Superoxide dismutase in diabetic polymorphonuclear leukocytes

The level of superoxide anion was found to be significantly elevated in polymorphonuclear leukocytes (PMNL) from diabetic subjects as compared with those from normal subjects. This elevation was attributed to the significant reduction in the activities of both cytoplasmic and mitochondrial superoxide dismutase (SOD), the effect being more pronounced in the cytoplasmic fraction. Although the content of copper decreased considerably in the diabetic PMNL, the decrease in the zinc content was less significant, with an insignificant alteration in the content of manganese. PMNL obtained from insulin-treated diabetic patients showed considerable alleviation of SOD levels. The implication of these results are discussed herein.     

Activated polymorphonuclear leukocytes enhance production of leukocyte microparticles with increased adhesion molecules in patients with sepsis

BACKGROUND: Leukocyte microparticles (MPs) derived from polymorphonuclear leukocytes (PMNLs) have been recently found to be activators of vascular endothelium in vitro. The precise role of leukocyte MPs has not been clarified in patients suffering severe insult. The objective of this study was to evaluate production of leukocyte MPs and expression of adhesion molecules on the MP surface in patients with sepsis. METHODS: Twenty-one patients with severe infection (fulfilling the criteria of sepsis with serum C-reactive protein > 10 mg/dL) and 21 healthy volunteers were included as study subjects. Production of leukocyte MPs, expression of CD11b on the MPs, and oxidative activity of PMNLs were measured by flow cytometry in the presence and absence of formyl-methionyl-leucyl-phenylalanine. CD11b expression was evaluated according to the MP size (more than, equal to, or less than 1.0 microm). Soluble E-selectin, thrombomodulin, and PMNL elastase were also measured in blood. RESULTS: Production of leukocyte MPs and superoxide production in PMNLs with and without formyl-methionyl-leucyl-phenylalanine increased significantly in patients with sepsis in comparison with production in normal volunteers. In patients with sepsis, expression of CD11b was also markedly enhanced on MPs less than 1.0 microm in diameter in comparison with expression in control subjects. Levels of soluble E-selectin, thrombomodulin, and PMNL elastase in blood were significantly increased in patients with sepsis. We succeeded in detecting leukocyte MPs visually by fluorescence microscopy. CONCLUSION: Activated PMNLs enhance production of leukocyte MPs with increased adhesion molecules in patients with sepsis. Activated leukocyte MPs may play a role in the pathogenesis of endothelial activation and leukocyte-endothelium interaction in the presence of sepsis.     

The production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with non-insulin dependent diabetes mellitus]

The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation with PMA, FMLP, aggregated IgG and phagocytosis were determined in 36 patients with non-insulin dependent diabetes mellitus (NIDDM). The H2O2 production of PMN after the stimulation was measured using by flow cytometry. The patients were divided into four stages as follows: (1) non-microalbuminuric stage, (2) microalbuminuric stage, (3) proteinuric stage without impairment of renal function (less than 1.2 mg/dl of serum creatinine) and (4) proteinuric stage with impairment of renal function (more than 1.3mg/dl of serum creatinine). The H2O2 production after stimulation with PMA or phagocytosis was significantly higher in patients with NIDDM than normal controls. And also, there is the tendency of an increase in the H2O2 production after stimulation with FMLP or aggregated IgG. This increase of the H2O2 production was observed in all four stages of NIDDM patients after the stimulation, especially in patients with renal failure associated with diabetic nephropathy. These results suggest that reactive oxygen species produced by PMN after stimulation under various conditions may play an important role in the progression and exacerbation of diabetic nephropathy.     

Energy expenditure in malnourished patients with colorectal cancer

To evaluate energy expenditure in patients who have colorectal cancer with varying stages of disease and to examine the possible determinants of energy expenditure in a group of patients with cancer who have the same type of tumor, we studied 73 patients with biopsy proven and pathologically staged adenocarcinomas of the colon and rectum. Resting energy expenditure (REE) was measured by indirect calorimetry and compared with predicted energy expenditure (PEE), which was calculated from the Harris-Benedict formulas. Nutritional and tumor characteristics were examined. Forty-nine percent of patients had abnormal REE (normal = PEE +/- 10%). One quarter of the patients were hypometabolic (REE less than 90% PEE). The abnormalities persisted despite normalization of REE to metabolic body size (kg0.75) or predicted values based on weight, height, age, and sex. There were no differences in nutritional status, as judged by the percent of weight loss and visceral protein levels, between those patients in the hypometabolic, normometabolic, or hypermetabolic categories, and there were no significant relationships between energy expenditure and the tumor burden. The mean duration of disease in the normometabolic group was 4.5 months, while the hypometabolic and hypermetabolic groups had mean durations of 9.5 and 14.2 months, respectively. The tumor site and duration of the disease are important variables in studies of energy expenditure in patients with cancer.     

