An (Anti)-Inflammatory Microbiota: Defining the Role in Inflammatory Bowel Disease?

While it is now accepted that the gut microbiota contribute to the genotype-environment-lifestyle interactions triggering inflammatory bowel disease (IBD) episodes, efforts to identify the pathogen(s) that cause these diseases have met with limited success. The advent of culture-independent techniques for characterizing the structure and/or function of microbial communities (hereafter referred to as metagenomics) has provided new insights into the events associated with the onset, remission and recurrence of IBD. A large number of observational and/or case-control studies of IBD patients have confirmed substantive changes in gut bacterial profiles (dysbiosis) associated with disease. These types of studies have been augmented by new profiling approaches that support the identification of more 'colitogenic' bacteria from numerically predominant taxa. Evidence of alterations in lesser abundant taxa such as the methanogenic archaea, to favor types that are more immunogenic, has also been forthcoming. Several recent longitudinal studies of patients with Crohn's disease have produced additional insights, including evidence for the role of 'anti-inflammatory' microbiota in providing a protective effect and/or promoting remission. In summation, the implications of dysbiosis and restoration of a 'healthy microbiota' in IBD patients requires definition beyond a taxonomic assessment of the changes in the gut microbiota during disease course. The available evidence does suggest that specific members of the gut microbiota can contribute either pro- or anti-inflammatory effects, and their ecological fitness in the large bowel affects the onset and recurrence of IBD. While metagenomics and related approaches offer the potential to provide novel and important insights into these microbiota and thereby the pathophysiology of IBD, we also need to better understand factors affecting the ecological fitness of these microbes, if new treatment of IBD patients are to be delivered.

Uridine and uracil transport in Escherichia coli and transport-deficient mutants.

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterize the transport mechanism of uracil and uridine. Mutant U-, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR-, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U-UR- isolated from a culture of mutant U-, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR-R- was isolated from a culture of mutant UR-, and is resistant to a high concentration of showdomycin. This mutant is defective for transport of intact uridine and addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U- and UR-, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these processes.

Mechanism of energy coupling for transport of deoxycytidine, uridine, uracil, adenine and hypoxanthine in Escherichia coli.

The transport processes for uridine, deoxycytidine, uracil, adenine and hypoxanthine require an energy source and are active under anaerobic or aerobic conditions. Inhibitory effects of cyanide, arsenate, carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol and N,N'-dicyclohexylcarbodiimide on the transport of uridine and deoxycytidine differ from the corresponding effects on the transport of uracil, adenine and hypoxanthine. The nature of these inhibitory effects supports the conclusion that uridine and deoxycytidine transport is energized either by electron transport or by ATP hydrolysis via (Ca2+ + Mg2+)-ATPase. The transport or uracil, adenine and hypoxanthine is dependent upon ATP or some high energy phosphate derivative of ATP, but is independent of (Ca2+ + Mg+)-ATPase and electron transport. Uptake of the ribose moiety of uridine by a mutant of Escherichia coli B, which lacks the transport system for uracil and intact uridine, is neither stimulated by energy sources nor inhibited by various inhibitors of energy metabolism under either aerobic or anaerobic conditions.

Randomized trial of vinorelbine compared with fluorouracil plus leucovorin in patients with stage IV non-small-cell lung cancer.

PURPOSE: This prospective randomized trial was performed to compare the effectiveness of intravenous vinorelbine tartrate with intravenous fluorouracil and leucovorin (5-FU/LV) on the primary end points of survival, quality of life (QOL), and relief of cancer-related symptoms in patients with advanced non-small-cell lung cancer (NSCLC). Secondary end points included tumor response rates and time to treatment failure. In addition, the safety of both treatment regimens was evaluated in this multicenter study. PATIENTS AND METHODS: Two hundred sixteen patients with stage IV NSCLC were enrolled onto this study from 18 centers. Vinorelbine was administered at a dose of 30 mg/m2/wk. 5-FU/LV was administered at a dose of 425 mg/m2 and 20 mg/m2, respectively, for 5 consecutive days every 4 weeks. Patients with progressive disease or toxicity were removed from study while responding and stable patients were continued on therapy. RESULTS: The median survival time of patients who received vinorelbine was 30 weeks, with 25% of patients alive at 1 year, compared with a median survival time of 22 weeks and 16% of patients alive at 1 year for those treated with 5-FU/LV (P = .03, log-rank test). This improvement in survival was associated with a higher objective response rate (12% v 3%) and time to treatment failure (10 weeks v 8 weeks) for vinorelbine versus 5-FU/LV. The dose-limiting toxicity of vinorelbine was granulocytopenia, with 54% of patients experiencing grade 3/4 granulocytopenia. Nonhematologic toxicity of vinorelbine was generally grade 1 or 2. The most common grade 3 toxicities were related to injection-site reactions. CONCLUSION: This trial confirms the efficacy of vinorelbine in patients with advanced NSCLC. The clinical activity and relatively favorable toxicity profile of this agent make it a reasonable and useful treatment option in the management of patients with this disease.

