Use of a FluoroType® System for the Rapid Detection of Patients with Multidrug-Resistant Tuberculosis-State of the Art Case Presentations.

According to the World Health Organization (WHO), there were 465,000 cases of tuberculosis caused by strains resistant to at least two first-line anti-tuberculosis drugs: rifampicin and isoniazid (MDR-TB). In light of the growing problem of drug resistance in Mycobacterium tuberculosis across laboratories worldwide, the rapid identification of drug-resistant strains of the Mycobacterium tuberculosis complex poses the greatest challenge. Progress in molecular biology and the development of nucleic acid amplification assays have paved the way for improvements to methods for the direct detection of Mycobacterium tuberculosis in specimens from patients. This paper presents two cases that illustrate the implementation of molecular tools in the recognition of drug-resistant tuberculosis.

Tuberculosis of the Urogenital Tract in Adults in a Tertiary Referral Center.

The genitourinary system is the main location of extrapulmonary tuberculosis. In Poland, it occupies the third place after tuberculosis of the pleura and lymph nodes. The aim of this study was to evaluate the prevalence and characteristics of tuberculosis in the urogenital tract in adult patients in a tertiary referral center in the years 2007-2015. The retrospective study included 87 patients, 42 women and 45 men. The average age was 62 ± 15 years. Changes in the urinary tract were diagnosed in 91% of women and 64% of men. Testicular tuberculosis was found in ten men, prostate tuberculosis in five, and in individual cases tuberculosis of the epididymis, scrotum, uterus, and the fallopian tube were found. The diagnosis was confirmed by bacteriological methods in 47% of patients, by histopathological in 41%, and by molecular methods in 23% of patients. In 84% of patients urological or gynecological interventions had to be applied. Patients were burdened with a number of urological diseases or diseases affecting other systems which hampered the diagnosis of tuberculosis. Antituberculosis treatment gave good results. Urogenital tuberculosis is a multivariate disease and a standard unified approach is impossible.

[Tuberculosis of the urinary tract of women suspected of pulmonary tuberculosis. A case report].

Diagnosis of tuberculosis requires bacteriological confirmation. The sputum is the material most frequently used for this purpose. In the case of extra-pulmonary tuberculosis, it is necessary to obtain other materials, such as urine, pleural fluid, pericardial effusion, ascites, cerebrospinal fluid, sample of tissue, etc. However, compared with sputum, these materials are much less likely to successfully culture mycobacteria. We described a case of a young woman with suspected pulmonary tuberculosis, however confirmation of the disease was obtained from urine culture. Despite hemoptysis and radiological changes typical for tuberculosis, the cultures of mycobacteria from respiratory tract were negative. The patient did not report any symptoms from urinary tract, and urinalysis was normal, ultrasound however revealed changes in the left kidney which were typical for tuberculosis. The mycobacterial culture of urine was positive. Antimycobacterial drugs were applied with a good effect.

Socio-economic status and the duration of pulmonary tuberculosis symptoms in women treated at the Mazovian Treatment Centre of Tuberculosis and Lung Diseases in Otwock.

INTRODUCTION: The prevalence of tuberculosis depends on various factors such as migration, homelessness, malnutrition, unemployment, bad life conditions and the aging of a society. The aim of this study was to evaluate tuberculosis in females treated at the Mazovian Treatment Centre of Tuberculosis and Lung Diseases (Mazowieckie Centrum Leczenia Chorób Pluc i Gruzlicy) in Otwock, regarding the context of demographic, social and professional status of female patients. The duration of the illness and the extent of radiographic changes were also taken into consideration. MATERIAL AND METHODS: The study was carried out retrospectively. The medical documentation that was evaluated concerned 100 women, aged between 20 and 92, hospitalized at the Mazovian Treatment Centre of Tuberculosis and Lung Diseases in Otwock in the years 2005 and 2006 due to bacteriologically confirmed tuberculosis. RESULTS: Most women with tuberculosis lived in cities (65%), 32% of the evaluated patients lived in villages and 3% were homeless. 1/3 of females were under 40 years of age, and 1/3 were over 60 years of age. Only 29% of the women were professionally active and 25% were unemployed. 60% of women were not married. 35% of women with tuberculosis were bringing up children and 7% had abandoned their offspring. More than 1/3 of women had had tuberculosis symptoms for more than half a year before tuberculosis was diagnosed. 40% of women with tuberculosis had small radiological changes (1 to 2 lung fields); however, 26% of them had extensive changes covering 4 to 6 lung fields. CONCLUSIONS: The majority of women with tuberculosis in the Mazovian district are single, over 40 years old, unemployed inhabitants of cities. 30% of women in the study group had had symptoms for more than 6 months before tuberculosis was diagnosed. 40% of women with tuberculosis had very extensive radiological changes covering 4 to 6 lung fields.

