Overweight HIV patients with abdominal fat distribution treated with protease inhibitors are at high risk for abnormalities in glucose metabolism - a reason for glycemic control.

BACKGROUND: In HIV patients, disorders in glucose metabolism seem to be side effects of highly active antiretroviral therapy (HAART) which may be favoured by obesity, abdominal fat accumulation and familial disposition for diabetes mellitus (DM). The aim of our study was to identify patients at high risk for abnormalities in glucose metabolism taking into account HAART, familial disposition for DM and anthropometric parameters. METHODS: Plasma glucose, insulin, c-peptide and insulin resistance (homeostasis model assessment, HOMA) were determined in 44 HIV patients [16 without HAART, 19 with protease inhibitors (PI), 9 without PI (non-PI)] and in 11 healthy subjects. Glucose tolerance was determined by standard procedures. Body mass index (BMI), triceps skin fold thickness and waist circumference were measured and the waist-to-hip-ratio was calculated. Familial disposition for DM was assessed by questionnaire. RESULTS: Impaired fasting glucose was observed in 28% of HAART-treated patients (21% with PI, 7% non-PI), in 13% of HAART-naive but none in healthy controls. 58% of PI, 44% of non-PI, 38% of HAART-naive and none of healthy controls had a HOMA-index > 2.5 which indicates insulin resistance. HAART-treated patients had significantly higher fasting glucose levels (PI: 97 +/- 11 mg/dL, p = 0.048; non-PI: 109 +/- 58 mg/dL, p = 0.009) compared to healthy controls (72 +/- 8 mg/dL). HOMA-Index was higher in PI treated patients (3.74 +/- 3.08) than in HIV negative controls (0.95 +/- 0.28, p = 0.018). The duration of HAART (p = 0.045), overweight and familial disposition for DM (p = 0.017) significantly affected fasting glucose among PI users. Waist circumference affected c-peptide (p = 0.046) concentration in these patients. CONCLUSION: HIV patients on long-term PI therapy with overweight and familial disposition for DM are at high risk to develop abnormalities of glucose metabolism. Thus, measurements of HOMA-Index, BMI and waist circumference should be routinely done especially in PI medicated patients.

Changes in TH1/TH2 immunity after endovascular and conventional infrarenal aortic aneurysm repair: its relevance for clinical practice.

OBJECTIVE: to evaluate local surgical trauma induced by endovascular (TPEG) and conventional infrarenal aortic aneurysm repair (AAA-C), the inflammatory response and changes in cell-mediated and antibody-mediated immunity as illustrated by the type-1/type-2 T-helper (TH1/TH2) cell balance were investigated. DESIGN: prospective study. PATIENTS AND METHODS: sixteen patients were included, eight patients underwent AAA-C and eight TPEG. Venous peripheral blood samples were collected 24h preoperatively and 24, 48, 72h, 5 and 7 days postoperatively. Besides the WBC, intracellular TH1/TH2 cytokines (IFN-gamma/IL-4) and the cell surface markers HLA-DR on monocytes and CD23 on B cells were measured by four colour flow cytometry. RESULTS: statistically significant higher values in the AAA-C group were demonstrated for neutrophiles. The TH1/TH2 immunobalance (expressed by forming the ratio of IFN-(gamma/IL-4 producing T cells as well as by the ratio of HLA-DR(pos) monocytes/CD23(pos) B-cells) showed a significant shift towards TH2 immunity in the AAA-C group whereas TPEG led to a significant lesser shift 24-72h after surgery (p < 0.05). CONCLUSIONS: TPEG leads to a minor distortion of the TH1/TH2 immunobalance. This implies that TPEG is a less stressing procedure, that is especially beneficial in patients whose conditions are considered less suitable for AAA-C due to age and serious comorbidity.

Increased susceptibility to apoptosis in circulating lymphocytes of critically ill patients.

