SHIP1, but not an AML-derived SHIP1 mutant, suppresses myeloid leukemia growth in a xenotransplantation mouse model.

Constitutive activation of the PI3K/AKT signaling pathway is found in ~50-70% of AML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP1) is a negative regulator of PI3K/AKT signaling in hematopoietic cells. SHIP1 knockout mice develop a myeloproliferative syndrome and concomitant deletion of SHIP1 and the tumor suppressor PTEN leads to the development of lethal B-cell lymphomas. In the study presented here, we investigated the role of SHIP1 as a tumor suppressor in myeloid leukemia cells in an in vivo xenograft transplantation model. NSG Mice transplanted with UKE-1 cells derived from a secondary AML showed a significantly extended lifespan after lentiviral-mediated overexpression of SHIP1 in comparison to the vector control cohort. In contrast, the AML-derived SHIP1Y643H mutant, which has a strongly reduced enzymatic activity showed a significant reversion of the SHIP1-induced prolongation of the survival time. In addition, the analysis of 290 AML patients revealed a correlation between expression of SHIP1 and overall survival of the AML patients. These results indicate that SHIP1 can act as a tumor suppressor in acute myeloid leukemia cells and that higher SHIP1 expression is associated with prolonged overall survival in AML patients. SHIP1 may be an interesting candidate for gene therapy.

Preclinical pilot study monitoring topical drug penetration and dermal bioavailability of a peptidase inhibitor from different galenic formulations into pig dermis, using cutaneous microdialysis.

BACKGROUND: Cutaneous microdialysis (CM) is an ex vivo technique that allows study of tissue chemistry, including bioavailability of actual tissue concentration of unbound drug in the interstitial fluid of the body. AIM: To test the penetration and dermal bioavailability of galenic formulations of the small-molecule IP10.C8, a dual-protease inhibitor of the dipeptidyl peptidase and aminopeptidase families. METHODS: Using CM, we tested the penetration and dermal bioavailability of IP10.C8 into the dermis and subcutis of pigs, and determined the tissue concentration of IP10.C8 enzymatically, using an enzyme activity assay (substrate Gly-Pro-pNA) and high performance liquid chromatography. RESULTS: Dermal bioavailability was enhanced by using microemulsion or the addition of the penetration enhancer oleic acid to a hydroxyethylcellulose (HEC) gel formulation. Dermal bioavailability was also enhanced when galenic formulations were prepared with higher pH (7.5 vs. 6.5) or higher drug concentration (5% vs. 1%) in HEC gel. CONCLUSION: It seems possible, using CM for topical skin penetration testing in anaesthetized domestic pigs, to test the bioavailability of newly designed drugs. However, the experimental time is limited due to the anaesthesia, and is dependent on drug recovery. Validation of this technique for routine use is challenging, and more experiments are needed to validate this preclinical set-up.

Membrane-bound proteindisulfide isomerase (PDI) is involved in regulation of surface expression of thiols and drug sensitivity of B-CLL cells.