PEG conjugates of potent α4 integrin inhibitors, maintaining sustained levels and bioactivity in vivo, following subcutaneous administration.

Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.

Selective and brain-permeable polo-like kinase-2 (Plk-2) inhibitors that reduce α-synuclein phosphorylation in rat brain.

Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of α-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition in vivo. One such compound significantly decreased phosphorylation of α-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.

Discovery of (R)-4-cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro-1H-pyrazolo[4,3-c]quinoline (ELND006) and (R)-4-cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro-2H-pyrazolo[4,3-c]quinoline (ELND007): metabolically stable γ-secretase Inhibitors that selectively inhibit the production of amyloid-ß over Notch.

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.

Discovery of a novel [3.2.1] benzo fused bicyclic sulfonamide-pyrazoles as potent, selective and efficacious γ-secretase inhibitors.

Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.

Correlation of N-acetyltransferase 2 genotype with isoniazid acetylation in Polish tuberculosis patients.

UNLABELLED: Isoniazid (INH), a key agent in the treatment of tuberculosis (TB), is metabolized primarily by the genetically polymorphic N-acetyltransferase 2 (NAT2) enzyme. Patients treated with INH can be classified as fast, intermediate, and slow acetylators. The objective of this study was to explore the relationship between NAT2 genotypes and the serum concentrations of INH. Blood samples from 130 patients were taken for the analysis, and plasma INH concentrations were determined by using the high-performance liquid chromatography (HPLC) technology. Acetylation genotype was determined on genomic DNA by using an allele-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Once the NAT2 genotypes were established, patients were classified into three categories: fast, intermediate, and slow acetylators. Of the 130 patients studied, 84 (64.6%) were slow, 39 (30%) were intermediate, and 7 (5.4%) were fast acetylators. Analysis of INH concentrations in the blood of patients receiving the approximate doses of the drug revealed that, at the time intervals examined, the average concentration of INH was 2- to 7-fold higher among slow acetylators compared to fast and intermediate acetylators. CONCLUSION: Determining mutations in the NAT2 gene enabled the identification of the INH acetylation type in patients and the genotyping results were consistent with the phenotype determined by methods of measurement of drug bioavailability.

[Socio-economic status and duration of TB symptoms in males treated at the Mazovian Treatment Centre of Tuberculosis and Lung Diseases in Otwock]. / Status spoleczno-ekonomiczny i czas trwania objawów u mezczyzn chorych na gruzlice leczonych w Mazowieckim Centrum Leczenia Chorób Pluc i Gruzlicy w Otwocku.

