Evolutionary origins of the placental expression of chromosome 19 cluster galectins and their complex dysregulation in preeclampsia.

INTRODUCTION: The dysregulation of maternal-fetal immune tolerance is one of the proposed mechanisms leading to preeclampsia. Galectins are key regulator proteins of the immune response in vertebrates and maternal-fetal immune tolerance in eutherian mammals. Previously we found that three genes in a Chr19 cluster encoding for human placental galectin-13 (PP13), galectin-14 and galectin-16 emerged during primate evolution and may confer immune tolerance to the semi-allogeneic fetus. MATERIALS AND METHODS: This study involved various methodologies for gene and protein expression profiling, genomic DNA methylation analyses, functional assays on differentiating trophoblasts including gene silencing, luciferase reporter and methylation assays. These methods were applied on placental specimens, umbilical cord blood cells, primary trophoblasts and BeWo cells. Genomic DNA sequences were analyzed for transposable elements, transcription factor binding sites and evolutionary conservation. RESULTS AND DISCUSSION: The villous trophoblastic expression of Chr19 cluster galectin genes is developmentally regulated by DNA methylation and induced by key transcription factors of villous placental development during trophoblast fusion and differentiation. This latter mechanism arose via the co-option of binding sites for these transcription factors through promoter evolution and the insertion of an anthropoid-specific L1PREC2 transposable element into the 5' untranslated region of an ancestral gene followed by gene duplication events. Among placental Chr19 cluster galectin genes, the expression of LGALS13 and LGALS14 is down-regulated in preterm severe preeclampsia associated with SGA. We reveal that this phenomenon is partly originated from the dysregulated expression of key transcription factors controlling trophoblastic functions and galectin gene expression. In addition, the differential DNA methylation of these genes was also observed in preterm preeclampsia irrespective of SGA. CONCLUSIONS: These findings reveal the evolutionary origins of the placental expression of Chr19 cluster galectins. The complex dysregulation of these genes in preeclampsia may alter immune tolerance mechanisms at the maternal-fetal interface.

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject's colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands-in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)-were significantly hypermethylated in tumor vs. normal tissues (P<0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-ß1 network-the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.

Pharmacokinetics of indinavir combined with low-dose ritonavir in human immunodeficiency virus type 1-infected children.

So far, no pediatric doses for indinavir combined with ritonavir have been defined. This study evaluated the pharmacokinetics of 400 mg of indinavir/m(2) combined with 125 mg of ritonavir/m(2) every 12 h (q12h) in 14 human immunodeficiency virus type 1-infected children. The area under the concentration-time curve from 0 to 24 h and the minimum concentration of drug in serum for indinavir were similar to those for 800 mg of indinavir-100 mg of ritonavir q12h in adults, while the maximum concentration of drug in serum was slightly decreased, with geometric mean ratios (90% confidence intervals in parentheses) of 1.1 (0.87 to 1.3), 0.96 (0.60 to 1.5), and 0.80 (0.68 to 0.94), respectively.

A phase III placebo-controlled study in advanced head and neck cancer using intratumoural cisplatin/epinephrine gel.

Patients with recurrent or refractory head and neck squamous cell carcinoma received cisplatin/epinephrine injectable gel or placebo gel injected directly into the clinically dominant tumour. The double-blind phase III trial comprised of up to 6 weekly treatments over 8 weeks, 4 weekly evaluation visits, and then monthly follow-up; open-label dosing began as needed after three blinded treatments. Tumour response was defined as complete (100% regression) or partial (50-99% regression) sustained for > or =28 day, and patient benefit as attainment of palliative or preventive goals prospectively selected by investigators and patients. With cisplatin/epinephrine gel, 25% (14 out of 57) of tumours responded (16% complete regression, 9% partial regression), vs 3% (one out of 35, complete regression) with placebo (P=0.007). Patient benefit was positively associated with target tumour response in the blinded period among cisplatin/epinephrine gel recipients (P=0.024): 43% (six out of 14) of responders benefited, vs 12% (five out of 43) of non-responders. The most frequent adverse event was pain during injection and the next most frequent was local cytotoxic effects consistent with the gel's mode of action. Systemic adverse events typical of intravenous cisplatin were uncommon. Intratumoural therapy with cisplatin/epinephrine gel provided safe, well-tolerated, effective palliative treatment for patients with locally advanced head and neck squamous cell carcinoma, who lack other satisfactory treatment options.

