OBJECTIVE

In this paper, effect of the Tolazoline as antagonist of the alpha-2 adrenergic receptors in patients with bronchial asthma and chronic obstructive bronchitis was studied, and also the effect of stimulation with Hexoprenaline of beta-2 adrenergic receptor after bronchi-constriction caused with Propranolol, and Acetylcholine.

METHODS

Lung function parameters are determined with Body plethysmography. In patients with bronchial asthma and chronic obstructive bronchitis was registered resistance (Raw), was determined the amount of intrathoracic gas volume (ITGV), and specific resistance was calculated as well (SRaw). Aerosolization was done with standard aerosolizing machine-Asema.

RESULTS

The study included a total of 21 patients. Two hours after the inhalation of Propranolol, in experimental group, it was applied the blocker of alpha-2 adrenergic receptors (Tolazoline 20 mg / ml with inhalator ways), which did not cause changes in bronchomotor tonus of tracheobronchial system (p>> 1.0). Meanwhile, at the same patient, stimulation of beta-2 adrenergic receptor with Hexoprenaline (2 inh x 0.2 mg) is associated with a significant decrease of the specific resistance of airways (SRaw, p < 0.01). Control group results show that after bronchi-constriction caused by Propranolol-aerosol (20 mg / ml) in patients with bronchial asthma and chronic obstructive bronchitis, an increase of specific resistance in airways was caused (SRaw, p < 0.01), which confirms the presence of hyper-reactive bronco-constrictor effects intermediated by vagal ways. Two hours after Propranolol, inhaled Hexorenaline has blocked the action of Propranolol, but not entirely. Furthermore, two hours after acetylcholine-aerosol (1 mg /ml) was applied, inhaled Ipratropium (2 inh x 1 mg) has fully blocked the action of chemical bronchoconstrictor mediators, causing a decline of specific resistance in the airways (SRaw; p < 0.01).

CONCLUSIONS

This suggests that primary mechanism, which would cause reaction in patients with increased bronchial reactibility, is prevalence of the cholinergic system over adrenergic one, and not the relationship in between alpha-2 and beta-2 adrenergic receptors.

During testicular maturation, both Sertoli cells (SCs) and germ cells (GCs) switch from an immature to a mature immunophenotype. The reexpression of markers of immaturity in adults has been reported in cancer and in other testicular pathologies, in men as well as in animal species. Naturally affected with testicular cancer, rabbits have long been used in human reproductive research, but reports on the expression of testicular cell markers in this species are few and data about the immunophenotype of normal postnatal SCs and GCs are lacking. The aim of this study was to investigate the immunophenotype of SCs and GCs in the rabbit, from neonatal to adult age, using the antibodies anti-Müllerian hormone (AMH), vimentin (VIM), CKAE1/AE3 (cytokeratins [CKs]), desmin (DES), inhibin alpha (INH-α), placental alkaline phosphatase (PLAP), and periodic acid-Schiff (PAS) staining. In SCs, VIM was constantly expressed, and AMH and CKs expression was limited to neonatal and prepubertal age, whereas DES, INH-α, PLAP, and PAS were constantly negative. GCs were negatively stained for PLAP, PAS, and for the other markers. Results revealed analogies with human testicular immunophenotype, suggesting that rabbits could represent a potential experimental model for the study of human testicular pathology.

Alpha-melanocyte-stimulating hormone (α-MSH) is processed from proopiomelanocortin (POMC) and acts on the melanocortin receptors, MC3 and MC4. α-MSH plays a key role in energy homeostasis. In the present study, to shed light on the mechanisms by which α-MSH exerts its anorectic effects, extracellular neuronal activity was recorded in the hypothalamus and the dorsal vagal complex (DVC) of anesthetized rats. We examined the impact of α-MSH on glucose-sensing neurons and gastric distension (GD) sensitive neurons. In the lateral hypothalamus (LHA), α-MSH inhibited 75.0% of the glucose-inhibited (GI) neurons. In the ventromedial nucleus (VMN), most glucose-sensitive neurons were glucose-excited (GE) neurons, which were mainly activated by α-MSH. In the paraventricular nucleus (PVN), α-MSH suppressed the majority of GI neurons and excited most GE neurons. In the DVC, among the 20 GI neurons examined for a response to α-MSH, 1 was activated, 16 were depressed, and 3 failed to respond. Nineteen of 24 GE neurons were activated by α-MSH administration. Additionally, among the 42 DVC neurons examined for responses to GD, 23 were excited (GD-EXC) and 19 were inhibited (GD-INH). Fifteen of 20 GD-EXC neurons were excited, whereas 11 out of 14 GD-INH neurons were suppressed by α-MSH. All these responses were abolished by pretreatment with the MC3/4R antagonist, SHU9119. In conclusion, the activity of glucose-sensitive neurons and GD-sensitive neurons in the hypothalamus and DVC can be modulated by α-MSH.

