Pharmacological Screening Identifies SHK242 and SHK277 as Novel Arginase Inhibitors with Efficacy against Allergen-Induced Airway Narrowing In Vitro and In Vivo.

Arginase is a potential target for asthma treatment. However, there are currently no arginase inhibitors available for clinical use. Here, a novel class of arginase inhibitors was synthesized, and their efficacy was pharmacologically evaluated. The reference compound 2(S)-amino-6-boronohexanoic acid (ABH) and >200 novel arginase inhibitors were tested for their ability to inhibit recombinant human arginase 1 and 2 in vitro. The most promising compounds were separated as enantiomers. Enantiomer pairs SHK242 and SHK243, and SHK277 and SHK278 were tested for functional efficacy by measuring their effect on allergen-induced airway narrowing in lung slices of ovalbumin-sensitized guinea pigs ex vivo. A guinea pig model of acute allergic asthma was used to examine the effect of the most efficacious enantiopure arginase inhibitors on allergen-induced airway hyper-responsiveness (AHR), early and late asthmatic reactions (EAR and LAR), and airway inflammation in vivo. The novel compounds were efficacious in inhibiting arginase 1 and 2 in vitro. The enantiopure SHK242 and SHK277 fully inhibited arginase activity, with IC50 values of 3.4 and 10.5 µM for arginase 1 and 2.9 and 4.0 µM for arginase 2, respectively. Treatment of slices with ABH or novel compounds resulted in decreased ovalbumin-induced airway narrowing compared with control, explained by increased local nitric oxide production in the airway. In vivo, ABH, SHK242, and SHK277 protected against allergen-induced EAR and LAR but not against AHR or lung inflammation. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. SIGNIFICANCE STATEMENT: Arginase is a potential drug target for asthma treatment, but currently there are no arginase inhibitors available for clinical use. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. Our new inhibitors show protective effects in reducing airway narrowing in response to allergens and reductions in the early and late asthmatic response.

Short Review: Investigating ARSACS: models for understanding cerebellar degeneration.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease that includes progressive cerebellar dysfunction. ARSACS is caused by an autosomal recessive loss-of-function mutation in the SACS gene, which encodes for SACSIN. Although animal models are still necessary to investigate the role of SACSIN in the pathology of this disease, more reliable human cellular models need to be generated to better understand the cerebellar pathophysiology of ARSACS. The discovery of human induced pluripotent stem cells (hiPSC) has permitted the derivation of patient-specific cells. These cells have an unlimited self-renewing capacity and the ability to differentiate into different neural cell types, allowing studies of disease mechanism, drug discovery and cell replacement therapies. In this study, we discuss how the hiPSC-derived cerebellar organoid culture offers novel strategies for targeting the pathogenic mutations related to ARSACS. We also highlight the advantages and challenges of this 3D cellular model, as well as the questions that still remain unanswered.

Thiazolidinediones Regulate the Level of ABC Transporters Expression on Lung Cancer Cells.

ATP binding cassette (ABC) transporters related to multidrug resistance (MDR) actively efflux various xenobio-tics from the cells across the cell membrane and decrease a drugs efficiency. Lung cancer is the leading cause of death among all types of cancer in the Czech Republic, and its incidence is still rising. Ciglitazone, rosiglitazone and troglitazone belonging to PPARγ agonist family (formerly used in diabetes mellitus treatment) were selected to investigate their capability to influence expression of ABC transporters on lung cancer cells. Therefore, the effect of PPARγ of agonists on transcription of following ABC transporters was investigated: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). We have investigated if these PPARγ agonists are substrates of ABC transporters using HL60 and HL60 derived cell lines (HL60-MDR1, HL60-MRP1, PLB-BCRP) by cytotoxicity test WST-1. We have mapped the changes in mRNA expression level of those transporters in A549 and HEK293 cells after PPARγ agonists treatment using quantitative reverse transcription real-time PCR (qRT-PCR). All three PPARγ agonists serve as substrates to at least one ABC transporter under study. PPARγ activation correlates with up-regulation of PTEN which may modulate the expression of ABC transporters through PI3K/ Akt signaling pathway. We have shown that rosiglitazone and troglitazone inhibit mRNA expression of MDR1 transporter in both cell lines whereas the expression of MRP1 in HEK293 cell was up-regulated after rosiglitazone treatment and the expression of MDR1 was upregulated after ciglitazone treatment.