Localization of enolase in the cancer nest of digestive organs

Gamma-enolase, otherwise called neuron specific enolase (NSE), is specific for neural and neuro-endocrine systems and is present in the tumors of these systems. Gamma-enolase is also reported to be present, however, in a localized form in the tissues of other nonneural systems in some cases of lung cancer in relatively large number of incidences. We examined immunohistologically specimens of digestive organ cancers. Two principal conclusions were obtained. First, gamma-enolase is not specific for neural and neuro-endocrine systems. Second, gamma-enolase is localized in the epithelium of cancers of some cases following the transformation into cancer.     

Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocytes in severely injured patients

BACKGROUND: Polymorphonuclear leukocyte (PMNL)-derived microparticles (MPs) have been recently reported as activators of vascular endothelium in vitro. The objectives of the present study were to evaluate the production of MPs in severely injured patients and to clarify the role of these MPs. METHODS: Thirty severely injured patients (mean Injury Severity Score of 27 +/- 11) and 21 healthy volunteers participated in the study. Blood samples were obtained serially at three time points: days 0 to 1, days 2 to 5, and days 6 to 12 after the trauma event. MP production, CD11b and CD62L expression on MPs, and oxidative activity in PMNLs were measured by flow cytometry in both the presence and absence of formylmethionyl-leucyl-phenylalanine. Expressions of CD11b and CD62L were differentially evaluated according to the size of the MPs (>or= or < 1.0 microm). Soluble E-selectin and thrombomodulin levels in blood, variables representative of systemic vascular endothelial damage, were also measured. RESULTS: Production of MPs with and without formylmethionyl-leucyl-phenylalanine and the oxidative activity in PMNLs (O ) were prominently increased on days 2 to 5 after trauma. CD62L expression was enhanced on MPs at all three time points, and CD11b expression was enhanced on MPs < 1.0 microm in diameter at all three time points. Soluble E2-selectin and thrombomodulin in blood did not change significantly between time points. CONCLUSION: Activated PMNLs enhance production of PMNL-derived MPs with increased adhesion molecule expression on days 2 to 5 after severe trauma. This response per se, however, may not progress to systemic vascular endothelial damage.     

Energy expenditure in malnourished cancer patients

It is widely believed that the presence of a malignancy causes increased energy expenditure in the cancer patient. To test this hypothesis, resting energy expenditure (REE) was measured by bedside indirect calorimetry in 200 heterogeneous hospitalized cancer patients. Measured resting energy expenditure (REE-M) was compared with expected energy expenditure (REE-P) as defined by the Harris-Benedict formula. The study population consisted of 77 males and 123 females with a variety of tumor types: 44% with gastrointestinal malignancy, 29% with gynecologic malignancy, and 19% with a malignancy of genitourinary origin. Patients were classified as hypometabolic (REE less than 90% of predicted), normometabolic (90-110% of predicted) or hypermetabolic (greater than 110% of predicted). Fifty-nine per cent of patients exhibited aberrant energy expenditure outside the normal range. Thirty-three per cent were hypometabolic (79.2% REE-P), 41% were normometabolic (99.5% REE-P), and 26% were hypermetabolic (121.9% REE-P) (p less than 0.001). Aberrations in REE were not due to age, height, weight, sex, nutritional status (% weight loss, visceral protein status), tumor burden (no gross tumor, local, or disseminated disease), or presence of liver metastasis. Hypermetabolic patients had significantly longer duration of disease (p less than 0.04) than normometabolic patients (32.8 vs. 12.8 months), indicating that the duration of a malignancy may have a major impact upon energy metabolism. Cancer patients exhibit major aberrations in energy metabolism, but are not uniformly hypermetabolic. Energy expenditure cannot be accurately predicted in cancer patients using standard predictive formulae.     

