokine flow cytometry after overnight stimulation of cells from peripheral blood and the peritoneal dialysate with Mycobacterium tuberculosis (MTB)-specific antigens revealed a 40-fold increase in MTB-specific CD4 + T cells expressing interferon-γ (IFN-γ) in peritoneal fluid compared with blood, which was indicative of active tuberculosis (TB). The presence of TB was later confirmed by polymerase chain reaction and growth of MTB in culture of the dialysate. The case illustrates the usefulness of MTB-specific immunodiagnosis for the rapid identification of peritoneal TB in PD patients.     

Characterisation of the cellular infiltrate in the foreign body granuloma of textile meshes with its impact on collagen deposition

Purpose

As part of the foreign body reaction, mesh filaments are surrounded by an infiltrate of inflammatory cells. Though macrophages are considered as being predominant, little is known about the origin of other cells.

Methods

On 55 meshes explanted from humans, we characterised the cells in the inflammatory infiltrate of the granuloma by immunohistochemistry using 10 cellular markers: CD3+ lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD34+ stem cells, CD45R0+ leucocytes, CD68+ macrophages, Mib1 for proliferation, Vimentin for mesenchymal origin, and Desmin for myocytes. Collagen deposits were analysed after staining with Sirius Red.

Results

More than 80 % of the cells in the infiltrate showed a positive expression of CD68, CD8, CD45R0 and Vimentin. CD4 and Desmin were seen in 30–80 % of the cells, unaffected by material or time. A score summarising the expression of all markers positively correlated significantly with an increased percentage of collagen type III (green) in the mesh wound. The analysis of collagen deposits was only affected to a small degree by size of area for investigation.

Conclusions

At the vicinity of the mesh filaments, the accumulated inflammatory cells represent a mixture of cells of various origins. The high expression of at least four markers requires co-expression of different surface markers and thus confirms the existence of multiple transition forms instead of dominance of just macrophages. This offers new options for interventions to attenuate the inflammatory reaction of mesh implants.