BACKGROUND AND AIMS: Lymphocyte apoptosis may influence immune responsiveness in systemic inflammation. Therefore, we investigated whether early signs of apoptosis (i.e., annexin-V binding and cell shrinkage) in peripheral lymphocytes were different among patients with severe sepsis, critically ill, nonseptic patients after major surgery, and healthy individuals. PATIENTS/METHODS: Ten patients with severe sepsis and ten critically ill, nonseptic patients after major surgery admitted to a surgical intensive care unit in a university hospital were included in the study. In addition, ten healthy blood donors were included for comparison. We investigated early signs of apoptosis using flow cytometric measurement of annexin-V binding to the cell surface and cell shrinkage of peripheral lymphocytes. RESULTS: The percentage of apoptotic lymphocytes determined as annexin-V positive and propidium iodide negative cells was increased in freshly prepared cells of patients with severe sepsis (11.4 +/- 0.5%) and critically ill, nonseptic patients after major surgery (18.5 +/- 2.0%) relative to healthy blood donors (4.4 +/- 0.5%) (P < 0.05). No significant difference between patients with severe sepsis and patients after major surgery were found. Annexin-V binding increased significantly after OKT-3 stimulation of lymphocytes in patients with severe sepsis (34.4 +/- 1.6%), patients after major surgery (33.8 +/- 3.4%), and healthy blood donors (21.1 +/- 2.8%). No significant difference among groups was detected following OKT-3 stimulation. Furthermore, freshly isolated peripheral lymphocytes of patients with severe sepsis and critically ill, nonseptic patients after major surgery revealed a significantly higher proportion of cell shrinkage than in healthy blood donors (55.0 +/- 2.2%, 21.5 +/- 2.4% vs 3.6 +/- 0.7%; P < 0.05). CONCLUSION: Circulating lymphocytes of critically ill patients show a high degree of early signs of cellular apoptosis. This may contribute to hyporesponsiveness of immune cells in systemic inflammation.

Soluble Fas and Fas-ligand in bladder cancer in vitro and in vivo.

Circulating soluble Fas (sFas) and expression of Fas-ligand on cancer cells are mechanisms of immune escape. The aim of the present study was to investigate expression and production of Fas and Fas-ligand on bladder cancer cell lines of different grade as a basic mechanism of their secretion in vivo. sFas and sFas-ligand serum levels of patients with different stage of bladder cancer were examined to determine the possible clinical use of these molecules as tumor markers. Bladder cancer cell lines RT4 (G1), RT112 (G1), T24 (G3) and SUP (G4) were analyzed by flowcytometry for Fas and Fas-ligand expression. To determine if the Fas-ligand gene is transcribed in these bladder cancer cell lines, RT-PCR was performed on mRNA extracted from these cell lines. Production of sFas and sFas-ligand was examined in cell culture supernatants of the cancer cells as well as in the serum of 62 patients with bladder cancer by a specific ELISA test. We demonstrate that Fas is expressed in similar levels on all human bladder carcinoma cell lines. In T24 (G3) and SUP (G4) cell lines we were able to detect the Fas-ligand protein, whereas no Fas-ligand protein could be found in RT4 and RT112 (G1) cells. Fas-ligand mRNA was expressed in all bladder cancer cell lines. Furthermore, all bladder cancer cell lines produce sFas but no sFas-ligand in spite of mRNA expression. The range of sFas levels in the serum of all patients with bladder cancer was large and did not show a correlation to the histopathological stage of bladder cancer. Although there is in vitro evidence that sFas and Fas-ligand play a role in bladder cancer, no correlation between the sFas and s Fas-ligand serum levels and the histopathological stage of bladder cancer could be found. Therefore, serum sFas and sFas-ligand have to date limited clinical relevance.

Bladder cancer cells acquire competent mechanisms to escape Fas-mediated apoptosis and immune surveillance in the course of malignant transformation.

