Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients.

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

MR spectroscopic imaging predicts early response to anti-angiogenic therapy in recurrent glioblastoma.

BACKGROUND: Determining failure to anti-angiogenic therapy in recurrent glioblastoma (GBM) (rGBM) remains a challenge. The purpose of the study was to assess treatment response to bevacizumab-based therapy in patients with rGBM using MR spectroscopy (MRS). METHODS: We performed longitudinal MRI/MRS in 33 patients with rGBM to investigate whether changes in N-acetylaspartate (NAA)/Choline (Cho) and Lactate (Lac)/NAA from baseline to subsequent time points after treatment can predict early failures to bevacizumab-based therapies. RESULTS: After stratifying based on 9-month survival, longer-term survivors had increased NAA/Cho and decreased Lac/NAA levels compared to shorter-term survivors. ROC analyses for intratumoral NAA/Cho correlated with survival at 1 day, 2 weeks, 8 weeks, and 16 weeks. Intratumoral Lac/NAA ROC analyses were predictive of survival at all time points tested. At the 8-week time point, 88% of patients with decreased NAA/Cho did not survive 9 months; furthermore, 90% of individuals with an increased Lac/NAA from baseline did not survive at 9 months. No other metabolic ratios tested significantly predicted survival. CONCLUSIONS: Changes in metabolic levels of tumoral NAA/Cho and Lac/NAA can serve as early biomarkers for predicting treatment failure to anti-angiogenic therapy as soon as 1 day after bevacizumab-based therapy. The addition of MRS to conventional MR methods can provide better insight into how anti-angiogenic therapy affects tumor microenvironment and predict patient outcomes.

Correlation Between Intraoperative and Pathological Findings for Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: This study aimed to examine the correlation between intraoperative and pathological findings for patients undergoing cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) and to determine their prognostic significance. METHODS: Pathological reports of all colorectal cancer (CRC) patients undergoing CRS/HIPEC between 2009 and 2016 were retrospectively reviewed. Pathological specimens lacking tumor cells were defined as negative pathological specimens (NPS). The intraoperative peritoneal cancer index (PCI) and pathological PCI (excluding NPS) were calculated separately. Receiver operating characteristic (ROC) curves were applied to compare the prognostic value of intraoperative and pathological scoring systems. RESULTS: For 108 CRC patients, 113 CRS/HIPEC procedures were performed. Of 959 pathological specimens examined, 178 (18.6%) were NPS. Overall, 78 procedures (69%) showed NPS. In 52 procedures (46%), the pathological PCI differed from the intraoperative PCI (∆PCI > 0). The ROC areas for intraoperative PCI and pathological PCI were similar in predicting 1-year overall survival (OS), 2-year OS, and 1-year disease-free survival (all p values not significant). However, for the patients with NPS, the number of positive specimens (containing tumor tissue) was superior to intraoperative PCI in predicting 2-year OS (ROC under the curve areas, 0.69 vs. 0.58, respectively; p = 0.012). In addition, a subgroup of 15 patients with a high ∆PCI (≥ 3) had a more favorable median OS than a matched group of 30 patients with similar intraoperative PCI and a ∆PCI of 0 (median survival not reached vs. 21.6 months, respectively; p = 0.05). CONCLUSIONS: In the majority of CRC CRS/HIPEC procedures, NPS may be found. Among patients with NPS, pathological correlation may have a prognostic significance.

Implications of Stoma Formation as Part of Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Formation of protective stoma as part of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) may be an effective tool in reducing anastomotic leak incidence. Our aim was to evaluate the incidence and implications of stoma formation during CRS-HIPEC and to examine whether a creation of protective stoma reduces the postoperative morbidity. METHODS: A cohort retrospective analysis of all CRS-HIPEC procedures performed between 2004 and 2016 was conducted. Predicting factors for stoma formation were assessed by comparing all patients who underwent stoma formation to those who did not; both groups were then restricted to cases with ≥2 bowel anastomoses and compared in terms of perioperative outcomes in order to determine whether protective stoma confers a morbidity benefit. RESULTS: One hundred and ninety-nine CRS-HIPEC procedures were performed on 186 patients. Thirty-four patients (17%) underwent stoma formation, 24 of them as protective stoma. Formation of a stoma was correlated with higher peritoneal carcinomatosis index score (13.6 ± 8 vs. 9.5 ± 7.7, p = 0.007), larger number of organs resected (p < 0.001), greater number of anastomoses (p < 0.001), prolonged operative time (8.1 ± 2.7 vs. 6.6 ± 2.2 h, p = 0.002), and prolonged hospital stay (12 vs. 8.5 days, p = 0.001). In procedures requiring ≥2 anastomoses, formation of protective stoma reduced the anastomotic leak rate (6 vs. 37%, p = 0.025), the morbidity rate (6 vs. 41%, p = 0.017), and reoperation rate (0 vs. 28%, p = 0.03). Overall, 15 patients (44%) underwent stoma reversal, 3 of whom had a complication treated non-operatively. CONCLUSIONS: Protective stoma should be considered in extensive CRS-HIPEC procedures requiring two or more bowel anastomoses in order to reduce the postoperative morbidity rate.