Indinavir did not increase the short-term risk of adverse cardiovascular events relative to nucleoside reverse transcriptase inhibitor therapy in four phase III clinical trials.

A retrospective person-time analysis of the randomized and non-randomized extension phases of four phase III trials was performed to assess the incidence of adverse cardiovascular events in 2680 HIV-infected patients receiving indinavir or nucleoside reverse transcriptase inhibitor therapy, or both. The observed rate of cardiovascular events was not increased in patients receiving indinavir-based regimens compared with therapy without a protease inhibitor. Extrapolation of these findings is limited by the brief length of therapy and the small number of cases.

Pharmacokinetic study of intralesional cisplatin for the treatment of hepatocellular carcinoma.

BACKGROUND: In the current study the authors examined the pharmacokinetics of direct intralesional injection of cisplatin/epinephrine/bovine collagen gel in patients with hepatocellular carcinoma and cirrhosis. METHODS: Six patients with cirrhosis and unresectable hepatocellular carcinoma received a direct intralesional injection (range, 6.7-26.7 mg) into their tumors under ultrasonographic guidance. The authors determined the total cisplatin (Pt) concentration in the plasma and urine and nonprotein-bound free Pt in plasma ultrafiltrate using flameless atomic absorption spectrometry. Data from individual patients were analyzed to calculate the pharmacokinetic parameters via a noncompartmental method for constant infusion. To demonstrate that the changes in pharmacokinetics are not related to the underlying cirrhosis, a similar methodology was applied to measure the pharmacokinetic parameters of four similar patients who were treated with cisplatin, 75 mg/m(2), as a 1-hour intravenous infusion. RESULTS: The time to attain maximum concentration of total Pt after intralesional injection was dose-dependent and ranged from 2-13 hours. The concentration-time curve was biphasic in nature. The initial half-life of total Pt in patients who received an intralesional injection varied with the cisplatin dose. The initial half-life for cisplatin doses < 15 mg was approximately 9 hours and the initial half-life at higher cisplatin doses (> 15 mg) was approximately 25 hours. The area under the curve (AUC) was dose-dependent with values ranging from 38-150 microm/mL x hour. Pharmacokinetic parameters for free Pt (ultrafiltrate) were significantly different. The time to attain maximum concentration (t-max) and terminal half-life were shorter and the average AUC was approximately 100-fold lower than total Pt. After the intravenous infusion of cisplatin, the t-max for total and free Pt was 1.3 hours and 1.1 hours, respectively. The terminal half-life and average AUC for total Pt was 194 hours and 247 microg/mL per hour, respectively, and its corresponding parameters for free Pt after intravenous infusion were much lower, similar to the findings for the intralesional injection. CONCLUSIONS: The prolonged t-max and initial half-life noted with the intralesional injection of cisplatin/epinephrine/collagen gel are consistent with its proclaimed ability to retain cisplatin at the tumor and delay its release in systemic circulation. The kinetics of intralesional cisplatin injection also suggest local sequestration of the drug in the injected site. Parameters of intravenous cisplatin infusion in cirrhotic patients are similar to those of patients from the historic control group.

Baseline human immunodeficiency virus type 1 phenotype, genotype, and RNA response after switching from long-term hard-capsule saquinavir to indinavir or soft-gel-capsule saquinavir in AIDS clinical trials group protocol 333.