Aequorin as Intracellular Ca2+ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment.

Natural proteins can be used in measuring intracellular Ca(2+) concentration. As one of the Ca(2+)- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca(2+) fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca(2+)]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca(2+) concentration in other types of non-adherent cells.

Developmental exposure to polychlorinated biphenyls or methylmercury, but not to its combination, impairs the glutamate-nitric oxide-cyclic GMP pathway and learning in 3-month-old rats.

Prenatal exposure to polychlorinated biphenyls (PCBs) or methylmercury (MeHg) contaminated food may affect brain development, leading to long-term alterations in cognitive function. Both types of contaminants, PCBs and MeHg, are often found together contaminating food, especially fish in some polluted areas. Exposure to combinations of neurotoxicants may exert different effects on the developing nervous system than exposure to individual contaminants. Developmental exposure (during pregnancy and lactation) to PCB126 or PCB153 impairs learning ability when the rats are 3 months old. Impairment of learning seems to be a consequence of impairment of the function of the glutamate-nitric oxide (NO)-cGMP pathway in brain in vivo. The aims of the present work were 1) to assess whether perinatal exposure to MeHg also affects the function of the glutamate-NO-cGMP pathway in brain in vivo analyzed by in vivo brain microdialysis and/or the ability to learn the Y maze task when the rats are 3 months old, and 2) to assess whether perinatal exposure to combinations of MeHg with PCB153 or PCB126 potentiates, decreases or does not modify the effects of the individual neurotoxicants. Perinatal exposure to PCB126, PCB153 or MeHg impaired the function of the glutamate-NO-cGMP pathway in cerebellum and learning ability. However, co-exposure to PCB126+MeHg or PCB153+MeHg inhibits the impairment of the pathway or learning ability. These results support that the function of this pathway modulates learning of the Y maze task. Moreover, they show that co-exposure to these PCBs and MeHg does not exacerbate, but reduces the effects on the ability to learn this task.

Chronic exposure to ammonia alters the modulation of phosphorylation of microtubule-associated protein 2 by metabotropic glutamate receptors 1 and 5 in cerebellar neurons in culture.

Hyperammonemia impairs signal transduction associated to glutamate receptors and phosphorylation of some neuronal proteins including microtubule-associated protein 2 (MAP-2). The aim of this work was to analyze the effects of hyperammonemia on modulation of MAP-2 phosphorylation by metabotropic glutamate receptors (mGluRs) in rat cerebellar neurons in culture. Hyperammonemia increased basal phosphorylation of MAP-2 (180%). Activation of mGluRs 1 and 5 with (S)-3,5-dihydroxyphenylglycine (DHPG) increased MAP-2 phosphorylation (170%) in control neurons but not in neurons exposed to ammonia. Activation of mGluRs 2 and 3 with (2S,3S,4S)-CCG/(2S, 1'S,2'S)-2-(carboxycyclopropyl)glycine increased slightly (25%) MAP-2 phosphorylation in neurons exposed to ammonia or not. Activation of mGluR5 with (+/-)-trans-azetidine-2,4-dicarboxylic acid increased MAP-2 phosphorylation (24%) in control neurons but decreased it by 56% in neurons exposed to ammonia. Activation of mGluR1 using 2-methyl-6-(phenylethynyl)pyridine and DHPG increased MAP-2 phosphorylation 183% in control neurons but only 89% in neurons exposed to ammonia. In control neurons mGluR1 activation greatly increases phosphorylation of MAP-2, while activation of mGluRs 5, 2 or 3 increased it slightly. Taken together, hyperammonemia reduces the increase in MAP-2 phosphorylation induced by mGluR1activation. Moreover, in neurons exposed to ammonia activation of mGluR5 reduces MAP-2 phosphorylation. These effects reflect significant alterations in signal transduction associated to mGluR1 and mGluR5 in hyperammonemia that may contribute to altered glutamatergic neurotransmission and to the neurological alterations in hyperammonemia and hepatic encephalopathy.