Mechanisms of resistance against Fas-mediated cell killing have been reported in different malignancies. However, the biological response of immune escape mechanisms might depend on malignant transformation of cancer cells. In this study we investigated different mechanisms of immune escape in 2 well-differentiated low-grade (RT4 and RT112) and 2 poorly differentiated high-grade (T24 and TCCSUP) bladder cancer cell lines. Fas, the receptor of Fas-ligand, is expressed and shedded by human transitional bladder carcinoma cell lines RT4, RT112, T24 and TCCSUP. Cytotoxicity and apoptosis assays demonstrate that in spite of the Fas expression, poorly differentiated T24 and TCCSUP cells are insensitive towards either recombinant Fas-ligand or agonistic apoptosis-inducing monoclonal antibody against Fas. In poorly differentiated T24 and TCCSUP cell lines we were able to detect marked Fas-ligand protein by flow cytometry and Western blot analysis. In grade 1 RT4 and RT112 cells only minor expression of Fas-ligand possibly because of proteinase action. Fas-ligand mRNA translation or post-translational processing seems to be regulated differentially in the cancer cell lines depending on malignant transformation. In co-culture experiments we show that poorly differentiated cells can induce apoptosis and cell death in Jurkat cells and activated peripheral blood mononuclear cells. This in vitro study suggests that bladder cancer cells can take advantage of different mechanisms of immune evasion and become more competent in avoiding immune surveillance during transformation to higher-grade malignant disease.

The clinical course of immune thrombocytopenic purpura in children who did not receive intravenous immunoglobulins or sustained prednisone treatment.

OBJECTIVE: To demonstrate the result of watchful waiting without specific therapy in unselected children with acute immune thrombocytopenic purpura (ITP). STUDY DESIGN: Between May 1992 and October 1999, 55 consecutive children (aged 2 months to 16 years; 28 boys and 27 girls) with acute ITP did not receive intravenously administered immune globulin G (IVIG) or sustained prednisone treatment. Patients with extensive mucosal bleeding were given prednisone, 2 mg/kg/d, for 3 days. RESULTS: In 37 of 55 patients the initial platelet count was <10,000/microL. Ten of these patients had active mucosal bleeding. Five additional patients with bleeding had platelet counts between 10,000 and 20,000/microL. Four patients were given a 3-day course of prednisone. Chronic ITP occurred in 7 (13%) of the patients; 29 patients achieved remission within 6 weeks, and 19 patients, between 6 weeks and 6 months. No life-threatening bleeding occurred, and no patient died. CONCLUSION: Most children with severe thrombocytopenia do not have active mucosal bleeding. This management approach, which did not administer specific therapy, avoided side effects, reduced cost, and was effective.

Modulation of cell surface markers on NK-like T lymphocytes by using IL-2, IL-7 or IL-12 in vitro stimulation.

Cytotoxicity and proliferation of NK-like T (CIK) cells are dependent on the continuous presence of exogenous cytokines, but it is not known which cytokine is optimal. Here, we compared the effect of exogenous interleukin 2 (IL-2), interleukin 7 (IL-7) or interleukin 12 (IL-12) on the generation of CIK cells in addition to IL-1, interferon-gamma and anti-CD3 antibodies. Cell surface markers important for cytotoxic activity and adhesion were defined and cytokines leading to their optimal expression were determined. The most important findings were: (a) IL-12 generates the most CD3/CD56-double-positive CIK cells, (b) the expression of LFA-1/CD11a which is important for cytotoxic activity is highest with IL-7, and (c) IL-7 also generates the most CD28-positive cells which may enhance T cell receptor co-stimulation. In summary, essential differences concerning antigen expression were found when generating CIK cells using IL-7 or IL-12 instead of IL-2. In particular, IL-12 may be of interest due to the high expansion of CD56 positive cells in CIK cell cultures and the important role of these cells in mediating cytotoxicity towards malignant tissues.

Strategies to evaluate metabolic stress and catabolism by means of immunological variables.

In this article, we outline how metabolic stress and catabolism are set off and influenced by the neuroendocrine network that interacts intensely with the immune system. When evaluating metabolic stress in individuals, the vast ocean of mediators, cell-surface markers and intracellular components that participate in metabolism and catabolic or anabolic changes make it necessary to focus on specific entities that may best mirror all these events. T cell responsiveness and factors that orchestrate the T helper type 1 and type 2 balance form an immunological mirror that can competently reflect catabolism and metabolic stress.