AIDS Clinical Trials Group protocol 333 was an open-label trial of a switch from saquinavir (SQV) hard capsules (SQVhc) to indinavir (IDV) or saquinavir soft-gel capsules (SQVsgc) after >48 weeks of prior treatment with SQVhc. Eighty-nine subjects received IDV or SQVsgc or continued to receive SQVhc and continued unchanged treatment with non-protease-inhibitor antivirals for 8 weeks. Subjects receiving SQVhc then switched treatment to IDV. Baseline drug susceptibility and protease gene sequencing were done; 12 codons related to IDV and SQV resistance were analyzed. After 112 weeks (median) of SQVhc, the fall in human immunodeficiency virus (HIV) type 1 RNA level from baseline was significantly greater with IDV and was inversely correlated with the number of protease substitutions. The number of substitutions also correlated with baseline CD4 cell count, HIV-1 RNA level, SQV experience, and drug susceptibility. Substitution at codon 10, which occurred only in isolates with >/=2 substitutions, was associated with blunted RNA response. IDV IC(50) correlated with HIV-1 RNA response after the switch to IDV but added little predictive power once the genotype was considered.

Randomized, double-blind trial comparing indinavir alone, zidovudine alone and indinavir plus zidovudine in antiretroviral therapy-naive HIV-infected individuals with CD4 cell counts between 50 and 250/mm3.

Treatment with indinavir has been shown to result in marked decreases in viral load and increases in CD4 cell counts in HIV-infected individuals. A randomized double-blind study to evaluate the efficacy of indinavir alone (800 mg q8h), zidovidine alone (200 mg q8h) or the combination was performed to evaluate progression to AIDS. 996 antiretroviral therapy-naive patients with CD4 cell counts of 50-250/mm3 were allocated to treatment. During the trial the protocol was amended to add lamivudine to the zidovudine-containing arms. The primary endpoint was time to development of an AIDS-defining illness or death. The study was terminated after a protocol-defined interim analysis demonstrated highly significant reductions in progression to a clinical event in the indinavir-containing arms, compared to the zidovudine arm (p<0. 0001). Over a median follow-up of 52 weeks (up to 99 weeks), percent reductions in hazards for the indinavir plus zidovudine and indinavir groups compared to the zidovudine group were 70% and 61%, respectively. Significant reductions in HIV RNA and increases in CD4 cell counts were also seen in the indinavir-containing groups compared to the zidovudine group. Improvement in both CD4 cell count and HIV RNA were associated with reduced risk of disease progression. All three regimens were generally well tolerated.

A randomized, controlled trial of indinavir, zidovudine, and lamivudine in adults with advanced human immunodeficiency virus type 1 infection and prior antiretroviral therapy.

A randomized, double-blind, multicenter study of indinavir, zidovudine, and lamivudine was conducted in 320 adults with human immunodeficiency virus type 1 (HIV-1) infection,

The elimination profiles of tenoxicam and hydroxytenoxicam in equine urine and serum after a 200-mg oral dose.

Tenoxicam (Mobiflex) was administered orally to four standardbred mares at a dose of 200 mg. Elimination profiles of tenoxicam and hydroxytenoxicam were generated based on quantitation of these analytes in urine and serum by liquid chromatography (LC) with ultraviolet detection. Tenoxicam was confirmed by LC-tandem mass spectrometry daughter ion mass spectra in the last postadministration sample in which tenoxicam was detected. The tenoxicam and hydroxytenoxicam urinary elimination profiles had the same shape for the same horse; however, each horse was significantly different from the others. One horse (Horse 15) showed a much broader and flatter elimination profile than the other horses. Each horse had a peak in analyte concentration at different collection times. The latest detection for both tenoxicam and hydroxytenoxicam was 29-31 h for all horses. The urinary tenoxicam limit of detection (LOD) and limit of quantitation (LOQ) were 0.3 and 0.4 microg/mL, respectively. The urinary hydroxytenoxicam LOD and LOQ were 0.6 and 0.8 microg/mL, respectively. Hydroxytenoxicam was found to be completely conjugated and tenoxicam completely unconjugated in equine urine. Serum elimination profiles of tenoxicam were measured to 120 h postadministration. Hydroxytenoxicam was not detected in postadministration serum. The last serum tenoxicam detection was at the 24-h collection time for all horses. The peak average concentration was 434.5 ng/mL at 3 h. The serum tenoxicam LOD and LOQ were 5.7 and 7.3 ng/mL.