INTRODUCTION: The incidence of tuberculosis depends on many factors, not only on health issues but also on socioeconomic factors. The aim of this study was to assess the duration of symptoms and the extent of radiological changes in men with bacteriologically confirmed pulmonary tuberculosis in relation to their socioeconomic status. MATERIAL AND METHODS: This was a retrospective study based on the analysis of 300 hospital records of patients hospitalised in 2004-2006 in the male ward of the Mazovian Treatment Centre of Lung Diseases and Tuberculosis in Otwock. In all patients, the diagnosis of tuberculosis was bacteriologically confirmed. We evaluated the duration of symptoms prior to hospitalisation, the extent of radiological changes and socioeconomic status. We also took into account the place of residence, professional activity, age and marital status. RESULTS: Among patients with TB hospitalised in the Mazovia Region, 74% were professionally inactive persons and 57% were unemployed. Patients population in cities and villages were similar, but as much as 10% of the patients hospitalised who were actively spreading bacilli in Mazovia Region were homeless. In the study group, 60% of the men were unmarried. In 63% of the patients symptoms of tuberculosis were present for more than two months. Chronic symptoms were reported more often in the unemployed (60%) and in single patients. As much as 81% of the patients at the initiation of treatment, had extensive radiological changes in 3 or more lung fields. Quite often sweeping pulmonary changes were observed in the homeless, unemployed and pensioners. Sputum smear-positive tuberculosis, was demonstrated in 87% of the examined patients. CONCLUSIONS: The incidence of tuberculosis observed in the Mazovia Region was especially observed in the unemployed, disabled and pensioners. Among these patients, many were homeless. The majority of patients in Mazovia Region at the start of treatment already had very extensive radiological changes and the symptoms were present with them for several weeks.

Synthesis of novel tetrahydro-1H-pyrazolo[4,3-c]pyridines via intramolecular nitrilimine cycloaddition.

The preparation of novel tetrahydro-1H-pyrazolo[4,3-c]pyridines is reported. Pivotal to the synthesis of these compounds was the development of mild reaction conditions to generate a highly functionalized nitrilimine capable of undergoing an intramolecular cycloaddition with a tethered alkyne. The desired cycloadduct was formed as an equal mixture of diastereomers.

Amino-caprolactam γ-secretase inhibitors showing potential for the treatment of Alzheimer's disease.

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aß(x-40) levels in FVB mice via a single 100 mpk IP dose are highlighted.

Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.

INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

[Immigrants treated for tuberculosis in Mazovian Center for Treatment of Lung Diseases and Tuberculosis in Otwock]. / Obcokrajowcy leczeni na gruzlice w Mazowieckim Centrum Leczenia Chorób Pluc i Gruzlicy w Otwocku.

INTRODUCTION: Migration of population contributes to the transmission of tuberculosis (TB), particularly multidrug-resistant tuberculosis. In the countries of Western Europe, the immigrants' inflow contributes to the deterioration of the epidemiological situation. Majority of newly detected TB cases in some countries were affirmed among immigrant and foreign born population. In Poland, this problem has not been investigated up to 2005. The aim of the study was the assessment of the occurrence of tuberculosis in foreigners treated in the Mazovian Centre for Treatment of Lung Diseases and Tuberculosis in Otwock. MATERIAL AND METHODS: This work had a retrospective character. The number of cases of tuberculosis in foreigners admitted between 2002 and 2007 was calculated from the data base of the Mazovian Centre for Treatment of Lung Diseases and Tuberculosis; 125 patients, whose basic demographic data, bacteriological status and the radiological changes suggested TB, were included in the study. RESULTS: The foreigners made up to 0.5-1.7% all tuberculosis cases treated in Mazovian Centre for Treatment of Lung Diseases and Tuberculosis. Among confirmed cases, twenty four nationalities were seen. Nationals of the Russian Federation (coming from the Republic of Chechnya) formed the biggest group (24%), followed by the Vietnamese (21%) and the Ukrainians (12%). Most of all cases were young men (77%; average age - 34 years). Children made up to 12% of all cases. Tuberculosis of lungs was predominating, and there were culture confirmed extrapulmonary locations in 13.6% of cases. Bacteriological confirmation was achieved in 53% of cases, but up to 22.7% cases were resistant to one of the antituberculosis medicines and 13.6% was multidrug-resistant. CONCLUSIONS: Despite the fact, that foreigners made up a small proportion among all the patient treated for tuberculosis in Mazovia, their number systematically increases. High proportion of multidrug-resistant tuberculosis reported in foreign-born cases is a concern.

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: structure-activity relationship of the aryl region.

The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.

Discovery of a novel sulfonamide-pyrazolopiperidine series as potent and efficacious gamma-secretase inhibitors.

Discovery of a series of pyrazolopiperidine sulfonamide based gamma-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.