Impaired inducibility of heat shock protein 70 in peripheral blood lymphocytes of patients with severe sepsis.

OBJECTIVE: To determine the extent of the potentially protective heat shock protein 70 response in peripheral blood lymphocytes of patients with severe sepsis after ex vivo lipopolysaccharide stimulation. DESIGN: Entry study of consecutive patients with severe sepsis, those who were critically ill or nonseptic after major surgery, and healthy blood donors. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Ten patients with diagnoses of severe sepsis; ten critically ill, nonseptic patients after major surgery; and ten healthy blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We investigated the ex vivo endotoxin-inducible expression of heat shock protein 70 in peripheral blood lymphocytes of patients with severe sepsis by means of flow cytometry. Only negligible amounts of inducible intracellular heat shock protein 70 accumulation (<4.2% of lymphocytes) could be detected in peripheral blood lymphocytes without lipopolysaccharide stimulation. The proportion of cells accumulating heat shock protein 70 after treatment with lipopolysaccharide was distinctly lower in patients with severe sepsis (p < .05) than in critically ill, nonseptic patients after major surgery and healthy blood donors (38.3+/-3.3%, 82.2+/-4.5%, and 70.9+/-3.9%, respectively; mean +/- SEM; n = 10). Patients with clinical signs of recovery from severe sepsis showed an increase in heat shock protein 70 expression. CONCLUSIONS: Inducibility of ex vivo heat shock protein 70 was impaired in peripheral blood lymphocytes of patients with severe sepsis. The impaired expression of the potentially protective heat shock protein 70 may contribute in vivo to immune dysfunction, because intact functioning of T and B lymphocyte responses is of central importance in resisting infection in severe sepsis. Monitoring of inducible heat shock protein 70 in peripheral blood lymphocytes may contribute to the evaluation of the immune consequences of severe sepsis.

Evaluation of immunological responses in patients with ovarian cancer treated with the anti-idiotype vaccine ACA125 by determination of intracellular cytokines--a preliminary report.

In a first clinical trial, 45 patients with advanced ovarian carcinoma and recurrences were treated with the murine monoclonal anti-idiotypic antibody (Ab2) designated ACA125 for active immunotherapy. The monoclonal antibody (MAb) ACA125 mimics a specific epitope of the tumor-associated antigen CA125 expressed by most malignant ovarian tumors. Patients with CA125-positive tumors are immunologically tolerant to CA125, which could be overcome by the use of an anti-idiotypic antibody as a surrogate for the tumor antigen CA125. An immunological response to the anti-idiotype ACA125 in these patients was associated with a statistically significant survival prolongation. Humoral immunity to ACA125 was assessed by induction of anti-anti-idiotypic antibodies (Ab3) directed against CA125. Using flow cytometric detection methods we observe alterations of the intracellular cytokines IFN-gamma, IL-2, and IL-4 at the single-cell level during the course of immunization. There was a strong increase of intracellular IFN-gamma and IL-2 characteristic for a Th1 cell type immune response after treatment with ACA125. A delayed induction of Th2 type response, which promotes antibody-mediated immunity by B cells, could also be detected. The understanding of the kinetics of Th1 and Th2 responses could be important to improve treatment schedules for effective immunotherapy with anti-idiotype vaccines.

Endoscopic vs conventional hernia repair from an immunologic point of view.

BACKGROUND: In this study we tried to estimate the local surgical trauma in patients undergoing endoscopic or conventional hernia repair via the changes in peripheral blood T cell subpopulations (i. e., T-helper 1 (TH1) and TH2 cells), recently shown to be recruited differentially to inflammatory sites. METHODS: Cells were identified flow-cytometrically by intracellular cytokine staining on a single cell level in 30 patients undergoing conventional (Shouldice) or total extraperitoneal patch (TEPP) hernia repair. RESULTS: The TH1 cells decreased postoperatively in Shouldice patients on an average of 20.8-31.4%, whereas in TEPP patients only a minor decline (mean, 7.8-9.2%) was observed. The TH2 cells did not change significantly in TEPP patients, and a small increase (mean, 7.7%) was detected in Shouldice patients. CONCLUSIONS: Our results suggest that the postoperative reduction in TH1 cells reflects local surgical trauma and can be helpful in evaluating different surgical procedures. When conventional and endoscopic hernia repair were compared, the latter proved less traumatizing.