The elimination profiles of oxaprozin in equine urine and serum after a 4.8-g dose.

A method for the extraction of oxaprozin from equine urine and serum and its quantitation by high-performance liquid chromatography-ultraviolet detection is presented. Confirmation of oxaprozin in postadministration extracts was accomplished by gas chromatographic- mass spectrometric analysis of methylated extracts or liquid chromatography with tandem mass spectrometry daughter ion mass spectra of underivatized extracts. Daypro, a formulation of oxaprozin, was administered orally at a dose of 4.8 g to four standardbred mares. Urine and serum samples were collected to 120 h postadministration. Base hydrolysis of equine urine before extraction resulted in an increase in the amount of oxaprozin measured, an indication of conjugation by ester formation. The urinary elimination profiles of each horse were significantly different from each other with more than one peak in oxaprozin concentration before the 29-31-h collection time. After this collection time, the differences between the oxaprozin urinary concentrations of each horse follow each other more closely. The peak average urinary concentrations of oxaprozin were 25.1 and 17.0 microg/mL at collection times of 8-10 and 18-22 h, respectively. The latest detection of oxaprozin in urine was at the last collection time of 119-121 h postadministration at a concentration close to the detection limit of approximately 0.1 microg/mL. The serum elimination profiles do not vary between horses as much as the urinary elimination profiles. The peak average serum concentration was 49.0 microg/mL at a collection time of 6 h postadministration. The latest detection was at the last collection time of 120 h. Oxaprozin is metabolized in the horse by hydroxylation. Two major urinary metabolites were isolated and identified as hydroxylated oxaprozin. The two urinary metabolites were isolated from equine postadministration urine and analyzed by mass spectrometry and proton nuclear magnetic resonance spectroscopy, which showed that the hydroxylation had occurred at the para positions of the two aromatic rings.

The urinary elimination profiles of diazepam and its metabolites, nordiazepam, temazepam, and oxazepam, in the equine after a 10-mg intramuscular dose.

A method for the extraction of diazepam and its metabolites (nordiazepam, temazepam, and oxazepam) from equine urine and serum and their quantitation and confirmation by liquid chromatography-tandem mass spectrometry is presented. Valium, a formulation of diazepam, was administered at a dose of 10 mg intramuscularly to four standard-bred mares. Diazepam is extensively metabolized in the horse to nordiazepam, temazepam, and oxazepam. Diazepam urinary concentrations were found to be less than 6 ng/mL. Nordiazepam was found to be mainly in its glucuronide-conjugated form and was measured out to a collection time of 53-55 h. Oxazepam and temazepam were entirely conjugated, and their urinary concentrations were measured out to collection times of 121 h and 77-79 h, respectively. Diazepam and nordiazepam were measured in equine postadministration serum out to collection times of 6 and 54 h, respectively. Oxazepam and temazepam were not detected in postadministration serum.

Maintenance antiretroviral therapies in HIV-infected subjects with undetectable plasma HIV RNA after triple-drug therapy. AIDS Clinical Trials Group Study 343 Team.