A multi-centre study of candidate genes for wheeze and allergy: the International Study of Asthma and Allergies in Childhood Phase 2.

BACKGROUND: Common polymorphisms have been identified in genes suspected to play a role in asthma. We investigated their associations with wheeze and allergy in a case-control sample from Phase 2 of the International Study of Asthma and Allergies in Childhood. METHODS: We compared 1105 wheezing and 3137 non-wheezing children aged 8-12 years from 17 study centres in 13 countries. Genotyping of 55 candidate single nucleotide polymorphisms (SNPs) in 14 genes was performed using the Sequenom System. Logistic regression models were fitted separately for each centre and each SNP. A combined per allele odds ratio and measures of heterogeneity between centres were derived by random effects meta-analysis. RESULTS: Significant associations with wheeze in the past year were detected in only four genes (IL4R, TLR4, MS4A2, TLR9, P<0.05), with per allele odds ratios generally <1.3. Variants in IL4R and TLR4 were also related to allergen-specific IgE, while polymorphisms in FCER1B (MS4A2) and TLR9 were not. There were also highly significant associations (P<0.001) between SPINK5 variants and visible eczema (but not IgE levels) and between IL13 variants and total IgE. Heterogeneity of effects across centres was rare, despite differences in allele frequencies. CONCLUSIONS: Despite the biological plausibility of IgE-related mechanisms in asthma, very few of the tested candidates showed evidence of association with both wheeze and increased IgE levels. We were unable to confirm associations of the positional candidates DPP10 and PHF11 with wheeze, although our study had ample power to detect the expected associations of IL13 variants with IgE and SPINK5 variants with eczema.

A multinational study of treatment failures in asthma management.

SETTING: Emergency rooms. OBJECTIVE: To assess quality of care and its determinants for asthma patients before emergency room treatment. DESIGN: Consecutive patients with acute severe asthma attending emergency rooms were questioned about the severity of their disease and treatment in the previous 4 weeks. Prescriptions of inhaled corticosteroids were recorded. Other outcomes included self-reported adherence to treatment and loss of work. RESULTS: Thirteen centres in 11 countries recruited 1156 patients. Only 36% of patients with persistent asthma had been prescribed an adequate dose of inhaled corticosteroids. This percentage improved in those receiving regular care from the same doctor (OR 2.86, 95%CI 1.38-5.96), and was at least as good for the 10% of patients receiving 'private' health care (OR 3.08, 95%CI 1.69-5.62). Forty-four per cent of patients had health insurance covering some asthma medications. These patients were more likely to be receiving adequate inhaled corticosteroids (OR 1.74, 95%CI 1.17-2.58), and reported better adherence than those without insurance (OR 3.00, 95%CI 1.64-5.50). Of those on adequate inhaled corticosteroids, 18% had lost work in each of the 4 previous weeks compared with 59% among those more than one treatment step below the recommended dose. CONCLUSIONS: Access to adequate treatment is critical for better management of asthma.

Pharmacological and molecular biological (RT-PCR) characterization of functional TP prostanoid receptors in immortalized human non-pigmented ciliary epithelial cells.

Immortalized human non-pigmented ciliary epithelial (NPE) cells (ODM-2) were shown to express the mRNA for the prostanoid TPalpha but not the TPbeta receptor using reverse transcription-polymerase chain reaction (RT-PCR). These TPalpha receptors were coupled to phospholipase C (PLC) and, thus, promoted phosphoinositide (PI) turnover. TP receptor agonists yielded the following potencies (EC50S) in the PI turnover assays: I-BOP = 8.2 +/- 1.1 nM; carbocyclic TA2 = 87.5 +/- 25.3 nM; U-44069 = 1.16 +/- 0.32 microM; U-46619 = 1.2 +/- 0.2 microM (n = 4-17). Agonists selective for other prostanoid receptor subtypes (e.g., fluprostenol and sulprostone) were inactive. The agonist effects of U-44619 and I-BOP were potently blocked, in an apparent non-competitive manner (ki = 53.9 +/- 12 nM; pA2s = 7.6-7.8; pKbs = 7.38), by the TP receptor-selective antagonist, SQ29,548, but were unaffected by other prostanoid receptor antagonists (e.g., AH6809, AL-8810). The PLC inhibitor (U73122) inhibited U-46619-induced PI turnover (IC50 = 4.3 +/- 0.6 microM). The functional potencies of the compounds stimulating or inhibiting the TP receptor-mediated PI turnover in the NPE cells correlated well with the TP receptor binding affinities of these compounds at human platelet TP receptors (r = 0.98). These studies have shown the presence of the mRNA for and the expression of functional TPalpha receptors coupled to PLC in human NPE cells. The TPalpha receptors on NPE cells may be responsible for inhibiting aqueous humor production and may help explain the intraocular pressure-lowering effects of certain TP agonists.