Evaluation of a new reagent for preserving fresh blood samples and its potential usefulness for internal quality controls of multichannel hematology analyzers.

We describe a new, easy-to-use reagent, Cyto-Chex (Streck Laboratories, Omaha, Neb), that preserves fresh whole blood in a non-cross-linking, nonformalin manner. Target values assigned to fresh blood were essentially met after preservation and storage of up to 31 days. Respective mean analytic inaccuracies and short-term intra-assay coefficients of variation (n = 30) were as follows: WBCs, 6.7% and 1.99%; RBCs, 0.7% and 0.76%; hemoglobin, -1.8% and 0.79%; hematocrit, -0.3% and 0.75%; mean corpuscular volume, -1.0% and 0.78%; and platelets, 6.9% and 3.12%. Linearity of dilution-sensitive analytes was satisfactory over a wide range of dilutions after preservation of blood samples. Ten independent laboratories using 10 different instruments determined day-to-day interassay imprecision during four 7-day periods after blood preservation. Mean interassay coefficients of variation for participating laboratories were WBC, 1.92%; RBC, 1.00%; hemoglobin, 1.29%; hematocrit, 2.00%; and platelets 3.29%. Cyto-Chex enables long-term monitoring of instrument accuracy and precision with retained blood specimens of healthy persons. Blood from patient cohorts with various hematologic disorders and with a wide range of numeric abnormalities and/or parameter aberrations can be preserved satisfactorily with this reagent. The reanalysis of preserved patient blood samples is an important adjunct to the use of commercial control material in quality control programs of multichannel hematology analyzers.

[Manifestations of sickle cell disease in adolescents and young adults. Clinical aspects and therapy references]. / Manifestationen der Sichelzellerkrankung bei Adoleszenten und jungen Erwachsenen. Klinik und Therapiehinweise.

In Germany about 300 sickle cell patients are being seen at more than 100 different hospitals. One third of these patients are adolescents and young adults. Since this is a congenital chronic disease, the majority of these teenagers and young adults are being cared for by pediatricians. Sickle cell disease in patients older than 15 years is characterized by the development of chronic organ damage, in addition to the occurrence of acute manifestations of disease such as pain crises, splenic sequestration, aplastic crises and Acute Chest Syndrome. Pediatricians who care for older sickle cell patients have to handle not only internal medicine problems but also to answer questions concerning pregnancy and contraception. In this paper the specific problems of adolescents and young adults with sickle cell disease are presented and suggestions are offered for the care of this group of patients.

[Changes in the cytokine concentration (Il-6, Il-8, Il-1ra) and their cellular expression of membrane molecules (CD25, CD30, HLA-DR) after surgical trauma]. / Veränderung der Zytokinkonzentration (IL-6, IL-8, IL-1 RA) und der zellulären Expression von Membranmolekülen (CD25, CD30, HLA-DR) nach operativem Trauma.

UNLABELLED: Elective surgical approaches and trauma cause changes in the production of different cytokines, an increased production of acute phase protein and changes in the expression of different cell surface markers. METHODS: In a prospective study we examined the C-reactive protein level, the production of the cytokines IL-6, IL-8 and IL-1 RA in 25 laparoscopic and 21 conventional cholecystectomies. In addition the cell surface markers CD25 and CD30 on different cell populations and HLA-DR on monocytes were measured. Statistical analysis was made by Student's-t-test and Mann-Whitney's rank sum test. RESULTS: The humoral markers showed a more distinct increase in patients operated on conventionally two and 24 hours after surgery, the differences between the two surgical approaches were significant (p < 0.01). The cell surface markers CD25 and CD30 showed the same reaction. The HLA-DR expression on monocytes was significantly lower in patients operated on conventionally. CONCLUSIONS: Elective surgical approaches cause changes in the immune system, which can be evaluated by the reaction of cytokines and cell surface markers. Laparoscopic cholecystectomies cause less activation of the immune system than conventional operations.

Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody-mediated immunity commensurate to the trauma.

BACKGROUND: Measuring serum cytokines, pituitary hormones, or acute phase proteins during or after surgery is not an optimal method for quantifying the impact of surgical procedures. In an effort to assess surgical stress by means of the immune response, we focused on changes in cell-mediated and antibody-mediated immunity as illustrated by the type 1/type 2 T-helper (Th1/Th2) cell balance. The sensitivity of this approach was evaluated by comparing laparoscopic and conventional cholecystectomy (LCE, CCE). METHODS: In a pragmatic prospective study 43 patients with symptomatic cholelithiasis were operated on either by LCE (n = 25) or CCe (n = 18). Blood sampling was done 24 hours before surgery, immediately before incision, and 2, 24, and 48 hours after surgery. Cell surface markers and cytokine production were used to characterize the Th1/Th2 balance and were measured by means of flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: Activation of Th2 cells evokes the production and secretion of interleukin-4 (IL-4), which up-regulates the expression of immunoglobulin E receptors (Fo epsilon RII, CD23) on B cells. Phytohemagglutinin-induced IL-4 production in freshly isolated peripheral blood mononuclear cells from patients increased more after CCE than LCE (IL-4, +41% versus +17%; p < 0.05). Also the expression of CD23 on B cells was higher after CCE than LCE (+146% versus +63%; P < 0.01). CD30, a membrane molecule that belongs to the tumor necrosis factor receptor superfamily and probably is an important indicator of Th2 activity, was more evaluated on T cells from patients who underwent CCE. The Th1 response, characterized by phytohemagglutinin-induced IFN-gamma secretion in peripheral blood mononuclear cells and up-regulation of human leukocyte antigen-DR expression on monocytes, was lower after CCE than after LCE. CONCLUSIONS: This study shows that surgical stress induces a shift in the Th1/Th2 balance toward Th2, suggesting that cell-mediated immunity is down-regulated and antibody-mediated immunity is up-regulated after surgery. The evaluation of this shift may be clinically meaningful and help quantify even less invasive surgical procedures. When comparing CCE and LCE in this not strictly randomized study, we found LCE to be the less stressful procedure.

[Pain crises in patients with sickle cell diseases. Pathogenesis, clinical aspects, therapy]. / Schmerzkrisen bei Patienten mit Sichelzellerkrankungen. Pathogenese, Klinik, Therapie.

About 70% of all patients with sickle cell disease suffer from pain crises. Pain crises are recurrent episodes of pain that range in severity from mild to severe, usually occur very abruptly and are often localized around joints. Pain crises are caused by vaso-occlusions in the vascular bed of the bone marrow, leading to necrosis, edema and increased pressure. For effective analgesia morphine or morphine analogues are often required. When treating a pain crisis the patient's complaints need to be taken seriously and analgesic therapy should be started promptly with analgesics in proportion to the severity of the patient's pain. With mild pain oral non-opioid analgesics are sufficient, in moderate pain they are given in combination with oral codeine. Severe pain requires IV morphine, also combined with a non-opioid analgesic. Intravenous morphine makes a thorough monitoring of ventilation and level of consciousness mandatory. Sickle cell patients do not become drug dependent if given morphine for adequate analgesia. While bone marrow transplantation has become an accepted treatment modality for sickle cell patients with severe pain crises, treatment with hydroxyurea to increase HbF levels and reduce incidence and severity of pain crises, however, is still experimental.

Enumeration of platelets by multiparameter flow cytometry using platelet-specific antibodies and fluorescent reference particles.