BACKGROUND: Combination antiretroviral therapy with indinavir, zidovudine, and lamivudine can suppress the level of human immunodeficiency virus (HIV) RNA in plasma below the threshold of detection for two years or more. We investigated whether a less intensive maintenance regimen could sustain viral suppression after an initial response to combination therapy. METHODS: HIV-infected subjects who had CD4 cell counts greater than 200 per cubic millimeter, who had been treated with indinavir, lamivudine, and zidovudine, and who had less than 200 copies of HIV RNA per milliliter of plasma after 16, 20, and 24 weeks of induction therapy were randomly assigned to receive either continued triple-drug therapy (106 subjects), indinavir alone (103 subjects), or a combination of zidovudine and lamivudine (107 subjects). The primary end point was loss of viral suppression, which was defined as a plasma level of at least 200 copies of HIV RNA per milliliter on two consecutive measurements during maintenance therapy. RESULTS: During maintenance treatment, 23 percent of the subjects receiving indinavir and 23 percent of those receiving zidovudine and lamivudine, but only 4 percent of those receiving all three drugs, had loss of viral suppression (P<0.001 for the comparison between triple-drug therapy and the other two maintenance regimens). Subjects with greater increases in CD4 cell counts during induction therapy, higher viral loads at base line (i.e., at the beginning of induction therapy), and slower rates of viral clearance were at greater risk for loss of viral suppression. The presence of zidovudine-resistance mutations in HIV RNA at base line was strongly predictive of the loss of viral suppression in subjects treated with zidovudine and lamivudine. CONCLUSIONS: The suppression of plasma HIV RNA after six months of treatment with indinavir, zidovudine, and lamivudine is better sustained by the continuation of these three drugs than by maintenance therapy with either indinavir alone or zidovudine and lamivudine.

Colicin V38 and microcin C38 produced by Escherichia coli strain 38.

Escherichia coli strain 38, an isolate from turkeys, has been previously shown to produce one or more broad-spectrum bacteriocins against other related enteric bacteria. Using a collection of E. coli strains that synthesized well-characterized colicins or microcins, along with a set of colicin/microcin-insensitive mutants, we were able to classify the bacteriocins produced by strain 38. We determined that strain 38 produced a microcin (microcin C38) and a colicin (colicin V38) and that the amount of microcin C38 depended on the type of media on which it was grown. Escherichia coli strain 38 was found to have cross-immunity with the microcin C7-producing strain MC4100 and with the colicin V-producing strain 4674. OmpF mutant cells were found to be insensitive to microcin C38, whereas colicin V38 was not active on cells that had a cir mutation. Both microcin C38 and colicin V38 were inactivated by proteases. Microcin C38 was stable at extremes of pH (pH 1.5 and pH 13) and heat (10 min at 98 C) conditions, whereas colicin V38 was not. In addition, microcin C38 was found to be active against a broader spectrum of gram-negative bacteria than was colicin V38.

A unique metabolite of nimesulide.

Nimesulide is a nonsteroidal anti-inflammatory drug recently detected in equine blood and urine samples taken at the race track. The detection of the drug in a blood sample led to the identification of an unknown thin-layer chromatographic (TLC) spot in track urine samples as a metabolite of nimesulide. Characterization of the unknown TLC spot and comparison with the synthesized compound shows that the unknown TLC spot is a previously unreported equine metabolite of nimesulide. The metabolite was identified as resulting from the reduction of the nitro group on nimesulide to an amino group. This reduced nitro metabolite (4-amino-2-phenoxy-methanesulfonanilide) is a major metabolite of nimesulide in the equine.

Genetic correlates of in vivo viral resistance to indinavir, a human immunodeficiency virus type 1 protease inhibitor.