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.

Pompe disease is an inherited disease caused by acid alpha-glucosidase (GAA) deficiency. A single center observational study aimed at assessing the prevalence of late-onset Pompe disease in a high-risk Brazilian population, using the dried blood spot test to detect GAA deficiency as a main screening tool. Dried blood spots were collected for GAA activity assay from 24 patients with "unexplained" limb-girdle muscular weakness without vacuolar myopathy in their muscle biopsy. Samples with reduced enzyme activity were also investigated for GAA gene mutations. Of the 24 patients with dried blood spots, one patient (4.2%) showed low GAA enzyme activity (NaG/AaGIA: 40.42; %INH: 87.22%). In this patient, genetic analysis confirmed two heterozygous mutations in the GAA gene (c.-32-13T>G/p.Arg854Ter). Our data confirm that clinicians should look for late-onset Pompe disease in patients whose clinical manifestation is an "unexplained" limb-girdle weakness even without vacuolar myopathy in muscle biopsy.

Immunohistochemical location and immunoblot of inhibin alpha-subunit peptides were analyzed in the testis of the Djungarian hamster from days 0-31 of postnatal development using a specific antibody. An intense immunoreaction was observed in the centrally located T1 prespermatogonia at day 0. The staining intensity decreased gradually in the spermatogonia when they make contact with the basal lamina at days 8-10. At days 13 and 15 there is no staining. Thereafter the immunoreactivity in Sertoli cells as well as in A spermatogonia gradually increased, being highest in sexually mature animals. The intensity of alpha-subunit staining in the seminiferous tubules was stage specific, being strongest at stages III and IV. Immunoblot analysis of testis homogenates with the anti-INH alpha 1-32 antibody showed several bands: 88K, 80K, and 43K in immature hamster testis (0-, 2-, 6-, 8-, or 10-day-old). In the adult hamster (31-day-old) 88K, 80K, 28K, and 20K bands were seen, but no 43K band. Dimeric inhibin was not detected. The 43-44K band most likely corresponds to the pro-alpha N alpha C, the 28K band to intermediate forms between alpha N alpha C and alpha C (alpha I alpha C), and the 20K band to mature alpha-subunit (alpha C). The shift from the immature pattern to mature occurs at about 20 days of age. Freezing of the samples was deleterious to alpha C, since it could be detected only in freshly homogenized samples. The results suggest that prespermatogonia produce predominantly monomeric alpha-subunit precursor pro-alpha N alpha C, whereas the mature Sertoli cells as well as A spermatogonia contain mainly monomeric alpha I alpha C. The alpha-inhibin precursors may act as auto-/paracrine regulators of spermatogenesis. Our results suggest that different alpha-subunit precursors, pro-alpha N alpha C and alpha I alpha C, might be involved in the differentiation and maintenance of spermatogenesis, respectively. The posttranslational processing of alpha-subunit precursors seems to play an important role in the physiology of reproduction.

Spermatogenesis is a complex developmental program in which interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model of Krabbe disease, characterised by the deficiency of galactosylceramidase, the enzyme that hydrolyses galactosylceramide and galactosylsphingosine. Galactosyl-alkyl-acyl-glycerol, a precursor of seminolipid, the most abundant glycolipid in spermatozoa, is also a substrate for galactosylceramidase. Altered sphingolipid metabolism has been suggested to be the cause of the morphological abnormalities reported previously in the spermatogenesis of Twitcher. However, given the frequency of infertility associated with neurological impairment, we hypothesised that an unbalanced hormonal profile could contribute to male infertility in this mutant. In order to clarify this issue, we investigated potential variations in the expression of hormones and hormone receptors involved in the regulation of spermatogenesis. Our data show that, in the brain of Twitcher mouse, gonadotrophin-releasing hormone (GnRH), LH and FSH gene expression is decreased, whereas expression of androgen receptor (AR) and inhibin ?A (INH?A) is increased. The changes in gene expression for the LH and FSH receptors and AR in the testes support the hypothesis that altered sphingolipid metabolism is not the only cause of Twitcher infertility.