Bile duct ligation plus hyperammonemia in rats reproduces the alterations in the modulation of soluble guanylate cyclase by nitric oxide in brain of cirrhotic patients.

Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from cirrhotic patients. The aim of this work was to assess whether an animal model of cirrhosis, bile duct ligation, alone or combined with diet-induced hyperammonemia for 7-10 days reproduces the alterations in NO modulation of sGC found in brains from cirrhotic patients. sGC activity was measured under basal conditions and in the presence of NO in cerebellum and cerebral cortex of the following groups of rats: controls, bile duct ligation without or with hyperammonemia and hyperammonemia without bile duct ligation. In cerebellum activation of sGC by NO was significantly lower in bile duct ligated rats with (12 +/- five-fold) or without (14 +/- six-fold) hyperammonemia than in control rats (23 +/- seven-fold). In cerebral cortex activation of sGC by NO was higher in rats with bile duct ligation with hyperammonemia (124 +/- 30-fold) but not without hyperammonemia (59 +/- 15-fold) than in control rats (66 +/- 11-fold). The combination of bile duct ligation and hyperammonemia reproduces the alterations in the modulation of soluble guanylate cyclase by NO found in cerebral cortex and cerebellum of cirrhotic patients while bile duct ligation or hyperammonemia alone reproduces the effects in cerebellum but not in cerebral cortex.

Chronic exposure to 2,5-hexanedione impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in rat brain in vivo.

2,5-Hexanedione is a neurotoxic metabolite of hexane. The mechanisms of its neurotoxicity remain unclear. We assessed whether chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway in primary cultures of cerebellar neurons and/or in the cerebellum of rats. Chronic exposure of cultured cerebellar neurons to 2,5-hexanedione (200 microM) reduced by approximately 50% NMDA-induced formation of cGMP. Activation of soluble guanylate cyclase by nitric oxide was reduced by 46%. This treatment reduced the content of neuronal nitric oxide synthase and soluble guanylate cyclase in neurons by 23 and 20%, respectively. In the cerebellum of rats chronically exposed to 2,5-hexanedione (in the drinking water) NMDA-induced formation of cGMP was reduced by 55% as determined by in vivo brain microdialysis. Activation of soluble guanylate cyclase by nitric oxide was reduced by 65%. The content of neuronal nitric oxide synthase and of soluble guanylate cyclase was reduced by 25 and 21%, respectively, in the cerebellum of these rats. The effects are the same in both systems, indicating that cultured neurons are a good model to study the mechanisms of neurotoxicity of 2,5-hexanedione. These results indicate that chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway at different steps both in cultured neurons and in cerebellum of the animal in vivo. The alteration of this pathway may contribute to the neurotoxic effects of 2,5-hexanedione.

Aluminium impairs the glutamate-nitric oxide-cGMP pathway in cultured neurons and in rat brain in vivo: molecular mechanisms and implications for neuropathology.

Aluminium (Al) is a neurotoxicant and appears as a possible etiological factor in Alzheimer's disease and other neurological disorders. The mechanisms of Al neurotoxicity are presently unclear but evidence has emerged suggesting that Al accumulation in the brain can alter neuronal signal transduction pathways associated with glutamate receptors. In cerebellar neurons in culture, long term-exposure to Al added 'in vitro' impaired the glutamate-nitric oxide (NO)-cyclic GMP (cGMP) pathway, reducing glutamate-induced activation of NO synthase and NO-induced activation of the cGMP generating enzyme, guanylate cyclase. Prenatal exposure to Al also affected strongly the function of the glutamate-NO-cGMP pathway. In cultured neurons from rats prenatally exposed to Al, we found reduced content of NO synthase and of guanylate cyclase, and a dramatic decrease in the ability of glutamate to increase cGMP formation. Activation of the glutamate-NO-cGMP pathway was also strongly impaired in cerebellum of rats chronically treated with Al, as assessed by in vivo brain microdialysis in freely moving rats. These findings suggest that the impairment of the Glu-NO-cGMP pathway in the brain may be responsible for some of the neurological alterations induced by Al.