The proteindisulfide isomerase (PDI), a multifunctional cytoplasmic enzyme with additional chaperone activity, has been shown recently, using monoclonal antibodies, to be located on the membrane of mature human B lymphocytes and B cell chronic lymphocytic leukemia (B-CLL) cells. Here, evidence is presented that this antigen exhibits catalytic activity as measured by the reductive degradation of insulin (release of A chain molecules) on intact B cells in patients suffering from B-CLL, as well as on JVM 13 cells (B-CLL cell line). More than 98% of these cells exhibited PDI activity which could be inhibited by bacitracin and also by monoclonal and polyclonal antibodies to PDI. Interestingly, surface PDI expression was strongly correlated in our study with protein-bound membrane SH groups. These surface protein thiols were specifically determined by using low concentrations of the chloromethyl-derivative based fluorescent probe 5-(and6)-(((4-chloromethyl)-benzoyl)amino)-tetramethyl-rhodamine (CMTMR) at low temperature in the presence of sodium azide in flow cytometry. The highest PDI and SH expression was found on B lymphocytes, particularly B-CLL cells. The mean fluorescence intensity (MFI) of CMTMR-positive B cells in the B-CLL line was up to 10-fold higher than that of controls, indicating a strong elevation of cell membrane-located protein thiols on malignant B cells. The link between PDI and SH expression on cell surfaces points to a functional interaction between the two. Treatment with bacitracin resulted in a strong inhibition of PDI and a dramatic increase in surface protein thiol expression of B-CLL cells. Similar effects could be observed by cell treatment with anti-PDI antibodies, indicating that this enzyme system plays a crucial role in the regulation of protein-bound SH groups. Interestingly, artificially induced protein thiol expression led to significantly higher cellular resistance to the cytostatic drugs chlorambucil, vinblastin, and cisplatin in vitro as measured by cell growth. These data suggest for the first time a regulatory effect of PDI on the surface protein thiol status of B cells. The increased expression of PDI may play a crucial role in SH-mediated protection and drug resistance in malignant B lymphocytes.

Evidence of a defective thiol status of alveolar macrophages from COPD patients and smokers. Chronic obstructive pulmonary disease.

In increasing numbers of pulmonary diseases an association with a loss of intracellular thiols, mainly glutathione, is postulated. Therefore, the quantitative measurement of thiols within different viable cells is a possible metabolic parameter for cellular function and defense capacity of all pulmonary immune cells including alveolar macrophages (AM), that are highly compromised by oxidative stress. In this study the cellular thiol content was determined using fluorochrom conjugated chloromethyl derivatives (5-chloromethylfluorescein diacetate, CMFDA) in flow cytometry. The procedure was evaluated in vitro using biochemical techniques for glutathione quantification. Based on this approach, AM obtained from bronchoalveolar lavage (BAL) of smokers and patients with chronic obstructive pulmonary disease (COPD) showed a significant thiol deficiency compared to a nonsmoker/non-COPD group. The cellular thiol expression of AM from smokers and COPD patients reached only 50 and 53% of the control group. Lowest thiol concentrations (47% of control) were detected within the smoker(+)/COPD(+) group. This intracellular thiol deficiency significantly correlated with reduced lung function (FEV(1), PaO(2)). With regard to the tightly regulated thiol metabolism of immune cells, these results imply the onset of functional disturbances in thiol deficient AM. The determination of the cellular thiol content of AM, obtained from BAL by flow cytometry, presents a simple and reliable tool to monitor the effect of therapeutic measures focusing on the stabilization of the cellular thiol status.

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-beta 1 (TGF-beta 1) in stimulated mouse splenocytes and thymocytes.

Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor beta 1 (TGF-beta 1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-beta 1 might have key functions in these processes.

Antioxidant and antiprotease status in peripheral blood and BAL fluid after cardiopulmonary bypass.

OBJECTIVE: Cardiopulmonary bypass (CPB) triggers systemic inflammation. Recent evidence suggests that metabolic and oxygenation management can affect the outcome of patients after cardiac surgery. We investigated the influence of oxidant/antioxidant and protease/antiprotease imbalance during the course of systemic and pulmonary inflammation. METHODS: In a study of 61 patients, we measured the intracellular thiol concentration, the intracellular activity of cathepsins and elastase, and the concentrations of secreted elastase, soluble alpha(1)-proteinase inhibitor (alpha(1)-PI), and secretory leukoprotease inhibitor (SLPI). Peripheral blood and BAL fluid (BALF) were obtained preoperatively and 2 h after CPB. RESULTS: A post-CPB depletion of thiol was found in blood granulocytes, lymphocytes, and monocytes, as well as BALF lymphocytes and macrophages. The degree of postoperative depletion correlated with PO(2) and blood glucose levels during CPB. Concomitant reduction of FEV(1) showed positive correlation with thiol depletion of blood monocytes and granulocytes. Elastase and cathepsin activities were increased in blood cells but not in lymphocytes or macrophages from BALF. The concentrations of secreted elastase were significantly increased in blood plasma but not in BALF. Enhanced antiprotease (alpha(1)-PI, SLPI) concentrations were measured in BALF but not in peripheral blood. CONCLUSIONS: The inflammatory response of the intra-alveolar compartment is clearly distinguishable from systemic inflammation. CPB causes a differentiated impairment of the antioxidant defense system as well as a protease/antiprotease imbalance in blood and BALF. Oxygenation under circumstances of CPB and concomitant pulmonary disease, as well as blood glucose metabolism, influence the antioxidative defense. Individual perioperative management of blood glucose and oxygenation could improve cellular defense systems in the peripheral blood and BALF and therefore result in a more favorable patient outcome.