We examined the correlations between changes in serum protein fractions and the prognosis of the patients. The levels of 21 protein components of the sera of 36 patients with maxillary sinus cancer were determined by a single radial immunodiffusion method before and after radiation therapy. The patients with maxillary sinus cancer were treated with combined intra-arterial infusion of bleomycin and external irradiation of 60 Co gamma-rays, and were concurrently treated with 5-fluorouracil at 200 mg/day p.o. The levels of the same protein components were also measured in 34 normal adult as a control. All patients were observed 5 years and 12 years after radiation therapy. In patients who had survived at least 5 years after radiation therapy, the Alb, Tf, Hx, IgG and IgM levels measured before radiation therapy were elevated significantly compared with those who had died within 5 years. In those who had survived at least 5 years, the Alb, Tf, Hx, IgG, IgM, IgA and I alpha I levels measured after radiation therapy were elevated significantly compared with those who had died within 5 years, and AT III was reduced. In cases of maxillary sinus cancer following a period of 5 to 12 years after radiation therapy, multiple regression analysis was used to determine whether increased concentrations of serum protein fractions were associated with good prognosis for the original disease, alpha 2HS. IgM, HX, alpha 1AT and alpha 1X before radiation therapy were positively correlated with survival, whereas AT III, Pmg, Cp, IgA, and alpha 1AG showed negative correlations. After radiation therapy, Pmg, Hx, Cp, Cl inh and Fib were found to be positive factors of survival rate, whereas alpha 2M, alpha 2Pl, I alpha 1, IgA, alpha 1AG and C3 were negative factors.

BACKGROUND

Inhibins are dimeric glycoproteins belonging to the TGF-beta1 family and are composed of an a-subunit (INH-alpha) and one of two possible beta-subunits (betaA or betaB; INH-betaA and INH-betaB). They have a substantial function in human reproduction and also seem to play an important role in endocrine-responsive tumors. Interestingly, there is an association between interferon and TGF-beta expression. However, this relationship has not been assessed in endometrial tissue regarding inhibin/activin expression. Therefore, the aim of this study was to determine expression changes of inhibin/activin subunits in the endometrial Ishikawa carcinoma cell line after stimulation with interferon-beta1a.

METHODS

The Ishikawa cell line was cultured until confluence was observed (after 2 days). After adding interferon-beta1a (1000 IE/ml) Ishikawa cells were immunohistochemical analyzed for INH-alpha, INH-betaA and INH-betaB subunits. Experiments were performed in triplicates. The immunohistochemical expression was analyzed with a semiquantitative score (IRS) and statistical analysis was performed.

RESULTS

Immunohistochemical reaction with INH-alpha could not be demonstrated in unstimulated cells, while it was significantly up-regulated in interferon-stimulated cells (p < 0.02). INH-betaA and INH-betaB were primarily observed during the mitotic phases of unstimulated cells. After stimulation their expression was significantly higher (p < 0.05 each) compared to controls and could be observed not only during mitotic phases but also in nonmitotic cells.

CONCLUSIONS

For the first time, we demonstrated a functional relationship between interferon and inhibin/activin subunits. The expression of INH-alpha, INH-betaA and INH-betaB were immunohistochemical significantly up-regulated in the Ishikawa endometrial cell line after stimulation with interferon-beta1a. Since INH-alpha is thought to be tumor suppressive in the mouse model, interferon-beta1a might activate its gene. It remains to be clarified if this effect can be used as therapeutic options in endometrial carcinomas.

OBJECTIVE

In this work, role of the adrenergic nerve system (<em>alpha</em>1 and beta2) in adjustment of the bronchomotor tonus in healthy people was researched.

METHODS

Parameters of the lung function are determined by Body plethysmography. Raw and ITGV were registered and SRaw was calculated as well. Aerosolization is done with standard aerosolizing machines - Asema.