Levobetaxolol (Betaxon) and other beta-adrenergic antagonists: preclinical pharmacology, IOP-lowering activity and sites of action in human eyes.

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.

Functional pharmacological evidence for EP2 and EP4 prostanoid receptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells.

The aim of these studies was to characterize the molecular pharmacology of the prostanoid receptors positively coupled to stimulation of adenylyl cyclase activity in immortalized human trabecular meshwork (TM-3) cells and to compare these results with that of the receptors in immortalized human nonpigmented epithelial (NPE) cells. In general, the TM-3 and NPE cells showed a similar profile with respect to their responses to various prostaglandin (PG) receptor agonists. The rank order of potency (EC50; means +/- SEM) for these compounds in the TM-3 cells was: PGE2 (124 +/- 21 nM) > 13,14-dihydro-PGE1 (430 +/- 110 nM) = PGE1 (522 +/- 345 nM) > 11-deoxy-PGE1 (1063 +/- 118 nM) = 16,16-dimethyl-PGE2 (1776 +/- 460 nM) = butaprost (1920 +/- 527 nM) >> PGD2 = PGI2 = PGF2alpha (n = 3 - 12). While the agonist profile indicated the presence of EP2 receptors, the effects of the EP4 receptor antagonists suggested the additional expression of EP4 receptors in both of these cells. Thus, the EP4 receptor antagonist, AH23848B, at a concentration of 30 microM, caused a dextral shift in the PGE2 concentration-response curves in both TM-3 and NPE cells coupled with a 20-28% decrease in the maximal response of PGE2, indicating apparent noncompetitive antagonism profiles. The antagonist potency of AH23848B in these cells was: Kb = 38.4 +/- 14.8 microM and 23.5 +/- 4.5 microM; -log Kb = 4.7. The other EP4 receptor antagonist, AH22921 (-log Kb = 4.1 - 4.7), was weaker than AH23848B. Taken together, these pharmacological studies have shown than TM-3 and NPE cells apparently contain functional EP2 and EP4 prostanoid receptors positively coupled to adenylyl cyclase.

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets.

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.

Endogenous EP4 prostaglandin receptors coupled positively to adenylyl cyclase in Chinese hamster ovary cells: pharmacological characterization.

The purpose of these studies was to investigate the pharmacology of E-series and selected prostaglandins of other classes on adenylyl cyclase activity in Chinese hamster ovary (CHO) cells expressing an endogenous prostanoid receptor and to compare these responses with those from immortalized human non-pigmented ciliary epithelial (NPE) cells containing the EP2 receptor. 11-deoxy-PGE2 was the most potent of the 16 prostanoid agonists tested for stimulating cAMP formation with a potency (EC50) value of 26 +/- 6 nM in the CHO cells. The endogenous ligand, PGE2, exhibited potencies of 40 +/- 7 nM (n = 24) in the CHO cells and 67 +/- 9 nM (n = 46) in the NPE cells. The EP2 receptor agonist, butaprost, produced an EC50 value of 212 +/- 58 nM (n = 4) in the NPE cells while being inactive (EC50 > 10,000 nM, n = 6) in the CHO cells. The EP4 receptor selective antagonists, AH22921 and AH23848B, at a concentration of 30 microM, caused a 2.2 +/- 0.5 (n = 4) and 8.2 +/- 2.7 (n = 4) fold rightward shift in the PGE2 concentration-response curves in the CHO cells, yielding apparent pKb values of 4.6 +/- 0.6 and 5.3 +/- 0.2 (n = 4), respectively. AH22921 and AH23848B were non-competitive antagonists at the CHO cell EP4 receptor, but did not shift the PGE2 concentration-response curves in the NPE cells containing the EP2 receptor. These studies have characterized the functional prostaglandin receptors in CHO cells pharmacologically and shown them to be consistent with the EP4 subtype.

