Targeting platinum resistant disease in ovarian cancer.

Ovarian cancer is an extremely aggressive disease in which the vast majority of patients face a very poor prognosis. Although most patients initially respond to current chemotherapeutic regimens that include a combination of platinum- based therapy (cisplatin/carboplatin) and paclitaxel, the vast majority of them quickly relapse and develop increased resistance to available treatments. Thus, intrinsic and acquired chemotherapy resistance is a major obstacle in the treatment of ovarian cancer patients. Consequently, the priorities for basic and translational ovarian cancer research need to include the identification of novel therapies directed against key molecular targets and signaling pathways in platinum resistant disease. At the same time, we need to develop novel systems for drug delivery aimed at increasing the efficacy and reducing the toxicity of platinum-based treatments. Improving the current responses to platinum chemotherapy is critical not only for achieving a better outcome clinically, including a longer survival, but also for allowing patients to have a better quality of life while in treatment.

An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer.

BACKGROUND: Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Changes in microRNA (miR) expression are seen in urothelial carcinogenesis in a phenotype-specific manner. We hypothesised that urinary miRs reflecting low- and high-grade pathways could detect bladder cancers and overcome differences in genetic events seen within the disease. METHODS: We investigated urinary samples (n=121) from patients with bladder cancer (n=68) and age-matched controls (n=53). Fifteen miRs were quantified using real-time PCR. RESULTS: We found that miR is stable within urinary cells despite adverse handling and detected differential expression of 10 miRs from patients with cancer and controls (miRs-15a/15b/24-1/27b/100/135b/203/212/328/1224, ANOVA P<0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance). CONCLUSION: The use of this panel in patients with haematuria would have found 94% of urothelial cell carcinoma, while reducing cystoscopy rates by 26%. However, two invasive cancers (3%) would have been missed.

The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype.

Although the latent membrane protein-1 (LMP1) of the Epstein-Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non-viral vector-based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkin's lymphoma cell lines. Strikingly, LMP1 down-regulated the expression of B-cell-specific genes including B-cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B-cell differentiation. Our data suggest that in EBV-positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed-Sternberg cells, including the loss of B-cell identity.

Urinary adenosine and aminoimidazolecarboxamide excretion in methotrexate-treated patients with psoriasis.

BACKGROUND: We hypothesized that low-dose methotrexate treatment for patients with psoriasis would block purine biosynthesis at the step catalyzed by aminoimidazolecarboxamide (AICA) ribotide transformylase and would inhibit adenosine metabolism as evidenced by increased urinary levels of AICA and adenosine, respectively. Eight patients collected a 24-hour urine specimen on the day before their methotrexate dose and the next day during their methotrexate dose. Eight age- and sex-matched controls also collected a 24-hour urine sample. Urinary AICA and adenosine were assayed by spectrophotometric and radioimmune assays, respectively; means are reported as micromole per millimole of creatinine and were compared by the paired t test (1-tailed). OBSERVATIONS: Mean AICA excretion increased from 1.30 micromol/mmol on the day before to 1.85 micromol/mmol on the day during methotrexate dosing (P<.01). Mean adenosine values increased from 0.68 to 1.07 micromol/mmol, (P<.03). Controls had mean AICA and adenosine levels of 1.29 and 0.50 micromol/mmol, respectively. During the day of methotrexate dosing, patients had higher mean AICA and adenosine levels when compared with controls (P<.01). Mean AICA levels increased from 1.36 to 2.06 micromol/mmol (P<.025), and mean adenosine levels increased from 0.72 to 1.25 micromol/mmol (P<.025) in 5 patients showing improvement in clinical disease activity. In contrast, 3 patients with no change or worsening in clinical disease activity had smaller increases. CONCLUSIONS: Methotrexate treatment of patients with psoriasis inhibits AICA ribotide transformylase and adenosine metabolism. Since adenosine is a T-lymphocyte toxin, it may be partially responsible for the immunosuppressive effect.

Rapid gas chromatographic analysis of drugs of forensic interest.

High-speed gas chromatographic (GC) screening for drugs of forensic relevance is performed using a commercial Flash GC instrument in which the chromatographic column is resistively heated at rates of up to 30 degrees C/s. Temperature programming conditions are varied in an experiment designed to evaluate trade-offs between resolution and analysis time for a mixture of 19 drugs of abuse. All 19 components can be separated with excellent resolution in 90 s. Specific analytes can be analyzed even faster; for example, amphetamine analysis is completed in less than 20 s. Case studies of confiscated street drugs containing amphetamine, cocaine, and heroin are analyzed to evaluate the retention time repeatability. Ten replicate injections over a 2-day period for 3 different drug samples achieved retention time relative standard deviations in the range of 0.48 to 0.81%.