RESULTS

Results gained shows that following the blockade of beta-2 adrenergic receptor with Propranolol (20 mg-aerosol), stimulation of alpha adrenergic receptor with Oxedrine (120 mg-aerosol) and blockage of these receptors with Tolazoline (20 mg-aerosol), does not change significantly the bronchomotor tonus of the tracheobronchial tree (p>> 0.1). Meanwhile, stimulation of the beta-2 adrenergic receptor with Hexoprenaline (2 inh × 0.2 mg) is associated with a significant increase of the peripheral resistance of the airways (p < 0.01).

CONCLUSIONS

This suggests that the activity of the <em>alpha</em>1-adrenergic receptor, unlike the activity of the beta2-adrenergic receptor in the healthy people smooth musculature, is not significant and as such is insufficient to oppose to the tonic activities of the cholinergic system.

Thrombotic events sometimes complicate erythropoietin therapy. Fibrinolytic system may play a role in their pathogenesis. The studies were performed on 22 chronically hemodialyzed patients with end-stage renal failure treated with recombinant human erythropoietin (rHuEPO, Eprex, Cilag) for 12 weeks. Alpha 2 antiplasmin (alpha 2AP), antithrombin III (AT III), C1 esterase inhibitor (C1 INH), plasminogen activator inhibitor (PAI) activities, alpha 2macroglobulin (alpha 2M), fibrinogen, fibrin monomers concentration and euglobulin clot lysis time (ECLT) were measured before and after 1, 2, 4, 8 and 12 weeks of rHEPO therapy. A fall in PAI activity (p < 0.05) was found after 1 week, while alpha 2AP and C1 INH activities decreased after 12 weeks of the therapy (p < 0.001 and p < 0.05, respectively). The activity of AT III, the main inhibitor of the coagulation cascade fell after 4 weeks of the treatment with rHuEPO (p < 0.001). Fibrin monomers concentration was found to be decreased after 12 weeks of rHuEPO administration. Fibrinogen and alpha 2M concentration showed no statistically significant changes during rHuEPO therapy. A decrease in plasma fibrinolytic inhibitor activities may be considered as a protective mechanism against thrombotic tendency observed during rHuEPO therapy.

Introduction: The exact risk of developing a thromboembolic event (TEE) while using complement 1 esterase inhibitors (C1-INHs) is currently undetermined for patients with hereditary angioedema (HAE). This systematic review aimed to define the potential risk of TEEs from these agents.

Areas covered: This evaluation covers publications examining or mentioning the risk of TEEs in association with C1-INHs. A systematic literature search was conducted utilizing PubMed, Scopus, and ProQuest. This review utilized search results through January 2020 and followed the PRISMA recommendations for a systematic review. Articles not available in English and animal or in-vitro studies were excluded. For inclusion, studies had to be open-label, randomized-controlled, cross-sectional, or clinical observational studies. A total of 13 studies met inclusion criteria and yielded 1716 patients receiving at least one dose of C1-INH, though only 41 incidences of thrombosis were documented.

Expert opinion: Significant heterogeneity exists in the available literature concerning both study design and the reporting of data; therefore, interpretation of thrombotic risk is difficult. TEEs are rarely reported in the literature, and they seem unlikely to occur in patients without underlying risk factors. Important risk factors include those found in the prescribing information of C1-INHs.

Keywords: C1 esterase inhibitors; clinical pharmacy services; embolism and thrombosis; hereditary angioedema; review; systematic.

Inhibins (INH) are dimeric glycoproteins composed of an alpha-subunit (INH-alpha) and one of two possible beta-subunits (INH-betaA or -betaB), with substantial roles in human reproduction and in endocrine-responsive tumours. The aim of the present study was the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in endometrial carcinoma cells of the cell line RL-95-2 after stimulation with estradiol and cortisol compared to unstimulated controls.

METHODS

Cells of the endometrial carcinoma cell line RL-95-2 were grown on quadriperm tissue slides and incubated with different concentrations (0.1 and 0.01 micromol/ml) of estradiol or cortisol. Expression of INH-alpha, betaA and betaB was analysed by immunocytochemistry with specific monoclonal antibodies directed against the inhibin subunits.