The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used for platelet counting cannot discriminate platelets from other cellular particles and precipitates that cause similar signals. Visual (chamber counting) methods are still frequently used in routine laboratories to verify low automated platelet counts (< 50 x 10/l) despite obvious technical and statistical drawbacks. The following report shows how platelet counts can be measured by multiparameter flow cytometry with the help of reference particles (fluorescent latex beads) and platelet-specific antibodies i.e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-alpha (CD42b) and anti-GP IIIa (CD61). The linearity of this method was highly satisfactory and the observed imprecision was within acceptable limits. At a platelet concentration of 10 x 10(9)/l the coefficient of variation (CV, n = 10) ranged from 5.3% (PCV = 0.456) to 5.6% (PCV = 0.148). Accuracy was evaluated by comparing results to the ICSH-selected method for platelet counting. The correlation of both methods was significant (P < 0.005) and Passing-Bablok's linear regression analysis showed no systematic differences between the two methods. Comparisons of this new platelet counting technique were also performed with routine visual methods, automated blood analysers (Technicon H-1, Sysmex E-5000) and a different flow cytometric method using only forward and side light scatter properties of platelets for their discrimination. The linear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 x 10(9)/l. At lower platelet concentrations, our new platelet counting technique correlated significantly only with the visual and the forward/side scatter methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C regulates IL-8 and fMLP induced cytoplasmic Ca2+ increase in human granulocytes by receptor modulation measurements by flow cytometry.

Changes in cytosolic free Ca2+ influence important granulocyte functions like chemotactic behavior, adherence to endothelia, and phagocytosis. In the following study we used a simple reproducible procedure involving flow cytometry in combination with the fluorescent dye Fluo-3 to measure Ca2+ changes in human granulocytes. The aim of our study was to investigate the involvement of protein kinase C in regulating cytosolic free Ca2+ concentrations after stimulation of cells with IL-8 and fMLP. Both reagents induced a 5-6 fold increase in cytosolic Ca2+. Experiments conducted in Ca(2+)-free media showed a minor 18-29% decrease in cytosolic Ca2+ response, suggesting that intracellular Ca(2+)-stores are the main source for Ca2+ release after fMLP or IL-8 stimulation. Activators of protein kinase C, phorbol myristate acetate (PMA) and 1-oleyl-2-acetyl-sn-glycerol (OAG), inhibited cytosolic Ca(2+)-increase completely when induced by IL-8 and by 68-82% in the case of fMLP. Staurosporine, an inhibitor of protein kinase C, was able to attenuate or even abolish the PMA/OAG-effect. Our results show that changes in cytosolic Ca2+ due to IL-8 and fMLP signalling can be regulated by protein kinase C in human granulocytes. This regulatory role of protein kinase C involves some form of receptor modulation (i.e. phosphorylation, internalization, shedding).

Qualitative and quantitative changes in platelets after coronary-artery bypass surgery may help identify thrombotic complications and infections.

We studied the effect of coronary-artery bypass surgery on blood cells and platelets. Hematological parameters of eighty-three patients were measured by an automated cell counting and sizing analyzer. Sampling time was from 24 h prior to 10 days after surgery. During this time leukocytes and platelets showed characteristic changes in numbers and size, whereas red blood cells revealed no typical modifications. Even though it seems clear that changes of hematological parameters occur after bypass surgery, it is important to be aware of the actual extent of such changes. Therefore the data of 50 patients who had had no post-operative clinical complications were combined to generate diagrams of those parameters that had changed in a characteristic fashion. The diagrams showing average changes, and 99% confidence intervals in mean platelet volume and platelet count were able to identify seven (out of 7) cases with complications up to 48 h before clinical signs were apparent. Complications ranged from mild (3 cases with infections) to severe (4 cases with thrombosis, embolic thrombosis and/or reinfarction). Diagrams showing changes in leukocyte parameters were able to identify only two cases with infections. Even though the number of cases is yet small, the results suggest that surveillance of platelet parameters may be useful in postoperative care. Furthermore, this study was able to confirm the recent findings of Trowbridge and Martin that an abnormal increase in platelet volume distribution width and low platelet counts found in patients with coronary heart disease may serve as good indicators for the prethrombotic state and the risk of myocardial infarction.