A method for isolating and culturing placental cells from failed early equine pregnancies.

Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL.

High performance liquid chromatographic enantioseparation development and analytical method characterization of the carboxylate ester of evacetrapib using an immobilized chiral stationary phase with a non-conventional eluent system.

Using multiple HPLC chromatographic modes and various chiral columns in the context of an automated screening system, a potential separation was initially identified for the methyl ester of evacetrapib and its stereoisomers using an immobilized polysaccharide-based HPLC column. The bonded nature of this column, the Chiralpak(®) IC, allows for enhanced separation development with a diverse solvent range not amenable to standard coated chiral stationary phases. The ternary eluent system ultimately identified provided isomer resolutions not obtainable via the more established hexane/alcohol or polar organic chromatographic modes. A systematic separation development process is described, first for the resolution of the isomers, and later incorporating five potential impurities. A robust separation system was eventually developed that effectively resolves all compounds within a reasonable analysis time.

Stereoselective high-performance liquid chromatography and analytical method characterization of evacetrapib using a brush-type chiral stationary phase: a challenging isomeric separation requiring a unique eluent system.

Using HPLC chiral separation screening, various columns representing the polysaccharide, macrocyclic antibiotic and brush classes were assessed in multiple chromatographic modes for the separation of evacetrapib, a potential cardiovascular drug, from its enantiomer, two diastereomers and several impurities. Screening data consistently pointed to the brush-type Whelk-O 1 chiral column as most promising for this task. A systematic separation optimization process is outlined using the (S,S) Whelk-O 1 chiral column, first for the resolution of the isomers, and later including six potential impurities. A relatively complex yet rugged separation system was eventually identified that effectively resolves all compounds within a reasonable analysis time, and should serve as an adequate tool for evacetrapib bulk drug enantiopurity measurement.

Investigation of the mechanism of racemization of litronesib in aqueous solution: unexpected base-catalyzed inversion of a fully substituted carbon chiral center.

Mitosis inhibitor (R)-litronesib (LY2523355) is a 1,3,4-thiadiazoline-bearing phenyl and N-(2-ethylamino)ethanesulfonamido-methyl substituents on tetrahedral C5. Chiral instability has been observed at pH 6 and above with the rate of racemization increasing with pH. A positively charged trigonal intermediate is inferred from the fact that p-methoxy substituent on the phenyl accelerated racemization, whereas a p-trifluoromethyl substituent had the opposite effect. Racemization is proposed to occur through a relay mechanism involving intramolecular deprotonation of the sulfonamide by the side chain amino group and attack of the sulfonamide anion on C5, cleaving the C5S bond, to form an aziridine; heterolytic dissociation of the aziridine yields an ylide. This pathway is supported by (1) a crystal structure providing evidence for a hydrogen bond between the sulfonamide NH and the amino group, (2) effects of substituents on the rate of racemization, and (3) computational studies. This racemization mechanism results from neighboring group effects in this densely functionalized molecule. Of particular novelty is the involvement of the side-chain secondary amino group, which overcomes the weak acidity of the sulfonamide by anchimeric assistance.

All-cause mortality in hospitalized HIV-infected patients at an acute tertiary care hospital with a comprehensive outpatient HIV care program in New York City in the era of highly active antiretroviral therapy (HAART).