AL-3138 antagonizes FP prostanoid receptor-mediated inositol phosphates generation: comparison with some purported FP antagonists.

The aim of this study was to pharmacologically characterize the antagonist properties of a novel prostaglandin F2alpha (PGF2alpha) analogue (11-deoxy-16-fluoro PGF2alpha; AL-3138) using a variety of second-messenger assays of prostaglandin receptor subtypes. A detailed comparison was made between AL-3138 and some purported FP receptor antagonists such as PGF2alpha dimethylamine, PGF2alpha dimethylamide, glibenclamide and phloretin using the FP receptor-mediated phosphoinositide turnover assay in A7r5 rat thoracic aorta smooth muscle cells and mouse Swiss 3T3 fibroblasts. The potency and efficacy of AL-3138 as an FP receptor agonist were: EC50 = 72.2 +/- 17.9 nM (Emax = 37%) (n = 3) in A7r5 cells and EC50 = 20.5 +/- 2.8 nM (Emax = 33%) (n = 5) in 3T3 cells. Being a partial agonist, the antagonist potency of AL-3138 against fluprostenol in A7r5 cells was determined to be: Ki = 296 +/- 17 nM (n = 3) and Kb = 182 +/- 44 nM (n = 5) (-log Kb = 6.79 +/- 0.1). AL-3138 exhibited very minimal or no antagonistic effects at EP2, EP4, DP and TP prostaglandin receptors. Both PGF2alpha dimethylamide and PGF2alpha dimethylamine were inactive as FP receptor antagonists, whereas phloretin and glibenclamide were very weak and had -log Kb values of 5.28 +/- 0.09 (n = 3) and 3.58 +/- 0.32 (n = 3), respectively. However, phloretin antagonized functional responses of EP2 and DP prostanoid receptors, and also the V1-vasopressin receptor. AL-3138 competed for [3H]PGF2alpha binding to FP receptors with a relatively high affinity (IC50high = 312 +/- 95 nM) matching its functional antagonist potency. In conclusion, AL-3138 is a more potent and selective FP receptor antagonist than glibenclamide, phloretin, PGF2alpha dimethylamide and PGF2alpha dimethylamine and is therefore a unique and novel pharmacological tool to help characterize FP receptor-mediated functions.

Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists.

Various prostaglandin agonists representing various classes of receptor subtypes were evaluated for their ability to stimulate adenylyl cyclase via the endogenous DP receptor in embryonic bovine tracheal (EBTr) cells. Two antagonists were used to block the agonist-induced cyclic AMP production. ZK118182 (EC50 = 16+/-4 nM), RS-93520 (EC50 = 23+/- 4 nM), SQ27986 (EC50 = 33+/-9 nM), ZK110841 (EC50 = 33+/-5 nM), BW245C (EC50 = 59+/-19 nM) and PGD2 (EC50=101+/-10 nM) (n = 4-70) were the most potent agonists. Whilst most compounds were full agonists (Emax = 100% relative to PGD2), BW245C was significantly more efficacious than PGD2 (Emax = 121+/-3%; P<0.001) and RS-93520 appeared to be a partial agonist (Emax = 64+/-9%; P<0.001). Agonists from the EP (e.g. enprostil; misoprostol; butaprost), FP (e.g. cloprostenol; fluprostenol; PHXA85), IP (iloprost; PGI2) and TP (U46619) prostanoid receptor classes were weak agonists or inactive in the EBTr cell assay system. The DP-receptor antagonist, BWA868C, showed a competitive antagonist profile with pA2 values of 8.00+/-0.02 and 8.14+/-0.13 in Schild analyses with two structurally different agonists, BW245C and ZK118182, respectively (n = 3). AH6809, another purported DP-receptor antagonist, weakly inhibited PGD2- and ZK 18182-induced cyclic AMP production (K(i)s = 808+/-193 nM and 782+/-178 nM, respectively). The current studies have characterized the DP receptor positively coupled to adenylyl cyclase in EBTr cells using a wide range of agonist and antagonist prostaglandins. These data support the utility of the EBTr cell line as a useful tool for the evaluation of DP receptor agonists and antagonists and for profiling other classes of prostaglandins.

A current appreciation of sites for pharmacological intervention in allergic conjunctivitis: effects of new topical ocular drugs.