RESULTS

Expression of INH-alpha and -betaB was higher in cortisol-stimulated RL-95-2 cells, whereas INH-betaA expression was lower. In contrast to these, INH-betaB expression was increased by estradiol while INH-alpha and -betaA were unchanged under estradiol treatment.

CONCLUSIONS

Expression of INH-subunits in RL-95-2 cells was described. Cortisol and estradiol showed an influence on INH expression. The RL-95-2 cell line could act as a useful model for the investigation of INH regulation, particularly for endometrial cancer.

The hypothesis for the present study is that the active immunization of female turkeys with inhibin (INH) would neutralize endogenous INH, and increase levels of circulating follicle stimulating hormone (FSH) and the number of preovulatory follicles, and subsequently enhance egg production. Two experiments were conducted with female turkeys in their first (30 wk of age) and second (62 wk of age) laying cycles. Treatment groups included control turkeys immunized with keyhole limpet hemocyanine (KLH) and experimental turkeys immunized with recombinant turkey inhibin alpha conjugated to KLH (rtINH), vasoactive intestinal peptide (VIP) conjugated to KLH or rtINH+VIP. Egg production increased (P < 0.05) in VIP and rtINH+VIP immunized birds, but not in rtINH immunized hens in comparison with a control group. A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a larger number of nongraded yellow follicles in rtINH-immunized (62.5%) and rtINH+VIP-immunized (73.5%) groups compared with that of controls, suggesting overstimulation by FSH. Anterior pituitary FSH beta subunit, LH beta subunit, and prolactin (PRL) mRNA contents were determined by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in laying hens at the end of the experimental period. Hens immunized with rtINH showed increased FSH beta subunit mRNA content, but no change in the content of LH beta subunit or PRL mRNA. Hens immunized with VIP or rtINH+VIP had significant increases in both pituitary LH beta subunit and FSH beta subunit mRNA contents, accompanied by a decline in PRL mRNA abundance. The magnitude of the increase in FSH beta subunit to INH immunoneutralization was greater in first-cycle hens than in second-cycle hens. These data suggest that active immunization of female turkeys with INH neutralizes endogenous INH and increases both circulating FSH and the number of preovulatory follicles. However, no significant increase in egg production was observed in INH-immunized hens. The data confirm previous reports that VIP immunoneutralization increases egg production in turkey hens and shows for the first time that it also increases FSH beta subunit and LH beta subunit gene expression.

Introduction: This study aimed to investigate the effect of EphB4/ephrinB2 signaling on orthodontically-induced root resorption repair and the possible molecular mechanism behind it.

Methods: Seventy-two 6-week-old male Wistar rats were randomly divided into 3 groups: blank control group, physiological regeneration group (PHY), and EphB4 inhibitor local injection group (INH). A root repair model was built on experimental rats of the PHY and INH groups. The animals in the INH groups received a daily periodontal local injection of EphB4 inhibitor NVP-BHG712, whereas the blank control group and PHY groups received only the vehicle.

Results: Histologic staining and microcomputed tomography analysis showed that root regeneration was inhibited in the INH group compared with the PHY group with a greater number of osteoclasts. Immunohistochemical staining showed active EphB4/ephrinB2 signaling activities during root regeneration. The cementogenesis-related factors cementum attachment protein, alkaline phosphatase, osteopontin, and runt-related transcription factor 2, and osteoclastic-related factors RANKL and osteoprotegerin were affected by regulated EphB4/ephrinB2 signaling.

Conclusions: These findings demonstrated that the EphB4/ephrinB2 signaling might be a promising therapeutic target for novel therapeutic approaches to reduce orthodontically-induced root resorption through enhancement of cementogenesis.

METHODS

In vitro and in vivo study of an isoniazid (INH) drug delivery system.

OBJECTIVE

To develop a system for the treatment of tuberculosis using a subcutaneous polymer implant with a large drug load released slowly over a long period. INH delivery by biodegradable poly-(alpha-hydroxy acid) polymers was evaluated using ground polymer and compression molded implants.

METHODS

Rate of drug release and structural stability of the implant in an aqueous environment were measured, as were in vivo evaluations of the duration of measurable levels of INH in serum and urine.