Diaphragmatic hernia of Morgagni.

Most cases of Morgagni hernia are asymptomatic and diagnosed incidentally on routine chest x-ray film, but they may occasionally become symptomatic. Symptomatic Morgagni hernias may present in many different ways, making the diagnosis challenging. We describe a patient with a Morgagni hernia, resulting in intractable nausea and vomiting, give a brief review of symptoms, note the different types of abdominal contents herniated, and describe the methods used to make the diagnosis.

Methotrexate in rheumatoid arthritis: folate supplementation should always be given.

Methotrexate is now the disease-modifying antirheumatic drug prescribed most frequently for the treatment of rheumatoid arthritis. Methotrexate is an antifolate that inhibits methylation reactions and reactions of amino acid, purine and pyrimidine metabolism. Toxic manifestations of methotrexate administration for rheumatoid arthritis (at relatively low doses compared with those used in cancer chemotherapy) include cytopenias, gastrointestinal intolerance, liver disease, pulmonary injury, central nervous system dysfunction, skin rashes and nodulosis. Delayed wound healing and increased risk for infections with opportunistic organisms also occur. Some of these toxic manifestations respond to supplementation with folates [folic acid or folinic acid (calcium folinate)]. The folate status of patients has been shown to be impaired after prolonged treatment with methotrexate, and poor baseline folate status is an independent risk factor for subsequent toxicity. Numerous studies have now documented that folic acid, even in high doses, and moderate doses of folinic acid are beneficial in preventing methotrexate toxicity without affecting efficacy. In this article we present guidelines and rationale for monitoring methotrexate therapy, and guidelines for folate supplementation during methotrexate therapy for rheumatoid arthritis. It is our recommendation that folic acid should be empirically supplemented in all patients at the initiation of methotrexate therapy. This regimen is associated with a high benefit : risk ratio and is likely to be cost effective.

Cofactor role for 10-formyldihydrofolic acid.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Differences in methotrexate and 7-hydroxymethotrexate inhibition of folate-dependent enzymes of purine nucleotide biosynthesis.

7-Hydroxymethotrexate (7-OH-MTX) is the major and, frequently the only, pteridine metabolite found in bone-marrow aspirates of patients chronically treated with low-dose oral methotrexate (MTX) [Sonneveld, Schultz, Nooter and Hahlen (1986) Cancer Chemother. Pharmacol. 18, 111-116]. The Ki values for MTX and 7-OH-MTX for avian liver 5-amino-imidazole-4-carboxamide ribonucleotide transformylase differ by 4.5-fold in favour of 7-OH-MTX as the better inhibitor, while Ki values for avian liver glycinamide ribonucleotide transformylase differ by 1.9-fold favouring MTX as the better inhibitor. Thus 7-OH-MTX possesses a different enzyme-inhibiting repertoire from its parent drug and this information may be useful in explaining the mechanism of action of low-dose MTX therapies used to treat autoimmune disease.

Antifolates in rheumatoid arthritis: a hypothetical mechanism of action.

The antifolates, methotrexate, aminopterin, 10-deazaaminopterin and sulfasalazine are clinically useful in the treatment of rheumatoid arthritis. Toxicity, rather than efficacy, appears to the the major factor limiting the usefulness of the classical antifolates (i.e., methotrexate and 10-deazaaminopterin). The fact that folate supplementation of methotrexate-treated rheumatoid arthritis patients reduces toxicity without altering efficacy also suggests that inhibition of the drug's target enzyme, dihydrofolate reductase, is not complete and not essential for efficacy. Since polyglutamates of methotrexate are direct inhibitors of thymidylate synthase and folate dependent enzymes of purine biosynthesis, the efficacy of this agent may involve blockade of these pathways. We hypothesize that blockage of aminoimidazole carboxamide ribotide transformylase, the folate dependent enzyme responsible for the insertion of carbon 2 into the purine ring, produces an immunosuppression mediated by secondary inhibition of adenosine deaminase, and S-adenosyl homocystein hydrolase by aminoimidazolecarboxamide metabolites. This mechanism of immunosuppression may explain the clinical effect of methotrexate, 10-deazaaminopterin, and possibly sulfasalazine. Since purine biosynthesis is a fundamental process, blockading this pathway may also decrease leukotriene production and interleukin-1 expression, which also could contribute to the efficacy of methotrexate.