Restoration of the cellular thiol status of peritoneal macrophages from CAPD patients by the flavonoids silibinin and silymarin.

During continuous ambulatory peritoneal dialysis (CAPD) the peritoneal immune cells, mainly macrophages, are highly compromised by multiple factors including oxidative stress, resulting in a loss of functional activity. One reason for the increase of inflammatory reactions could be an imbalance in the thiol-disulfide status. Here, the possible protective effects of the antioxidant flavonoid complex silymarin and its major component silibinin on the cellular thiol status were investigated. Peritoneal macrophages from dialysis fluid of 30 CAPD patients were treated with silymarin or silibinin up to 35 days. A time-dependent increase of intracellular thiols was observed with a nearly linear increment up to 2.5-fold after 96 hours, reaching a maximum of 3.5-fold after 20 days of culture. Surface-located thiols were also elevated. The stabilization of the cellular thiol status was followed by an improvement of phagocytosis and the degree of maturation as well as significant changes in the synthesis of IL-6 and IL-1ra. Furthermore, the treatment of peritoneal macrophages with flavonoids in combination with cysteine donors resulted in a shortened and more efficient time course of thiol normalization as well as in a further increased phagocytosis. In addition, GSH-depletion in thiol-deficient media simulating CAPD procedures led to intracellular thiol deficiency similar to the in vivo situation. It is concluded that treatment with milk thistle extracts silymarin and silibinin alone or, more effectively in combination with cysteine donors, provide a benefit for peritoneal macrophages of CAPD-patients due to a normalization and activation of the cellular thiol status followed by a restoration of specific functional capabilities.

CD26/dipeptidyl peptidase IV in lymphocyte growth regulation.

DP IV/CD26 is involved in regulation of DNA synthesis and proliferation as well as production of cytokines of hematopoietic cells under various conditions. Inhibition of DNA synthesis in T lymphocytes, B lymphocytes, NK cells and myelomonocytic cells as well as of the production of IL-2, IL-6 TNF alpha, IL-1, IL-10, IL-12, IL-13, IFN-gamma, GM-CSF are not due to apoptosis of these cells. DP IV/CD26 inhibitors induce TGF-beta 1 mRNA synthesis and latent protein release demonstrating a crucial role of TGF-beta 1 in mediating CD26 function. X-X-Pro peptides as HIV-Tat protein strongly inhibit DP IV enzymatic activity and suppress DNA synthesis. This group of peptides may represent a class of natural DP IV/CD26 ligands and effectors, respectively. Hyperphosphorylation of p56lck as well as protein tyrosine phosphorylation of a number of proteins in T lymphocytes can be modulated by DP IV inhibitors. These data suggest that enzymatic activity or, at least in part, the active site of DP IV are both essential for its regulatory function in lymphocytes. Further work is required to determine the natural ligands, i.e. substrates and effectors, which are play the central role in DP IV/CD26 action in T cell growth and to understand the molecular mechanism of the early steps of this fundamental process.

John F. Fulton, coccidioidomycosis, and penicillin.