PURPOSE: The overall mortality rate among human immunodeficiency virus (HIV)-infected patients has significantly declined in the era of highly active antiretroviral therapy (HAART). However, little is known about the causes of death for HIV-infected patients who are hospitalized in acute care hospitals. METHODS: A retrospective chart review of hospitalized HIV-infected patients from 2004 to 2008 was undertaken. RESULTS: Among 9,101 hospitalized HIV-infected patients, 237 deaths were identified, with an overall mortality rate of 237/9,101 (2.6 %). The mortality rate did not differ from year to year (2-3 %). Charts for 208 patients were available for review and were analyzed. The following medians were noted: age 49 years, CD4+ T cell count 137 cells/µL, HIV viral load (VL) log10 3.93, length of stay 16 days. The proportion of men were 71.6 %, African Americans (AAs) were 62.5 %, and HAART use was 52.4 %, with an overall good adherence rate of only 17.3 %. The major causes of death were non-acquired immunodeficiency syndrome (AIDS)-related illness (81.7 %, 170/208): sepsis (34.6 %, 72/208), non-recurrent bacterial pneumonia (19.7 %, 41/208), cardiac disease (5.8 %, 12/208), liver disease (4.3 %, 9/208), and non-AIDS-related malignancy (4.3 %, 9/208). The major causes of death due to AIDS-related illness (18.3 %, 38/208) were: Pneumocystis jirovecii pneumonia (4.8 %, 10/208) and AIDS-related encephalopathy, including progressive multifocal leukoencephalopathy/cryptococcal meningitis/cerebral toxoplasmosis (3.4 %, 7/208). Mortality due to AIDS-related illnesses was associated with younger age (median age 44 vs. 50 years, p = 0.001), female sex (44.7 vs. 24.7 %, p = 0.013), and lower CD4+ T cell counts (median 10 vs. 66, p = 0.001). CONCLUSION: The mortality rate in our hospitalized HIV-infected patients remained low. Non-AIDS-related illnesses were the major causes of death, with sepsis being the most common. Low CD4+ T cell count and female sex were associated with deaths due to AIDS-related illness. Poor adherence to HAART was also noted in those patients to whom treatment was offered in the outpatient setting. Further prospective studies are needed in order to better define the epidemiology and outcomes for hospitalized HIV-infected patients in the era of HAART.

Genetic analysis of NR0B1 in congenital adrenal hypoplasia patients: identification of a rare regulatory variant resulting in congenital adrenal hypoplasia and hypogonadal hypogonadism without testicular carcinoma in situ.

There have been few testicular histology reports of adult patients with congenital adrenal hypoplasia/hypogonadal hypogonadism (AHC/HH), but Leydig cell hyperplasia has been observed, an indicator of the possibility of malignant transformation. We aimed to define the basis of AHC/HH in 4 pedigrees of different ethnic backgrounds. One patient was elected to have testicular biopsy which was examined for evidence of carcinoma in situ (CIS). NR0B1 mutation analysis was performed by sequence analysis. NR0B1 expression was investigated by RT-PCR. Testicular biopsy sections were stained with HE or immunostained for OCT3/4, an established marker of CIS. We identified NR0B1 variants in the 4 AHC pedigrees: pedigree 1 (United Arab Emirates), c.1130A>G predicting p.(Glu377Gly); pedigree 2 (English Caucasian), c.327C>A predicting p.(Cys109*); pedigree 3 (Oman), a 6-bp deletion of a direct repeat, c.857_862delTGGTGC predicting p.(Leu286_Val287del); pedigree 4 (English Caucasian), c.1168+1G>A, a regulatory variant within the NR0B1 splice donor site. This last male patient, aged 30 years, presented with evidence of HH but incomplete gonadotrophin deficiency, following an earlier diagnosis of Addison's disease at 3 years. Hormonal therapy induced virilisation. Testicular biopsy was performed. The c.1168+1G>A variant abrogated normal splicing of testicular mRNA. Histological examination showed poorly organised testicular architecture and absence of spermatozoa. Morphological analyses and the absence of immunohistochemical staining for OCT3/4 excluded the presence of malignant germ cell cancer and its precursor lesion, CIS. These studies add to the knowledge of the types and ethnic diversity of NR0B1 mutations and their associated phenotypes, and provide insight into the assessment and interpretation of testicular histology in AHC and HH.

Eosinophilic oesophagitis: an unsuspected aetiology for dysphagia in an HIV-positive patient.

Patients with HIV/AIDS are often afflicted with oesophageal disorders. Opportunistic infections such as candidiasis, herpes simplex, cytomegalovirus, mycobacterial infections, Kaposi sarcoma or lymphoma involving the oesophagus, motility disorders and reflux oesophagitis are the usual culprits. Eosinophilic oesophagitis (EE), a recently recognized entity, is an important cause of dysphagia, food impaction and chest discomfort. We report the case of an HIV-infected man who had persistent dysphagia for six months despite treatment with proton pump inhibitor. He was diagnosed with EE after having endoscopic evaluation and biopsy of his oesophagus and was successfully treated with swallowed fluticasone. This case represents the first reported case of EE in an HIV-infected individual.