A meal conference format to teach ambulatory nutrition concepts.

Third-year medical students participated in a program using a meal conference approach to teach ambulatory nutrition concepts called "Building Better Health Through Nutrition." The series of three interactive presentations was given during the required family medicine clerkship. A pretest and posttest were used to measure acquisition of nutrition knowledge. There was a statistically significant (P less than 0.05) average increase in posttest compared to pretest scores. Seventy percent of students rated the meal conference approach as "effective" or "very effective" and 76% stated that the series expanded their knowledge of nutrition's role in clinical medicine. We conclude that the meal conference format is an effective way to teach nutrition during the clinical years in medical school.

Homocysteine levels in patients with rheumatoid arthritis treated with low-dose methotrexate.

Plasma homocysteine levels were determined in patients who participated in a randomized, double-blind placebo-controlled trial of folate supplementation (1 mg/day) during methotrexate therapy for rheumatoid arthritis. Plasma and red blood cell folate levels before methotrexate therapy were significantly negatively correlated with homocysteine levels. Homocysteine levels were not significantly correlated with the initial C1 index (an assay that measures the folate status of blood mononuclear cells) or the C1 index during methotrexate therapy. There was no significant difference in homocysteine levels between pretreatment and levels drawn at 3 or 6 months. Initial homocysteine levels were predictive of toxicities, such as gastrointestinal intolerance and elevations of liver enzymes in the placebo group. There was no significant correlation between occurrence of toxicity and initial homocysteine levels in the folic acid-supplemented group. Homocysteine levels were not predictive of the efficacy of methotrexate therapy. We conclude that plasma homocysteine levels are correlated with plasma and red blood cell folate levels before methotrexate therapy but is not correlated with folate status in blood mononuclear cells.

Chemotaxonomic differentiation of legionellae by detection and characterization of aminodideoxyhexoses and other unique sugars using gas chromatography-mass spectrometry.

Legionellae have been differentiated previously by analyzing their carbohydrate contents by gas chromatography with flame ionization detection. In the present study, total ion mode gas chromatography-mass spectrometry (GC-MS) was used to detect a number of unusual sugars, including one that is structurally related to O-methyldideoxyheptoses. Increased sensitivity and selectivity for carbohydrate detection was achieved by selected ion-monitoring GC-MS. Two of the uncommon sugars previously discovered in the legionellae (X1 and X2) were identified as quinovosamine and fucosamine, respectively. Legionella pneumophila contained rhamnose and quinovosamine but not the quinovosamine isomer fucosamine. Tatlockia micdadei and Legionella maceachernii contained large amounts of rhamnose, fucose, and fucosamine but not quinovosamine. These two species were the only legionellae studied that contained another unusual sugar that is referred to as X3, pending determination of its structure. Fluoribacter dumoffi, Fluoribacter bozemanae, and Legionella anisa were varied in their carbohydrate contents, both within and between species, but could be distinguished from L. pneumophila and the T. micdadei and L. maceachernii group. Fluoribacter gormanii was unique among the legionellae in that it lacked both quinovosamine and fucosamine. Legionella jordanis contained other unusual carbohydrates in addition to quinovosamine. GC-MS may have wide application in the differentiation of bacterial species.

Methotrexate therapy in rheumatoid arthritis patients diminishes lectin-induced mononuclear cell proliferation.

Methotrexate (MTX) is an anti-folate drug used in cancer chemotherapy because of its anti-proliferative effects. However, it is unclear whether the anti-proliferative effects of MTX contribute to the efficacy of low-dose MTX in the treatment of rheumatoid arthritis (RA). To date, either no change or a paradoxical increase in lectin-induced proliferation has been observed in cultures of peripheral blood mononuclear cells (PBMC) from MTX-treated RA patients (RA + MTX). In these earlier studies, high folate-containing media and tritiated thymidine (3H-TdR) were used. Our studies were designed to test the hypothesis that the use of a culture medium with a low folate content along with tritiated deoxyuridine (3H-UdR) permits detection of diminished phytohemagglutinin (PHA)-induced proliferative responses of PBMC from RA + MTX. The data demonstrate decreased PHA-induced cellular proliferation of cultured PBMC from RA + MTX compared with controls. When comparing the PBMC proliferative responses in high vs low folate medium, a significantly greater increase (P less than 0.05) in proliferation occurs in the cells from RA + MTX cultured in the high folate medium. This suggests that an in vivo folate-deficient state of the cells from RA + MTX may be corrected in vitro when a high folate medium is used in culture. We conclude that the use of 3H-UdR and a medium containing folate within the normal range of plasma folate levels eliminates artifacts associated with the use of high folate medium and 3H-TdR, which obscures the anti-proliferative effect of MTX in RA patients.