Increased chromium uptake in polymorphonuclear leukocytes from burned patients

Following thermal injury neutrophil function is severely impaired and thought to be hypometabolic; however, the host is considered to be hypermetabolic. To further investigate the metabolism and the function of neutrophils following thermal injury, neutrophil migration and chromium uptake were studied using radio-labelled neutrophils. Random and directed migration were found to be significantly reduced compared to control values. Neutrophil lysozyme content was also reduced in these burn cells while serum lysozyme from the same patients was significantly elevated over control values. These data suggest lysozyme is released by the neutrophil into the circulatory system. The influx of chromium in cells from burned patients was much greater than the influx in normal cells used in studies for chemotaxis. Influx of chromium over time and over varying concentrations of chromium was linear (r2 = 0.90) in cells from burned patients and normals. Cells from burned patients, however, took up more chromium than normals. Influx velocity of chromium was also determined and found to be greater in burn cells than normal cells. Since it has been shown that chromium influx is an energy-dependent reaction it is suggested that cellular energy stores are being depleted by the influx of chromium. Whether this is a response to an intracellular deficit or uncoupling of metabolic pathways is not known at this time.     

Laparoscopic methods of creating fistulas in cancer of the digestive organs

Modified methods of making external fistulas for laparoscopy in 72 patients with tumors of digestion organs are described. An assessment of such fistulas with respect to their functional role, localization of the tumor process and other conditions is given.     

Oxidative metabolism and phagocytosis of polymorphonuclear leukocytes in patients with chronic renal failure

The respiratory burst activity (generation of hydrogen peroxide) of peripheral polymorphonuclear leukocytes (PMN) in both phorbol myristate acetate (PMA)-stimulated and unstimulated states and phagocytosis were assessed using flow cytometry on 46 patients with chronic renal failure: 33 patients undergoing chronic hemodialysis (CHD), 8 patients who have never been on dialysis (nonhemodialysis; NHD), 5 patients undergoing continuous ambulatory peritoneal dialysis (CAPD); these patients were compared with 27 normal control subjects. In patients just before the initiation of dialysis, impaired hydrogen peroxide production by PMA-stimulated PMN and depressed phagocytosis were noted, which was restored to the control levels by hemodialysis. A mild but significant reduction of hydrogen peroxide production in a PMA-stimulated state was found in NHD patients, and an inverse correlation was noted between the impairment of this function and the degree of diminished renal function. There was no significant difference between CAPD patients and controls in hydrogen peroxide production by PMA-stimulated PMN. Decreased hydrogen peroxide production by unstimulated PMN was observed in both CHD and CAPD patients. These findings may explain, at least partly, the enhanced susceptibility to bacterial and fungal infections of these patients.     

Interaction between erythropoietin and peripheral polymorphonuclear leukocytes in hemodialysis patients

The effect of erythropoietin (EPO) on the oxidative stress and inflammation caused by polymorphonuclear leukocytes (PMNLs) in chronic hemodialysis (HD) patients was investigated in vivo and in vitro. The studies were performed on isolated PMNLs from peripheral blood of healthy controls and HD patients before and following 6 weeks of EPO treatment. The oxidative stress was expressed by the rate of superoxide release from phorbol 12-myristate 13-acetate stimulated PMNLs, and the inflammatory state was evaluated by in vitro PMNL survival, in addition to white blood cell and PMNL counts of the enrolled subjects. Following 6 weeks of EPO treatment, in HD patients, the rate of superoxide release from PMNLs as well as WBC and PMNL counts fell significantly when compared with the pretreatment values. PMNLs from HD patients and healthy controls incubated in vitro with increasing amounts of EPO displayed a significant reduction in their rates of superoxide release and a significant improvement in survival. We have concluded that EPO interacts with PMNLs, attenuating their primed state in HD patients, thus reducing oxidative stress and the extent of inflammation. To the best of our knowledge, this attenuation of the primed state of PMNLs by EPO is a new finding.     

Superoxide anion production by leukocytes exposed to post-ischemic skeletal muscle.

Superoxide anion (O2-) and polymorphonuclear leukocytes (PMNs) have been implicated in the genesis of skeletal muscle ischemia-reperfusion (I-R) injury, but the source of (O2-) has not been established. We studied PMNs as a potential source of O2- using a ferricytochrome reduction assay in 5 anesthetized dogs. Using a gracilis muscle model of I-R, 6 hours of ischemia was followed by 2 hours of reperfusion. The contralateral muscle served as control. Prior to ischemia and after 0.5 and 2.0 hours of reperfusion, PMNs were separated from the gracilis venous effluent of ischemic (I) and control (C) muscles. Central venous samples were also obtained prior to surgical preparation and after reperfusion. Assays for O2- were performed with and without zymosan (Z) activation. Results are expressed as nmol O2-/2 x 10(6) PMNs +/- SEM. Baseline production of O2- was 0.49 +/- 0.54 in central venous samples; Z increased the values to 6.77 +/- 2.13. After 2 hrs of reperfusion, central O2- was 1.57 +/- 0.75, which increased to 7.1 +/- 1.04 with Z. Gracilis venous samples O2- values with and without Z are reported in Table I. One way measures of analysis of variance showed no significant (p > 0.05) differences between samples. Our results demonstrate that PMNs are not the sole source of O2- in the pathophysiology of skeletal muscle I-R injury. PMN associated injury may be mediated by mechanisms other than O2- production.     