Indinavir (IDV) (also called CRIXIVAN, MK-639, or L-735,524) is a potent and selective inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease. During early clinical trials, in which patients initiated therapy with suboptimal dosages of IDV, we monitored the emergence of viral resistance to the inhibitor by genotypic and phenotypic characterization of primary HIV-1 isolates. Development of resistance coincided with variable patterns of multiple substitutions among at least 11 protease amino acid residues. No single substitution was present in all resistant isolates, indicating that resistance evolves through multiple genetic pathways. Despite this complexity, all of 29 resistant isolates tested exhibited alteration of residues M-46 (to I or L) and/or V-82 (to A, F, or T), suggesting that screening of these residues may be useful in predicting the emergence of resistance. We also extended our previous finding that IDV-resistant viral variants exhibit various patterns of cross-resistance to a diverse panel of HIV-1 protease inhibitors. Finally, we noted an association between the number of protease amino acid substitutions and the observed level of IDV resistance. No single substitution or pair of substitutions tested gave rise to measurable viral resistance to IDV. The evolution of this resistance was found to be cumulative, indicating the need for ongoing viral replication in this process. These observations strongly suggest that therapy should be initiated with the most efficacious regimen available, both to suppress viral spread and to inhibit the replication that is required for the evolution of resistance.

Culture variability associated with the U.S. Environmental Protection Agency Tuberculocidal Activity Test Method.

Tuberculosis continues to be a major world health threat. The etiologic agent is among the vegetative organisms most resistant to chemical disinfection. Tuberculocidal efficacy testing for regulatory approval of chemical germicides has evolved considerably over the past decade. A method currently in use is the Environmental Protection Agency Tuberculocidal Activity Test Method, a suspension test using a Mycobacterium bovis culture grown under specific conditions and stored frozen until used. Differing tuberculocidal label claims on products with similar formulations have raised questions concerning the equivalence of test suspensions prepared by different laboratories. Five M. bovis suspensions from laboratories currently performing this test were compared against a battery of three disinfectants at a single test site. A significant difference between test cultures was found, with two of the five exhibiting a significant difference from the other three and also from each other. There was a significant culture-by-disinfectant interaction, indicating that the five cultures did not respond in a consistent manner across the different disinfectants used. However, these differences were due to cultures that were not prepared in accordance with the standard procedure or otherwise did not meet the test suspension criteria. In addition, a 0.55% sodium hypochlorite solution was found to be a sensitive indicator of culture variability. These data reinforce the need to adhere to published procedures and guidelines when growing and preparing a tuberculocidal test suspension and shed light on the variables associated with this type of testing.

Pneumocystis carinii pneumonia: a major complication of immunosuppressive therapy in patients with Wegener's granulomatosis.

The risk factors and clinical and laboratory parameters in Pneumocystis carinii pneumonia in patients with Wegener's granulomatosis have not been well characterized. We undertook a retrospective chart review of all patients with a diagnosis of Wegener's granulomatosis and P. carinii pneumonia who were followed at the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The chart review focused on clinical, laboratory, and roentgenologic evidence of P. carinii pneumonia. Eleven cases of P. carinii pneumonia were diagnosed in some 180 patients with Wegener's granulomatosis, for an overall incidence of approximately 6%. All patients developed P. carinii pneumonia either during the initial course of treatment or during therapy for recurrent Wegener's granulomatosis. All patients were receiving daily glucocorticoids and a second immunosuppressive therapy. Lymphocytopenia was noted in all patients, with a mean +/- SEM total lymphocyte count of 303 +/- 69 cells/microL. All patients tested (10 of 11) were seronegative for human immunodeficiency virus (HIV) infection. Eight presented with worsening chest roentgenograms compared with baseline, whereas three presented with normal chest roentgenograms. We conclude that P. carinii is a common opportunistic pathogen in patients with Wegener's granulomatosis receiving immunosuppressive therapy. Therapeutic immunosuppression (daily glucocorticoids and immunosuppressive agents) and the resultant lymphocytopenia, as well as the lymphocyte and monocyte functional abnormalities caused by glucocorticoids, may be the most likely factors predisposing to P. carinii pneumonia in patients with Wegener's granulomatosis. Based on our data, all patients with Wegener's granulomatosis should be given chemoprophylaxis against P. carinii while they are receiving daily glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)