Biomedical subjects
Mark Holmes
Publications and source records attributed to Mark Holmes.
Practice-based clinical research.
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Increased intracellular pro- and anti-inflammatory cytokines in bronchoalveolar lavage T cells of stable lung transplant patients.
BACKGROUND: Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increased expression of T-cell proinflammatory cytokines. We have recently shown that peripheral blood T-cell proinflammatory cytokine production was significantly reduced in stable lung transplant patients consistent with immunosuppression therapy. However, analysis of inflammatory cytokine profiles in bronchoalveolar lavage (BAL) T cells may be more relevant than peripheral blood T cells for assessing graft status. METHODS: To investigate the immunomodulatory effects of currently used immunosuppressive regimens on BAL T-cell cytokine production, whole blood and BAL from stable lung transplant patients and control volunteers was stimulated in vitro and cytokine production by CD8+ and CD4+ T-cell subsets determined using multiparameter flow cytometry. RESULTS: There was a significant decrease in T-cell proinflammatory cytokine production in BAL compared with blood from control subjects but not transplant patients. Anti-inflammatory cytokine IL-4 was increased in BAL compared with blood from both groups. There was a significant increase in IFNgamma, IL-2, IL-4, TGFbeta, and TNFalpha production by CD8 T cells and IFNgamma and TNFalpha production by CD4 T cells in BAL from transplant patients compared with controls. CONCLUSIONS: We have shown decreased T-cell pro- and anti-inflammatory cytokine production in BAL compared with blood in control subjects but not in stable lung transplant patients. Current immunosuppression protocols have limited effect on T-cell proinflammatory cytokine production in BAL but do upregulate anti-inflammatory cytokines IL-4 and TGFbeta. Drugs that effectively reduce T-cell proinflammatory cytokine production in BAL may improve current protocols for reducing graft rejection in these patients.
Poststorage leuko-depleted plasma inhibits T-cell proliferation and Th1 response in vitro: characterization of TGFbeta-1 as an important immunomodulatory component in stored blood.
BACKGROUND: Poststorage, leuko-depleted blood transfusions have been associated with increased postoperative infections and improved allograft survival compared with prestorage leukocyte-depleted blood transfusion. Although the mechanism of this phenomenon remains to be fully elucidated, it is clear that the immunomodulatory effect is mediated by leukocytes/platelets or their products. METHOD: The aim of this study was to investigate the in vitro effects of pre- and poststorage leuko-depleted plasma (LDP) and buffy coat LDP on T-cell proliferation and cytokine synthesis using multiparameter flow cytometry. RESULTS: In cell cultures exposed to prestorage LDP and buffy coat LDP there were no significant changes compared with fresh blood. In cell cultures exposed to poststorage LDP, T-cells showed reduced expression of CD69, CD25 (IL-2Ralpha), CD122 (IL-2Rbeta) and CD132 (IL-2Rtau) and production of TNF-alpha and IL-2 but there was no significant alteration for IFN-tau or IL-4. Changes in cytokine/cytokine receptor synthesis and T-cell proliferation were shown to be directly proportional to poststorage LDP concentration. Some of these changes were characteristic of TGFbeta-1. Addition of TGFbeta-1 neutralising antibody to poststorage LDP, negated the immunosuppressive effect on PHA-stimulated PBMC cultures. CONCLUSIONS: The decrease in T-cell proliferation and Th1 cytokines TNF-alpha and IL-2, may be one basis of altered immunoregulation resulting in increased rates of certain types of infections and increased graft tolerance reported in patients receiving poststorage LD blood transfusions. TGFbeta-1 is a major immunomodulatory component of poststorage LD blood transfusions.
Up-regulation of interleukin-8, interleukin-10, monocyte chemotactic protein-1, and monocyte chemotactic protein-3 in peripheral blood monocytes in stable lung transplant recipients: are immunosuppression regimens working?