Two important realizations about pathophysiological mechanisms involved in allergic conjunctivitis have led to novel drug discovery efforts and new topical ocular medications for prevention and treatment of this prevent allergic disease. The first of these, interspecies and intraspecies mast cell heterogeneity, was established in the mid-1980's by investigators working in the field of asthma. It is now appreciated that secretory responses as well as effects of pharmacological agents differ depending upon the mast cell population studied. Two types of human mast cells, the tryptase containing (T) and the tryptase/chymase containing (TC) mast cells, have been characterized in a variety of tissues. Significantly, Irani et al. (1) demonstrated by immunohistochemical staining that the mast cells present in conjunctival tissues from patients with allergic conjunctivitis were 100% TC. Functional responses of human conjunctival mast cells to a variety of secretagogues (2) were consistent with their classification as TC or connective tissue type mast cells. Importantly, the studies by Miller et al. mentioned above allowed the harvesting and preparation of human conjunctival mast cells for use in drug screening studies. Utilization of these cells has led to the identification of Patanol, the most effective human conjunctival mast cell stabilizer available for topical use in allergic conjunctivitis (3). These same studies demonstrated the lack of mast cell stabilizing activity for cromolyn and nedocromil in these connective tissue type, TC containing, human conjunctival mast cells. Similar lack of effect was noted with these drugs on human skin mast cell degranulation (4). The second important discovery in the area of allergic conjunctivitis has been the demonstration that conjunctival epithelial cells may contribute to the perpetuation of the allergic response. A report from Gamache et al. (5) identified cytokines produced by human conjunctival epithelial cells following treatment with a number of stimuli. Significantly, Sharif et al. (6) subsequently identified functional histamine H1 receptors on these same cell types. Recently, Weimer et al. (7) have shown that exposure of human conjunctival epithelial cells to histamine leads to the production of pro-inflammatory cytokines IL-6 and IL-8. Importantly, treatment of the epithelial cells with drugs that possess histamine H1 antagonist properties prevents cytokine production. It is noteworthy that first generation anti-histamines antazoline and pheniramine are not potent inhibitors of histamine-stimulated cytokine synthesis in intact epithelial cells, while newer anti-histamines Emadine and levocabastine are more potent. Surprisingly, Patanol was more potent as an inhibitor of histamine-stimulated cytokine production by the epithelial cells than would be predicted from its histamine H1 antagonist affinity. These inhibitory effects on conjunctival epithelial cell production of pro-inflammatory cytokines may contribute to enhanced clinical activity noted with these recently approved drugs.

Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.

PURPOSE: To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies. METHODS: Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays. RESULTS: The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. CONCLUSIONS: The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.

Inhibition of histamine-induced human conjunctival epithelial cell responses by ocular allergy drugs.

OBJECTIVE: To evaluate the effects of topical ocular drugs with histamine H1-antagonist activity on histamine-stimulated phosphatidylinositol turnover and interleukin (IL) 6 and IL-8 secretion from human conjunctival epithelial cells. METHODS: Primary human conjunctival epithelial cell cultures were stimulated with histamine in the presence or absence of test drugs. Phosphatidylinositol turnover was quantified by ion exchange chromatography and cytokine content of supernatants by enzyme-linked immunosorbent assay. RESULTS: Antazoline hydrochloride, emedastine difumarate, levocabastine hydrochloride, olopatadine hydrochloride, and pheniramine maleate attenuated histamine-stimulated phosphatidylinositol turnover and IL-6 and IL-8 secretion. Emedastine was the most potent in ligand binding, phosphatidylinositol turnover, and IL-6 secretion, with dissociation constant and 50% inhibitory concentrations of 1-3 nmol/L. Olopatadine, antazoline, and pheniramine exhibited similar H1-binding affinities (32-39 nmol/L). However, olopatadine was approximately 10-fold more potent as an inhibitor of cytokine secretion (50% inhibitory concentration, 1.7-5.5 nmol/L) than predicted from binding data, while antazoline and pheniramine were far less potent (20- to 140-fold) in functional assays. Levocabastine (dissociation constant, 52.6 nmol/L) exhibited greater functional activity (50% inhibitory concentration, 8-25 nmol/L) than either antazoline or pheniramine. CONCLUSIONS: Histamine-stimulated phosphatidylinositol turnover and cytokine secretion by human conjunctival epithelial cells are attenuated by compounds with H1-antagonist activity. However, antihistaminic potency alone does not predict anti-inflammatory potential. Olopatadine, emedastine, and levocabastine were notably more potent than pheniramine and antazoline. CLINICAL RELEVANCE: Selected topical ocular drugs with antihistaminic activity may offer therapeutic advantages to patients with allergic conjunctivitis by inhibiting proinflammatory cytokine secretion from human conjunctival epithelial cells.

Pharmacology of [3H]-pyrilamine binding and of the histamine-induced inositol phosphates generation, intracellular Ca2+ -mobilization and cytokine release from human corneal epithelial cells.

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.