RESULTS

Factors that influenced the suitability of an implant in an in vitro system included polymer molecular weight and crystallinity, polymer and drug particle size, drug loading dose, and press temperature and pressure. The implant characteristics that most closely approached optimal conditions include a polymer of 100% L-lactide with low intrinsic viscosity, polymer particle size <75 micron, and INH particle = 126-180 micron, INH loading dose not to exceed 46%, and press conditions of 70 degrees C and 345000 kPa. Studies of subcutaneous implants in rabbits and baboons show that INH is released from the implant for 15 to 26 weeks.

CONCLUSIONS

An INH-containing polymer was developed that was structurally stable in an aqueous environment and that released INH over a period of at least 15 weeks. Studies with infected animals will be necessary to determine the dose required for prophylaxis and treatment of active disease.

(<em>A</em>bstractText>The aim of the study is to detect the pattern of genetic mutation, i.e., <em>Inh</em> <em>A</em> or Kat G or both (<em>Inh</em> <em>A</em> and kat G) in isoniazid (<em>INH</em>) monoresistant mycobacteria and to correlate with the pattern in multidrug-resistant (MDR) isolates.</<em>A</em>bstractText>(<em>A</em>bstractText>In this study, a quantitative research approach was used. The research design was descriptive observational study. The study was conducted at the Department of Respiratory Medicine, JLN Medical College, <em>A</em>jmer, Rajasthan, and Intermediate Referral Laboratory, State TB Demonstration Centre, <em>A</em>jmer. <em>A</em> total of 298 samples found to have resistant strains of Mycobacterium tuberculosis were enrolled with purposive sampling.</<em>A</em>bstractText>(<em>A</em>bstractText>The mean age of patients was 40.27 ± 13.82 years. There were 250 (83.9%) males, while 48 (16.1%) were females. One hundred ninety-two (64.4%) were resistant for <em>INH</em> only, while the rest were resistant to both <em>INH</em> as well as rifampicin (MDR-tuberculosis). The most common mutation in <em>INH</em> monoresistance was kat G (125; 65.1%) as compared to <em>inh</em> <em>A</em> (54; 28.1%) and both <em>inh</em> <em>A</em> and kat G (13; 6.7%). <em>A</em>mong kat G mutations, the most common gene pattern was the absence of WT (S315T) and the presence of MUT1 (S315T1).</<em>A</em>bstractText>(<em>A</em>bstractText>Knowledge about mutation patterns of different <em>INH</em> resistant strains is important in the present era where there is a provision of separate regimens for <em>INH</em> monoresistant TB. Since these mutations are very closely related to high- or low-degree resistance to <em>INH</em>, the therapeutic regimens cannot be generalized.</<em>A</em>bstractText>

The influence of four different membrane oxygenators (HF 4000, BOS-CM 50, CML 2, Maxima) on leucocyte count, concentrations of PMN-elastase, clotting factor XII, AT-III, C1-INH, alpha 2-antiplasmin and C3a was registered before, during and after CPB with pulsatile and nonpulsatile flow in 80 male patients aged between 36 and 67 years. With all systems tested, there was a drop in the concentrations of clotting factor XII, AT-III, C1-INH and alpha 2-antiplasmin in the early extracorporeal circulation (ECC) phase, exceeding the average hematocrit reduction accounted for by dilution. This drop was the least distinct with CML 2 systems, both with pulsatile and nonpulsatile perfusion, indicating system-inherent influences. Leucocyte cound and PMN-elastase concentration rose significantly during ECC irrespective of oxygenator tested of flow type applied. The rise in leucocyte count even continued for about 4 h after ECC. During the first 40 min of ECC, these changes were paralleled by a significant rise in C3a concentration, suggesting complement activation as a main cause for PMN activation. However, there is reason to suppose involvement of further mechanisms operating in PMN activation, since the elevated C3a-concentrations began to fall off while leucocyte count and PMN-elastase concentrations were still increasing.