[Comparison of Th1 and Th2 response in the blood of tuberculous patients and healthy contacts]. / Odpowiedz Th1-Th2 u chorych na gruzlice oraz osób zdrowych pozostajacych z nimi w stycznosci.

INTRODUCTION: Th1 response is known to play a dominant role in the resistance to tuberculosis. Nevertheless, IFN gamma levels are frequently increased in tuberculous patients, especially at the site of the disease. It is also possible that the shift toward Th2 response is responsible for the loss of resistance. The aim of this study was to compare the Th1 function of peripheral blood cells and the levels of antimycobacterial antibodies in the serum of culture positive tuberculosis patients and healthy tuberculosis (Tb) contacts. The correlation between the levels of antimycobacterial antibodies and Th1 function of blood cells was also evaluated. MATERIAL AND METHODS: The material consisted of 51 tuberculous patients and 20 healthy persons, close contacts of tuberculosis patients. The ability of peripheral blood cells to secrete IFN gamma and IL-2 was estimated in whole blood cultures with PHA, PWM and tuberculin. The levels of IFN gamma and IL-2 in the supernatants of cultures was estimated via a commercial ELISA test. The levels of antimycobacterial antibodies was measured with commercial immunoenzymatic kits detecting IgG antibodies against 38 kDa + 16 kDa and IgG, IgA and IgM antibodies to 38 kDa + lipoarabinomannan (LAM). RESULTS: No difference was found in the secretion of IFN gamma and IL-2 after stimulation with PHA and PWM between the patients and contacts. The secretion of IFN gamma after stimulation with tuberculin was even greater in tuberculous patients than in contacts. The levels of IgG and IgA (38 kDa + LAM) were higher in tuberculous patients than in contacts. There was a negative correlation between the level of IgG anti 38 kDa + LAM and the ability of peripheral blood cells to secrete IFN gamma after non-specific stimulation in patients with tuberculosis. CONCLUSIONS: Our study confirms the hypothesis that it is not the diminished production of Th1 cytokines, but rather the parallel overproduction of Th2 cytokines, which are essential in the development of tuberculosis.

Identification of gamma-secretase inhibitor potency determinants on presenilin.

Production of amyloid beta peptides (Abeta), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Abeta, BACE1 and gamma-secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of gamma-secretase. In comparing presenilin selectivity of several classes of gamma-secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising gamma-secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu(172), Thr(281), and Leu(282) in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu(172), Thr(281), and Leu(282) identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Abeta production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for gamma-secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of gamma-secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of gamma-secretase are feasible, and such inhibitors may allow differential inhibition of Abeta peptide production and Notch signaling.

Preparation and optimization of a series of 3-carboxamido-5-phenacylaminopyrazole bradykinin B1 receptor antagonists.

The B1 receptor is an attractive target for the treatment of pain and inflammation. A series of 3-carboxamido-5-phenacylamino pyrazole B1 receptor antagonists are described that exhibit good potency against B1 and high selectivity over B2. Initially, N-unsubstituted pyrazoles were studied, but these compounds suffered from extensive glucuronidation in primates. This difficulty could be surmounted by the use of N-substituted pyrazoles. Optimization efforts culminated in compound 41, which has high receptor potency and metabolic stability.

Structure-chiroptical properties relationship in oxabicyclic beta-lactam derivatives.

The relationship between molecular structure of 5-dethia-5-oxacephams and clavams and their chiroptical properties was investigated by means of X-ray diffraction analysis, molecular modeling calculations and circular dichroism spectroscopy. It was found that the amide chromophore of the beta-lactam unit in these compounds is nonplanar with nitrogen atom having a pyramidal configuration. It was also found that the helicity of the lactam moiety in investigated oxacephams and clavams is controlled by the absolute configuration at the C-6 and C-5 carbon atom, respectively. Thus, the applicability of helicity rule correlating a positive (negative) torsional angle of the beta-lactam subunit O=C-N-C with a negative (positive) sign of the n-->pi* CE, previously applied to oxacephams, is now extended to clavams.