When the late Dr. John F. Fulton contracted severe pulmonary coccidioidomycosis in January, 1942, a metastatic lesion posed the threat of further progression and fatal dissemination. The possibility that an untested and generally unavailable antibiotic, penicillin, might be of value in Fulton's illness led his physician, Dr. John Bumstead, to appeal directly to Fulton to obtain this antibiotic, but ostensibly for the benefit of another patient succumbing to hemolytic streptococcal infection. While of no value for Fulton, penicillin was highly successful in the treatment of his other patient and soon of a second one with staphylococcal sepsis and pneumonia. This penicillin, administered in March, 1942, was the first clinical trial of penicillin under the control of the Office of Scientific Research and Development. The unique contribution of Dr. Fulton and of his illness to this event is described.

The comparative ultrastructure of fibrin induced by thrombin and by staphylocoagulase.

Fibrin induced by the action of thrombin and by staphylocoagulase was studied by transmission electron microscopy. Periodic striations were consistently observed in the negatively stained preparations of both fibrins. When 4200 major periods in the thrombin fibrin system were measured the mean length was 228 A. For 3666 major periods in the coagulase fibrin system the mean length was 223 A. While the T test analysis of these values gave a value of 10, it is noteworthy that the differences are well within the scatter of periodicity reported in the literature for thrombin-induced fibrin. Gross inspection of the preparations indicated that the coagulase-induced fibrin had a knottier appearance and was accompanied by a greater amount of background debris than the thrombin-induced fibrin.

Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase).

The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.

A molecular mechanism for the cleavage of a disulfide bond as the primary function of agonist binding to G-protein-coupled receptors based on theoretical calculations supported by experiments.

A model of the binding site of delta-opioids in the extracellular region of the G-protein-coupled opioid receptor based on modelling studies is presented. The distance between Asp288 and the disulfide bridge (Cys121-Cys198) formed between the first and second extracellular loops was found to be short. This model is consistent with site-directed mutagenesis studies. The arrangement of the ligands found in the receptor led to the development of a reaction mechanism for the cleavage of the disulfide bond catalysed by the ligands. Semi-empirical quantum chemical PM3 and AM1 calculations as well as ab initio studies showed that the interaction between the carboxylic acid side chain of aspartic acid and the disulfide bond leads to the polarization of, and withdrawal of a proton from, the protonated nitrogen of the ligand to one of the sulfur atoms. A mixed sulfenic acid and carboxylic acid anhydrate is formed as an intermediate as well as a thiol. The accompanying cleavage of the disulfide bond may produce a conformational change in the extracellular loops such that the pore formed by the seven-helix bundle opens allowing entrance of the ligand, water and ions into the cell. Cleavage of the disulfide bond after opioid administration was demonstrated experimentally by flow-cytometric measurements employing CMTMR and monobromobimane-based analyses of membrane-located thiols. The suggested mechanism may explain, in a consistent way, the action of agonists and antagonists and is assumed to be common for many G-protein coupled receptors.

Nephrocutaneous fistula diagnosed by computed tomography.

We present an unusual case of isolated nephrocutaneous fistula secondary to renal calculi with perirenal infection. The usefulness of computed tomography (CT), with its depiction of the extent of involvement and its characterization of the disease process, is described and the literature is reviewed.

gamma-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias.

The expression of the ectoenzyme gamma-glutamyl transpeptidase (EC2.3.2.2., gamma GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to be gamma GT positive. The highest expression was measured on monocytes (CD14/gamma GT+ cells: 60%). Within the subsets of T lymphocytes (CD3/gamma GT+ cells: 18%) we saw no clear differences between CD4+ and CD8+ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, alpha-IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of gamma GT+ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression of gamma GT+ cells in the total MNC populations (B-CLL: 57%, CML: 62% gamma GT+ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, the gamma GT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-alpha. These data suggest a possible protective role of gamma GT in MNC and a regulatory function of this enzyme in the development of CML.