Isoni<em>a</em>zid (<em>INH</em>), <em>a</em> first-line drug in <em>a</em>nti-tuberculosis ther<em>a</em>py, is known to be potenti<em>a</em>lly h<em>a</em>rmful <em>a</em>nd is <em>a</em>ssoci<em>a</em>ted with numerous side effects especi<em>a</em>lly in the blood <em>a</em>nd liver. In the course of our previous investig<em>a</em>tions, 1,2,3-thi<em>a</em>di<em>a</em>zole cont<em>a</em>ining hydr<em>a</em>zone (compound <b>3</b>) showed excellent <em>a</em>ntimycob<em>a</em>cteri<em>a</em>l <em>a</em>ctivity <em>a</em>g<em>a</em>inst <em>a</em> referent str<em>a</em>in <i>M. tuberculosis</i> H37Rv (MIC v<em>a</em>lue 0.39 μM), low cytotoxicity, <em>a</em>nd did not h<em>a</em>ve toxic effects when <em>a</em>dministered by or<em>a</em>l or intr<em>a</em>peritone<em>a</em>l routes to experiment<em>a</em>l <em>a</em>nim<em>a</em>ls (selectivity index SI > 1979, LD₅₀>2000 mg/kg b.w.) wh<em>a</em>t reve<em>a</em>led its suit<em>a</em>bility for further explor<em>a</em>tion. In the present study compound <b>3</b> w<em>a</em>s chosen to determine its effects on the liver <em>a</em>nd kidney functions in fem<em>a</em>le mice. The compound w<em>a</em>s <em>a</em>dministered or<em>a</em>lly for 14 d<em>a</em>ys <em>a</em>t three doses (100, 200, <em>a</em>nd 400 mg/kg b.w.). The qu<em>a</em>ntity of m<em>a</em>londi<em>a</em>ldehyde (MDA), the level of reduced glut<em>a</em>thione (GSH), blood hem<em>a</em>tologic<em>a</em>l <em>a</em>nd biochemic<em>a</em>l p<em>a</em>r<em>a</em>meters were <em>a</em>ssessed, <em>a</em>nd urine <em>a</em>n<em>a</em>lysis w<em>a</em>s c<em>a</em>rried out. As <em>a</em> positive control <em>INH</em> w<em>a</em>s used or<em>a</em>lly <em>a</em>t <em>a</em> dose of 50 mg/kg b.w. The investig<em>a</em>ted compound <b>3</b> did not <em>a</em>ffect the urine <em>a</em>nd serum hem<em>a</em>tologic<em>a</em>l <em>a</em>nd biochemic<em>a</em>l p<em>a</em>r<em>a</em>meters <em>a</em>s <em>INH</em> did, comp<em>a</em>red to those of the control mice. The new compound did not <em>a</em>ffect signific<em>a</em>ntly the MDA qu<em>a</em>ntity <em>a</em>nd m<em>a</em>int<em>a</em>ined its level ne<em>a</em>r to the control v<em>a</em>lues, though lower by 36% (<i>p</i> < 0.05) th<em>a</em>n in the <em>INH</em> tre<em>a</em>ted <em>a</em>nim<em>a</em>ls. At the higher doses, 200 <em>a</em>nd 400 mg/kg, it depleted the GSH content by 25% (<i>p</i> < 0.05), comp<em>a</em>red to the control. However, its level rem<em>a</em>ined 47% (<i>p</i> < 0.05) higher th<em>a</em>n in the <em>INH</em> tre<em>a</em>ted <em>a</em>nim<em>a</em>ls.

BACKGROUND

C1-inhibitor (C1-INH), a serine protease inhibitor in plasma plays a central role in the cross-talk among the complement, coagulation, fibrinolytic and kallikrein-kinin systems. However, previous reports indicate thrombotic risks in children following supraphysiological dosing with C1-INH.

OBJECTIVE

To investigate the role of supraphysiological C1-INH concentrations in clot development with and without addition of Escherichia coli (E. coli) in fresh human whole blood using thromboelastometry.

METHODS

Blood was collected in citrate tubes, and C1-INH (3.0 to 47.6μM) or human serum albumin (HSA) was added as a control. Activated partial thromboplastin time (aPTT) was analysed in the plasma. The analyses non-activated thromboelastometry (NATEM), extrinsic (EXTEM) or intrinsic thromboelastometry (INTEM) were performed using rotational thromboelastometry.

RESULTS

C1-INH increased aPTT 1.8-fold (p< 0.05), whereas HSA had no effect. C1-INH increased NATEM clotting time (CT) from 789s to 2025 s (p< 0.05) in a dose-dependent manner. C1-INH reduced the NATEM alpha angle from 47 to 28° (p<0.05) and increased the NATEM clot formation time from 261s to 595s (p< 0.05). E. coli significantly reduced the NATEM CT after 120min of incubation. C1-INH prevented E. coli-induced activation (p< 0.05). C1-INH significantly increased the INTEM CT (p< 0.05), but had no effect on EXTEM CT. C1-INH (47.6μM) significantly reduced fibrinolysis measured as NATEM and EXTEM lysis indices LI60.