(S)-N-{3-[1-cyclopropyl-1-(2,4-difluoro-phenyl)-ethyl]-1H-indol-7-yl}-methanesulfonamide: a potent, nonsteroidal, functional antagonist of the mineralocorticoid receptor.

A novel, potent series of indole analogs were recently developed as MR antagonists, culminating in 14. This compound represents the first MR antagonist in this class of molecules, exhibiting picomolar binding affinity and in vivo blood pressure lowering at pharmaceutically relevant doses.

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems.

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.

Comparison of separation efficiency of early phase active pharmaceutical intermediates by steady state recycle and batch chromatographic techniques.

Chromatographic resolution of enantiomers has become the accepted method for generation of active pharmaceutical intermediates (APIs) in early phase development. Continuous processes such as simulated moving bed (SMB) are recognized as alternative approaches for manufacturing of larger quantities of enantiomerically pure compounds. Steady state recycle (SSR) technology was initially described in 1998 [C.M. Grill, L. Miller, J. Chromatogr. A 827 (1998) 359] and has recently become a common technique in some laboratories. Batch chromatography, SSR, and SMB processes should be considered "phase appropriate" technologies. Phase appropriate technology refers to the scale of separation required in a step of the drug development process. SSR is phase appropriate technology for producing 100 g to several kilogram quantities of material. In this report, we will describe development and scale up of chiral separations utilizing the SSR technique for six different APIs utilizing several different chiral stationary phases and compare the efficiencies of both SSR and batch chromatographic techniques.

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography.

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.

Lethal dose and reproductive parameters of p-nonylphenol in rats.

Male reproductive abnormalities may be due to an increased level of maternal estrogens affecting the developing fetus. Man is exposed to environmental estrogens in multiple ways: diet, drinking water, air, and skin, para-Nonylphenol (p-NP), an alkylphenol, has estrogenic properties. This study was performed to evaluate the potency and workable concentrations before reproductive tests could be performed. The oral LD50 (median lethal dose) value for rats was 1475 mg/kg. For both males in the reproductive test the litter size after long-term treatment at 50 mg/kg p-NP was smaller than that in the breeding records. Five females did not conceive at all.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds.

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC.

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high-performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pKa values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids.

A city-wide outbreak of a multiple-drug-resistant strain of Mycobacterium tuberculosis in New York.

SETTING: Incident patients with active tuberculosis (TB) resistant to two or more drugs in New York City hospitals in 1992. OBJECTIVE: To examine the New York-wide distribution of Public Health Research Institute (PHRI) strain W of Mycobacterium tuberculosis, an extremely drug-resistant strain identified by a 17-band Southern hybridization pattern using IS6110, during the peak tuberculosis year of 1992. We also compared strain W with other strains frequently observed in New York. DESIGN: Blinded retrospective study of stored M. tuberculosis cultures by restriction fragment length polymorphism (RFLP) DNA fingerprinting, and chart review. RESULTS: We found 112 cultures with the strain W fingerprint and 8 variants in 21 hospitals among incident patients hospitalized in 1992. Almost all isolates were resistant to four first-line drugs and kanamycin. This single strain made up at least 22% of New York City multiple-drug-resistant (MDR) TB in 1992, far more than any other strain. Almost all W-strain cases were acquired immune deficiency syndrome (AIDS) patients. The cluster is the most drug-resistant cluster identified in New York and the largest IS6110 fingerprint cluster identified anywhere to date. CONCLUSION: Because recommended four-drug therapy will not sterilise this very resistant strain, there was a city-wide nosocomial outbreak of W-strain TB in the early 1990s among New York AIDS patients. Other frequently seen strains were either also very resistant, or, surprisingly, pansusceptible. Individual MDR strains can be spread widely in situations where AIDS and TB are both common.