[Complications in the use of blood transfusions--alloimmunization in polytransfused patients]. / Komplikacija primanja transfuzija krvi--aloimunizacija politransfundovanih bolesnika.

INTRODUCTION: The objective of this paper was to examine the frequency of red blood cell (RBC) alloantibodies in polytransfused hematologic patients. MATERIAL AND METHODS: Blood samples of 2669 polytransfused hematologic patients were examined on clinical significant alloantibodies using antibody screening and identification according to Standards of AABB Technical Manual (1). Available medical charts were reviewed for sex, age and medical history and total number of given transfusions. RESULTS: During a three year period blood samples of 2669 polytransfused hematologic patients were examined for RBC alloantibodies. Alloantibodies were detected in 48 cases with the incidence of 1.79%. 36 patients (1.35%) developed single antibody while in 12 patients (0.45%) multiple antibodies were detected. Antibodies were registered more frequently in females than in males (37:17). In patients with single antibody next specificity was detected: anti-D (38.89%), anti-K (22.22%), antibodies to antigens MNSs system (22.22%), while anti-Le, anti-Fy and anti-P specificity was detected in 13.89%. Patients with multiple antibodies had specificity to Rhesus, Kell, Duffy, MNSs, Lewis and P blood group systems. All patients received more than 10 RBC transfusions. CONCLUSION: The incidence of alloimmunization was 1.79%. Sensibilization occurred more frequently in females than in males. Usually, the discovered alloantibodies were clinically significant and made problems in pretransfusion testings and required special efforts in blood selection for transfusion. For patients with the risk of frequent transfusions we suggest to include blood transfusion charts with complete phenotyping against antigens in Rhesus, Kell, Kidd and MNSs blood group systems and the data of all received transfusions.

Genetic variation at the apoB 3'hypervariable region in a Serbian population.

We investigated common length polymorphism caused by a variable number of tandem repeats in the hypervariable region located at the 3' end of the human apolipoprotein B gene in 696 Serbian (Belgrade area) unrelated individuals of both genders. After using the polymerase chain reaction to amplify this polymorphic region, 17 different alleles, containing 22-54 repeats, were distinguished. The bimodal distribution and the heterozygosity index (average 0.71) obtained in both genders are similar to those reported for other Caucasian populations. However, the HVE34 allele was found to be the commonest in both female and male samples. There was also a lower frequency of the HVE > 36 alleles than in other Caucasian populations studied.

Inhibition of release of tumor necrosis factor-alpha from human vascular tissue and smooth muscle cells by glucocorticoids.

OBJECTIVES: Based on our previous study that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from human vascular tissue and smooth muscle cells, we tested the hypothesis that release of TNF could be inhibited by pretreatment with glucocorticoids. DESIGN: Prospective, repeated-measures analysis of concentration-response relationships. SETTING: Academic anesthesiology research laboratory. SUBJECTS: Segments of internal mammary artery and saphenous vein were obtained during coronary artery bypass surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Confluent human smooth muscle cells, cultured from saphenous vein and internal mammary artery, were exposed to 20 micrograms/mL of bacterial lipopolysaccharide following pretreatment for 18 hrs with either 0.1, 1.0, or 10.0 microM of dexamethasone. At 1, 3, 6, 18, and 24 hrs, the culture medium was removed and analyzed for biologically active TNF-alpha using the L929 cell cytotoxicity assay. Smooth muscle cells exposed to bacterial lipopolysaccharide but not treated with dexamethasone served as controls. In control internal mammary cells, bacterial lipopolysaccharide stimulated TNF-alpha release in a time-dependent manner to a peak of 36 +/- 2.3 U/mg of cell protein at 6 hrs, compared with 0.7 +/- 0.3 U/mg of cell protein in cells not exposed to lipopolysaccharide. Dexamethasone inhibited bacterial lipopolysaccharide-stimulated release at all time points in a concentration-dependent manner. For instance, at 6 hrs, TNF-alpha was 12 +/- 2.2, 6.9 +/- 1.7, and 2.3 +/- 0.9 U/mg of cell protein for cells pretreated with 0.1, 1.0, and 10.0 microM of dexamethasone, respectively (p < .05 vs. control). In separate experiments, segments of internal mammary artery and saphenous vein were obtained from five patients who received 1 g of methylprednisolone intravenously during induction of anesthesia, and from seven patients who did not receive methylprednisolone. Bacterial lipopolysaccharide induced release of TNF-alpha from vascular tissues of untreated patients in a time-dependent manner (e.g., 733 +/- 44 U/g of tissue at 6 hrs in saphenous vein). In contrast, in patients treated with methylprednisolone, bacterial lipopolysaccharide did not stimulate release from vascular tissues incubated for up to 24 hrs. CONCLUSIONS: These results indicate that human vascular tissue, particularly the smooth muscle cell, may be a source of TNF-alpha and that glucocorticoids inhibit release stimulated by bacterial lipopolysaccharide.