D-alanine as a chemical marker for the determination of streptococcal cell wall levels in mammalian tissues by gas chromatography/negative ion chemical ionization mass spectrometry.

A gas chromatography/mass spectrometry method using selected ion monitoring with negative ion detection and methane chemical ionization was employed to quantitate a marker for bacterial peptidoglycan, D-alanine, in mammalian tissues. D-Alanine originating from bacterial peptidoglycan was obscured by substantial amounts of D-alanine generated by racemization from L-alanine present in tissue protein. To overcome this problem, samples were enzymatically treated and hydrolyzed in deuterated hydrochloric acid. Newly formed D-alanine derived from protein was labeled with deuterium and bacterial D-alanine remained unlabeled, enabling differentiation by the molecular weight increase. Butyl heptafluorobutyryl derivatives of the D- and L-amino acids were separated on a fused silica capillary column coated with Chirasil-val. The amounts of bacterial D-alanine found in livers of arthritic rats were consistent with previously reported levels of other carbohydrate-derived markers for bacterial peptidoglycan-polysaccharide complexes.

Nutrition education for medical students: evaluation of the relative contribution of freshman courses in biochemistry and nutrition to performance on a standardized examination in nutrition.

An examination previously developed and used for assessment of nutrition knowledge of medical students in the Southeastern Regional Medical-Nutrition Education Network was used to compare the effectiveness of a basic medical biochemistry course and a 58-hour required nutrition course. The examination was administered to a cohort of freshman students upon entry to medical school, after biochemistry, and then after nutrition. Two other student groups took the examination at the end of the sophomore and senior years, respectively. In the freshman cohort, mean nutrition knowledge scores increased slightly after biochemistry, (52% to 56%), which contained 37 nutrition-related lectures. The mean score of the cohort was 75% after the nutrition course. The sophomores scored 75% and the seniors 73%. These findings suggest 1) basic science courses such as biochemistry cannot be relied upon to add significantly to nutrition knowledge, and 2) a required freshman course can be an effective way to introduce basic and clinical nutrition with good retention of knowledge in subsequent years.

Creation of a regional medical-nutrition education network.

The Southeastern Regional Medical-Nutrition Education Network (SER-MEN) was developed to coordinate and improve nutrition education in a consortium of the medical schools in Alabama, Florida, Georgia, and South Carolina. SERMEN's central office is at the Medical College of Georgia with the testing office at the University of Alabama at Birmingham. Students, faculty, and consultants in nutrition, education, and computer networking work together on projects on each campus that are coordinated and planned through semiannual meetings. A standardized examination was developed with the Nutrition Test-Item Bank to assess nutrition knowledge at various years of medical students from network schools. Each SERMEN school is connected to a microcomputer system at the central office that provides access to a data base of nutrition education and resources on each campus for developing curricula and syllabi. Funding has been provided by societies, foundations, and government agencies.

Cefazolin versus cefamandole for prophylaxis during total joint arthroplasty.

A prospective, randomized, double-blind comparison of cefazolin versus cefamandole was carried out to evaluate safety and efficacy and to determine bone and serum antibiotic concentrations in patients undergoing total joint arthroplasty. Dosages were 1 g of cefazolin before surgery followed by 500 mg every eight hours for six doses, versus 2 g of cefamandole before surgery followed by 1 g every eight hours for six doses. Intraoperative doses were given during prolonged procedures. No significant adverse drug reactions were clearly attributable to either drug. Among 48 patients receiving cefazolin there was one postoperative wound infection and one distant site infection. Among 49 patients receiving cefamandole, there were two postoperative wound infections and two distant site infections. No deep wound infections occurred in either group during at least 48 months of follow-up study. In hip specimens removed at surgery, the mean antibiotic concentrations were 1.6 +/- 1.4 micrograms/g for cefazolin recipients, compared with 5.7 +/- 5.9 micrograms/g for cefamandole recipients (p less than .001). In knee specimens, the mean antibiotic concentrations were 0.64 +/- 0.57 microgram/g for cefazolin recipients compared to 3.8 +/- 3.4 micrograms/g for cefamandole recipients (p = .004). Cefazolin given at one-half the dose of cefamandole appeared to be equally safe and effective but resulted in lower bone concentrations of antibiotic.