BACKGROUND: Alveolar macrophages are a major source of inflammatory cytokines and chemokines involved in the pathogenesis of lung transplant rejection and are derived from blood monocytes that migrate to the lung. Levels of monocyte cytokines and chemokines that may be relevant in transplant rejection have not previously been determined in transplant recipients. We hypothesized that production of these inflammatory mediators by blood monocytes may be up-regulated despite the use of potent immunosuppression therapy. METHOD: To investigate this, whole blood from 9 stable lung transplant recipients and 12 control volunteers was stimulated with lipopolysaccharide in vitro, and intracellular chemokine and cytokine production were determined with multiparameter flow cytometry. RESULTS: Monocyte production of chemokines interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and MCP-3, and anti-inflammatory cytokine IL-10 were significantly increased in the lung transplant group, but IL-6, tumor necrosis factor-alpha, IL-1alpha, IL-12, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and transforming growth factor-beta levels were unchanged. CONCLUSIONS: Because MCP-3 is a major chemoattractant for leukocytes to sites of antigenic challenge and is a natural ligand for MCP-1 receptor, this novel finding has important implications for the pathogenesis of lung transplant rejection. We now provide evidence that current immunosuppression protocols have a limited effect on monocyte inflammatory cytokine production and do not adequately suppress monocyte IL-8, MCP-1, and MCP-3 chemokine production. Drugs that modulate the action of these chemokines may improve current protocols for reducing graft rejection. Intracellular chemokine and cytokine analysis with flow cytometry may be a more accurate indicator of immunosuppression than drug levels in these patients.
Evaluation of particle size distribution of albuterol sulfate hydrofluoroalkane administered via metered-dose inhaler with and without valved holding chambers.
BACKGROUND: Administration of inhaled medications via metered-dose inhaler (MDI) to pediatric patients younger than 4 years usually requires use of a holding chamber or spacer with an attached face mask. OBJECTIVE: To determine the particle size distribution and overall dose of albuterol from the albuterol sulfate hydrofluoroalkane delivered in conjunction with 2 US-marketed valved holding chambers (VHCs) compared with the dose delivered via MDIs without VHCs. METHODS: Cascade impaction methods with high-performance liquid chromatography were used to evaluate the fine particle mass (FPM) of albuterol administered without and with the use of 2 commercially available VHCs. RESULTS: Particle size distributions for the 2 VHCs and the control were similar. The mean FPM values for the 2 VHCs and the control were 32, 28, and 30 microg, respectively. Statistical comparison of the FPM shows a similar profile when differences from the albuterol hydrofluoroalkane without a spacer were evaluated. CONCLUSIONS: In vitro results obtained under these test conditions demonstrate that all the FPM values for the VHCs tested were within 15% of the control range, a difference that is unlikely to be clinically meaningful. These results do not warrant a change in the recommended dose of albuterol hydrofluoroalkane administered when using the VHCs tested. The use of an MDI in conjunction with a VHC provides a reasonable therapeutic approach for administration of albuterol hydrofluoroalkane to young children and other patients who have difficulty administering the MDI alone.
Increased peripheral blood T-cell apoptosis and decreased Bcl-2 in chronic obstructive pulmonary disease.
Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease, usually associated with cigarette smoking. Stimulated peripheral blood T cells from patients with COPD have an increased propensity to undergo apoptosis. The mitochondrial apoptotic pathway is regulated by pro-apoptotic proteins (including p53 and Bax) as well as anti-apoptotic proteins (e.g. Bcl-2) and cytokines (IL-2, IL-4 and IL-7). We hypothesized that alterations in expression of these apoptosis-related proteins, cytokines and cytokine receptors may be important in determining the susceptibility of T cells to undergoing apoptosis in COPD. We further hypothesized that inhaled corticosteroids (GCS) contribute to the increased rates of T-cell apoptosis observed in COPD. The process of apoptosis (assessed by Annexin V and ssDNA staining), as well as Bcl-2, Bax, p53, IL-2, IL-4 and receptors IL-7R, IL-4R and IL-2Rgamma were investigated in PHA-stimulated peripheral blood-derived T cells, using flow cytometry. Fifteen patients with COPD receiving inhaled GCS (four of who received additional prednisolone), eight patients with COPD receiving symptom control medication, and 16 control subjects were studied. T cells (CD4(+) and CD8(+)) from GCS-treated COPD patients showed an increased propensity to undergo apoptosis, associated with significantly decreased Bcl-2 and IL-7 receptor expression. No significant differences were observed for the COPD patients who were receiving symptom control medication. These findings may suggest a negative peripheral effect of inhaled GCS on the immune system in COPD, although the clinical significance of these effects remains uncertain.
Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease.
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a serious, chronic inflammatory disease of the airway associated with cigarette smoking. Leucocytes are involved in the inflammatory process in the airways in COPD. There is a need for accurate characterization of cellular populations in bronchoalveolar lavage (BAL) due to variation in the predominant cell types reported, which were investigated mostly with manual counting techniques. METHODS: Bronchial brushings and BAL were obtained from human subjects undergoing fiber optic bronchoscopy. Flow cytometry was applied to identify various cell types. Quenching of autofluorescence of BAL-derived alveolar macrophages was achieved with eta-octyl beta-D-galactopyranoside and crystal violet. Comparisons of cell counts obtained with flow cytometric and manual counting methods were performed. RESULTS: Correlation analysis showed that manual cell counting methods overestimated the percentage of macrophages when compared with flow cytometric methods (R2 = 0.54). There was also a small tendency by manual counting to underestimate the percentage of lymphocytes and neutrophils. Using flow cytometry, the percentage and absolute numbers of alveolar macrophages and lymphocytes in BAL were not significantly different between patients with COPD and control subjects. The percentage and absolute numbers of neutrophils were higher in BAL from patients with moderate to severe COPD. CONCLUSIONS: This novel flow cytometric assay for identification of various cell types from heterogenous samples of BAL and bronchial brushing will allow further investigation of cell characteristics, such as cytokine production and receptor expression, and an accurate evaluation of apoptosis for different cell types and provide a rationale for urgently required effective treatments for COPD.
Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells.
Chronic obstructive pulmonary disease is a highly prevalent, complex disease, usually caused by cigarette smoke. It causes serious morbidity and mortality and costs the global community billions of dollars per year. While chronic inflammation, extracellular matrix destruction and increased airway epithelial cell apoptosis are reported in chronic obstructive pulmonary disease, the understanding of the basic pathogenesis of the disease is limited and there are no effective treatments. We hypothesized that the accumulation of apoptotic airway epithelial cells chronic obstructive pulmonary disease in could be due to defective phagocytic clearance by alveolar macrophages. There have been no previous studies of the phagocytic capacity of alveolar macrophages in chronic obstructive pulmonary disease using physiologically relevant apoptotic airway epithelial cells as phagocytic targets. We developed a phagocytosis assay whereby cultured 16HBE airway epithelial cells were induced to apoptosis with ultraviolet radiation and stained with mitotracker green. Alveolar macrophages from bronchoalveolar lavage from eight control and six chronic obstructive pulmonary disease subjects were analysed following 1.5 h incubation with apoptotic airway epithelial cells, then staining with macrophage marker anti CD33. CD33+/mitotracker green + events (i.e., alveolar macrophages which had phagocytosed apoptotic airway epithelial cells) were analysed using flow cytometry. Phagocytosis of polystyrene microbeads was investigated in parallel. A significantly reduced proportion of alveolar macrophages from chronic obstructive pulmonary disease subjects ingested apoptotic airway epithelial cells compared with controls (11.6 +/- 4.1% for chronic obstructive pulmonary disease versus 25.6 +/- 9.2% for control group). Importantly, the deficiency was not observed using polystyrene beads, suggesting that the failure to resolve epithelial damage in chronic obstructive pulmonary disease may result, at least partially, from specific defects in phagocytic ability of alveolar macrophages to ingest apoptotic airway epithelial cells.