A multi-institutional outbreak of highly drug-resistant tuberculosis: epidemiology and clinical outcomes.

OBJECTIVE: To investigate a multi-institutional outbreak of highly resistant tuberculosis and evaluate patient outcome. DESIGN: Epidemiologic investigation of every tuberculosis case reported in New York City. SETTING: Patients cared for at all public and nonpublic institutions from January 1, 1990, to August 1, 1993 (43 months). PATIENTS: We reviewed medical and public health records and conducted clinical, epidemiologic, drug susceptibility, and restriction fragment length polymorphism (RFLP) analyses. A case was defined as tuberculosis in a patient with an isolate resistant to isoniazid, rifampin, ethambutol hydrochloride, and streptomycin (and rifabutin, if sensitivity testing included it), and, if RFLP testing was done, a pattern identical to or closely related to strain W. MAIN OUTCOME MEASURES: Patient survival and the conversion of sputum cultures from positive to negative. RESULTS: Of the 357 patients who met the case definition, 267 had identical or nearly identical RFLP patterns; isolates from the other 90 patients were not available for RFLP testing. Among these 267 patients, 86% were human immunodeficiency virus (HIV)-infected, 7% were HIV-negative, and 7% had unknown HIV status. All-cause mortality was 83%. Epidemiologic linkages were identified for 70% of patients, of whom 96% likely had nosocomially acquired disease at 11 hospitals. Survival was prolonged among patients who received medications to which their isolate was susceptible, especially capreomycin sulfate, and among patients with a CD4+ T-lymphocyte count greater than 0.200 x 10(9)/L (200/microL). Treatment with isoniazid and a fluoroquinolone antibiotic was also independently associated with longer survival. CONCLUSIONS: This outbreak accounted for nearly one fourth of the cases of multidrug-resistant tuberculosis in the United States during a 43-month period. Most patients had nosocomially acquired disease, were infected with HIV, and unless promptly and appropriately treated, died rapidly. With appropriate directly observed treatment, especially combinations including an injectable medication, even severely immunocompromised patients had culture conversion and prolonged, tuberculosis-free survival.

Tuberculosis in health care workers at a hospital with an outbreak of multidrug-resistant Mycobacterium tuberculosis.

OBJECTIVE: Investigate reports of tuberculosis in health care workers employed at a hospital with an outbreak of multidrug-resistant Mycobacterium tuberculosis. DESIGN: Case series of tuberculosis in health care workers, January 1, 1989, through May 31, 1992. Antimicrobial susceptibility testing and restriction fragment length polymorphism analysis of M tuberculosis isolates. Longitudinal analysis of cumulative tuberculin skin test surveillance data. Assessment of infection control. The patients consisted of 361 health care workers who had either serial tuberculin skin tests or tuberculosis. RESULTS: Six health care workers, the largest number linked to one multidrug-resistant tuberculosis outbreak, had disease due to M tuberculosis that matched the outbreak strain from hospitalized patients. The two who were seropositive for human immunodeficiency virus died, one of tuberculous meningitis and the other of multiple causes including tuberculosis. The estimated risk of a skin test conversion was positively associated with time and increased by a factor of 8.3 (1979 to 1992). In 1992 the annual risk for workers in the lowest exposure occupational group was 2.4%. In comparison, nurses and housekeepers had relative risks of 8.0 (95% confidence interval, 3.2 to 20.3) and 9.4 (95% confidence interval, 2.7 to 32.3), respectively. Laboratory workers had a relative risk of 4.2 (95% confidence interval, 1.1 to 15.5). Tuberculosis admissions increased, but the hospital had inadequate ventilation to isolate tuberculosis patients effectively. There were lapses in infection control practices. CONCLUSIONS: Health care workers who were exposed during a hospital outbreak of multidrug-resistant tuberculosis had occupationally acquired active disease. The human immunodeficiency virus-infected health care workers with tuberculosis had severe disease and died. The risk of skin test conversion increased during the study period, and higher exposure occupations had elevated risk. Effective infection control is essential to prevent the transmission of tuberculosis to health care workers.