Reactive oxygen species production by monocytes and polymorphonuclear leukocytes during dialysis

Intradialytic production of reactive oxygen species (ROS) by monocytes and polymorphonuclear leukocytes (PMNL) was examined separately in six hemodialysis patients. Samples obtained 15 minutes after initiation of dialysis with new cuprophane membranes demonstrated significantly increased (P less than 0.05) ROS production in both cell populations as measured by the fluorescence of a specific intracellular marker (2',7'-dichlorofluorescein diacetate [DCFH-DA]) assayed by flow cytometry. Granulocytes harvested during dialysis also showed decreased responsiveness to exogenous C5a and F-Met-Leu-Phe (FMLP) at 15, 30, and 60 minutes after initiation of dialysis (P less than 0.05). Our data suggest that hemodialysis with cuprophane membrane is associated with monocyte and PMNL activation as shown by production of ROS coincident with peak activation of the complement cascade; these granulocytes become refractory to further stimulation with C5a and FMLP during dialysis.     

Role of polymorphonuclear leukocytes in experimental lung injury--chemotaxis and active oxygen metabolite production

To clarify the role of polymorphonuclear leucocytes (PMNs) in acute lung injury, acute experimental lung injury was produced by intravenous injection of endotoxin to male Hartley guinea pigs. White blood cell (WBC) counts in the peripheral blood, total nucleated cell counts and PMNs' population in the lung lavage fluid, chemotaxis and chemiluminescence of the PMNs in the blood and in the lung lavage fluid were studied. Results were as following 1. WBC counts in the blood decreased after injection of endotoxin. In the lung lavage fluid, total nucleated cell counts and the differential counts of PMNs increased with time. 2. The chemotaxis of PMNs in the blood decreased significantly (647 +/- 118 cells/5 high-power-fields (5HPF) in no treatment group (NT group), vs 256 +/- 120 cells/5HPF in 6 hours after endotoxin injection group (6h group), p less than 0.01), but that in the lung lavage fluid increased significantly (93 +/- 63 cells/5HPF in NT group, vs 334 +/- 24 cells/5HPF in 6h group p less than 0.01). 3. The chemiluminescence of the PMNs in the blood increased (3.64 +/- 2.41 counts/cell in NT group, vs 51.2 +/- 32.9 counts/cell in 6h group, p less than 0.01), and that in the lung lavage fluid increased (1.89 +/- 0.94 counts/cell NT group, vs 59.2 +/- 49.1 counts/cell in 6h group, p less than 0.01). We concluded that increased chemotaxis of PMNs contributed to the influx of PMNs into the alveolar spaces after endotoxin injection. As the pMNs in the alveolar spaces had increased ability to produce active oxygen metabolite, they might be involved in the progression of endotoxin-induced acute lung injury.     

Predictive value for survival of lymphocytes and polymorphonuclear leukocytes in patients with sepsis.

To study their value in predicting prognosis, tests were performed on peripheral blood lymphomononuclear cells and polymorphonuclear leukocytes in 34 critically ill patients with sepsis. Intially, the number of lymphomononuclear cells was reduced by 32% compared with healthy control subjects and was 42% lower in those who died than in survivors. The values remained low in those who died. The numbers of T and B cells, determined by rosette formation using sheep and mouse erythrocytes, did not change during the period of observation. Intially, K cell activity was decreased by 48% compared with normal activity. In those younger than 65 years, K cell activity was 68% lower in patients who later died than in survivors. It returned to normal at 20 to 30 days and decreased in those who died. Polymorphonuclear leukocyte adherence was decreased by 50% compared with healthy control subjects and tended to be lower in those who died. Chemotactic migration of polymorphonuclear leukocytes and intracellular killing of Staphylococcus aureus and Pseudomonas aeruginosa were not impaired. It was concluded that the lymphomononuclear cell count, K cell activity and adherence of polymorphonuclear leukocytes were decreased in patients with sepsis and that the values were useful in predicting prognosis.