CONCLUSIONS

Supraphysiological C1-INH concentrations have dose-dependent anticoagulant effects in human whole blood in vitro. At very high levels C1-INH also inhibits fibrinolysis.

OBJECTIVE

Isoniazid (INH) is one of the effective antituberculosis (TB) drugs used for TB treatment. However, most of the drug-resistant Mycobacterium tuberculosis (MTB) clinical strains are resistant to INH, a first-line antituberculous drug. Certain metabolic enzymes such as adenosylhomocysteinase (Rv3248c), universal stress protein (Rv2623), nicotinamide adenine dinucleotide (reduced)-dependent enoyl-acyl carrier protein reductase (Rv1484), oxidoreductase (Rv2971), dihydrofolate reductase (Rv2763c), pyrroline-5-carboxylate dehydrogenase (Rv1187) have been identified to bind INH-nicotinamide adenine dinucleotide (INH-NAD) and INH-nicotinamide adenine dinucleotide phosphate adducts coupled to Sepharose resin. These enzymes are reported to be involved in many important biochemical processes of MTB, including cysteine and methionine metabolism, mycobacterial growth regulation, mycolic acid biosynthesis, detoxification of toxic metabolites, folate biosynthesis, etc. The truncated INH-nicotinamide adenine dinucleotide (oxidized) adduct, 4-isonicotinoylnicotinamide, isolated from urine samples of human TB patients treated with INH therapy is proposed to have antimycobacterial activity.

METHODS

To understand the mechanism of interaction of the truncated INH-NAD adduct, binding energy studies were carried out on the aforementioned six enzymes with known three-dimensional structures using AutoDock4.2.

RESULTS

In silico docking analysis of these MTB enzymes with the truncated INH-NAD adduct showed favorable binding interactions with docking energies ranging from -5.29 to -7.07 kcal/mol.

CONCLUSIONS

Thus, in silico docking study revealed that the INH-NAD adduct, which is generated in vivo after INH activation, may undergo spontaneous hydrolysis to form the truncated INH-NAD adduct and further binds and inhibits multiple enzymes of MTB, in addition to InhA, confirming that INH is an effective anti-TB drug acting at multiple enzymes. Further analysis of amino acid residues in the active site of INH-NAD-binding proteins showed the probable presence of catalytic triad in four enzymes possibly involved in INH binding to the enzyme.

It is urgent to understand the regulatory mechanism of drug resistance in widespread bacterial pathogens. In Mycobacterium tuberculosis, several transcriptional regulators have been found to play essential roles in regulating its drug resistance. In this study, we found that an ArsR family transcription regulator encoded by Rv2642 (CdiR) responds to isoniazid (INH), a widely used anti-tuberculosis (TB) drug. CdiR negatively regulates self and adjacent genes, including arsC (arsenic-transport integral membrane protein ArsC). CdiR directly interacts with INH and Cd(II). The binding of INH and Cd(II) both reduce its DNA-binding activity. Disrupting cdiR increased the drug susceptibility to isoniazid, whereas overexpressing cdiR decreased the susceptibility. Strikingly, overexpressing arsC increased the drug susceptibility as well as cdiR. Additionally, both changes in cdiR and arsC expression caused sensitivity to other drugs such as rifamycin and ethambutol, where the minimal inhibitory concentrations in the cdiR deletion strain were equal to those of the arsC-overexpressing strain, suggesting that the function of CdiR in regulating drug resistance primarily depends on arsC. Furthermore, we found that Cd(II) enhances bacterial resistance to INH in a CdiR-dependent manner. As a conclusion, CdiR has a critical role in directing the interplay between Cd(II) metal ions and drug susceptibility in mycobacteria.

<strong cl<em>a</em>ss="sub-title"> Keywords: </strong> <em>a</em>rsC; cdiR; ArsR; Tr<em>a</em>nscription<em>a</em>l regul<em>a</em>tor; drug susceptibility.