Up-regulation of production of TGF-beta and IL-4 and down-regulation of IL-6 by apoptotic human bronchial epithelial cells.
Human bronchial epithelial cells secrete cytokines that play a role in immune responses in the lung. However, the roles of these cytokines in regulating epithelial repair following acute lung injury are largely unknown. Responses to injury include hyperplasia of epithelial cells and squamous metaplasia. The resolution stage is characterized by discontinuation of hyperplasia. Apoptosis is considered to be the most efficient mechanism of removal of unwanted cells without causing inflammation. The presence of TGF-beta1 increases apoptosis, induces squamous metaplasia and inhibits proliferation of airway epithelial cells. Interleukin-4 increases the ability of macrophages to phagocytose epithelial cells and produce inflammatory cytokines. The purpose of this study was to investigate the hypothesis that apoptotic lung epithelial cells produce cytokines, which could act in an autocrine manner to control hyperplasia and induce squamous differentiation following acute lung injury. A bronchial epithelial cell line (16 HBE) was used as an in vitro model, to study the production of TGF-beta, IL-4 and IL-6 by lung epithelial cells undergoing apoptosis. Apoptotic and live cells were sorted on the basis of bright and negative staining with FITC-conjugated Annexin V, respectively. Intracellular IL-6, TGF-beta and IL-4 was measured using flow cytometric techniques. Electron microscopy, immunohistochemistry and ELISA were used as supportive techniques. Apoptotic cells produced significantly more TGF-beta and IL-4 (but less IL-6) than viable cells. Increased production of TGF-beta and IL-4 by epithelial cells undergoing apoptosis may contribute to the inhibition of proliferation, squamous metaplasia, and reduction of the inflammatory response in acute lung injury.
The effect of glycerol hyperhydration on olympic distance triathlon performance in high ambient temperatures.
The purpose of this study was to examine the effect of prior glycerol loading on competitive Olympic distance triathlon performance (ODT) in high ambient temperatures. Ten (3 female and 7 male) well-trained triathletes (VO2max = 58.4 +/- 2.4ml kg(-1) min(-1); bestODTtime = 131.5 +/- 2.6 min) completed 2 ODTs (1.5-km swim, 40-km bicycle, 10-km run) in a randomly assigned (placebo/ glycerol) double-blind study conducted 2 weeks apart. The wet-bulb globe temperature (outdoors) was 30.5 +/- 0.5 degrees C (relative humidity: 46.3 +/- 1.1%; hot) and 25.4 +/- 0.2 degrees C (relative humidity: 51.7 +/- 2.4%; warm) for day 1 and day 2, respectively. The glycerol solution consisted of 1.2 g of glycerol per kilogram of body mass (BM) and 25 ml of a 0.75 g x kg(-1) BM carbohydrate solution (Gatorade) and was consumed over a 60-min period, 2 hours prior to each ODT. Measures of performance (ODT time), fluid retention, urine output, blood plasma volume changes, and sweat loss were obtained prior to and during the ODT in both the glycerol and placebo conditions. Following glycerol loading, the increase in ODT completion time between the hot and warm conditions was significantly less than the placebo group (placebo 11:40 min vs. glycerol 1:47 min; p < .05). The majority of the performance improvement occurred during the final 10-km run leg of ODT on the hot day. Hyperhydration occurred as a consequence of a reduced diuresis (p < .05) and a subsequent increase in fluid retention (p < .05). No significant differences were observed in sweat loss between the glycerol and placebo conditions. Plasma volume expansion during the loading period was significantly greater (p < .05) on the hot day when glycerol appeared to attenuate the performance decrement in the heat. The present results suggest that glycerol hyperhydration prior to ODT in high ambient temperatures may provide some protection against the negative performance effects of competing in the heat.
Latinos, immigrants, and the uninsured.
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