[Use and preparation of thrombocyte concentrates]. / Priprema i cuvanje koncentrata trombocita.

Blood component transfusion therapy is an achievement of up-to-date transfusiology and is possible due to plastic equipment, centrifuge with air conditioning and modern technology which enables splitting blood into its components. Platelet concentration is a preparation of platelets gained from one unit of the whole blood and is used in therapy of patients with thrombocytopenias and thrombocytopathies. It can be preserved, depending on plastic bags, from 1 to 5 days with rotation on 22 degrees Celsius. This paper presents two methods of platelet concentration preparation (out of thrombocytes rich plasma and buffy coat) contribution of thrombocytes in concentrations and contamination of concentration with leucocytes, as well as changes during 72 hours of concentration preservation. One hundred units of thrombocytes had an average volume 51.42 ml. Platelet concentration gained out of thrombocyte rich plasma was 0.98 x 1011/l, and 0.80 x 109/l leucocytes. The number of thrombocytes in concentrations from buffy coat was 0.82 x 101/l, and 0.10 x 109/l leucocytes. Plasma pH after preparation was 7.21, and three days of conservation later it was 6.68. During these 72 hours of preserving the concentration there has not been a significant decrease in regard to the number of thrombocytes.

[Transfusion aspects of viral hepatitis C]. / Transfuzioloski aspekti virusnog hepatitisa C.

The investigation comprised 83 patients with acute non-A, non-B hepatitis who were treated at the Clinic of Infectious and Dermatovenerology Diseases in Novi Sad in the period 1986-1991. They were receiving blood transfusions and/or blood products from blood donors who had not been previously tested for presence of hepatitis C virus (HCV). In order to establish diagnosis anti-HCV ELISA screening tests from various producers were used. Out of 83 blood screening assays 17 patients (20.48%) were found to be anti-HCV seropositive. Out of these 17 seropositive patients 3 patients were intravenous drug-addicts (IVDA), while 14 patients (16.68%) previously received blood transfusions, whereas out of these 7 patients underwent open heart surgery. Only 2 blood donors, out of 48 whose blood was used for therapeutical purposes, were found to have anti-HCV antibodies after blood screening assays. The blood with detected anti-HCV antibodies caused acute post transfusion hepatitis C in 6 blood recipients. The obtained results show that comprehensive and thorough blood screening assays should be performed in all blood donors in order to prevent incidence of post transfusion C hepatitis. The Blood Transfusion Center in Novi Sad introduced compulsory viral screening assays for all blood donors on November 15, 1994, and since then 15019 blood donors have been tested. Only 38 (0.25%) were found to be anti-HCV seropositive, and were excluded from further blood donation and are under constant control. Now it is impossible to transmit HCV to patients who receive blood transfusion and/or blood products, and at the same time anti-HCV seropositive donors are prevented to develop chronic hepatitis and adverse complications due to HCV presence.