Hypoglycemic coma associated with benign pleural mesothelioma.

A case of hypoglycemic coma and benign pleural mesothelioma is described. Serum insulin levels, as measured by insulin radioimmunoassay, were appropriately suppressed and consistent with hypoglycemia. Assay of the tumor showed insulin to be undectable. The mechanisms for hypoglycemia probably included increased glucose consumption by the tumor and, more important, the inhibition of lipolysis and hepatic gluconegenesis caused by tumor release of L-tryptophan and its metabolites and/or possibly nonsuppressible insulin-like activity, soluble in acid-ethanol(NSILA-s).

HLA antigen frequencies among patients with juvenile chronic arthritis and amyloidosis: a brief report.

Amyloidosis is seen in a small number of patients with juvenile chronic arthritis (JCA). In order to determine whether particular HLA markers might predispose to the development of amyloid in JCA a group of 45 patients with amyloidosis confirmed by biopsy was typed for the HLA-A, B, C and DR loci. The results confirmed previous smaller studies that no HLA antigen detected by standard serological techniques was associated specifically with the development of amyloidosis. Those antigens which showed an altered frequency (ie. DR4 and DRw8) were known to be associated with the different types of JCA onset represented in this group.

The disease concept of alcoholism: its impact on women's treatment.

The disease concept of alcoholism has had a dynamic impact on attitudes, policies, and treatment orientations. Despite its demonstrable effects on changing behaviors and belief systems, it remains both an acceptable and controversial entity amongst researchers and treatment personnel alike. This report provides a current focus of the disease concept as its applies to the characteristics and experiences of women alcoholics and their treatment. It especially targets the need for empowerment in this population and questions whether this important trait can be developed by the disease concept's emphasis on illness and the sick role, which inadvertently validates the feelings of powerlessness and helplessness of these women who already have been intensively socialized in dependent and subordinate roles.

RP-HPLC tryptic mapping of IgG1 proteins with post-column fluorescence derivatization.

Peptide mapping is an important analytical technique widely used to study the primary structure of proteins. In quality control settings, it is employed as an identity test to probe for small changes in protein primary structure. A great challenge in peptide mapping is to minimize the detection limit for peptides due to the low detectability of smaller peptides based on their ultraviolet absorbance. The detection of peptide fragments can be enhanced by pre-or post-column derivatization with fluorescent tags. The use of post-column o-pthalaldehyde (OPA) and fluorescamine chemistries for on-line derivatization of peptide fragments from the RP-HPLC tryptic maps of several IgG1 monoclonal antibodies was explored. This paper describes the simple and sensitive peptide mapping technique for structural confirmation of proteins using picomoles of samples by post-column fluorescence derivatization. A comparison of UV and fluorescence detection of a peptide map is also presented. The method includes post column OPA derivatization of tryptic peptides from RP-HPLC tryptic maps with fluorescence detection. The conclusion reached that fluorescence detection gave relative detectability for tryptic peptides that range from 10- to 100-fold better than those observed with UV detection. The sensitivity of the peptide map increased by about 200-500 fold, i.e. peptide maps could be obtained using 2-5 pmol of digest instead of 1 nmol of digest. A roughly equal fluorescence response for all peptides (equal peak areas) was generally observed.

Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody. We have used this method routinely for over 1 year to support bioprocess development and test production lots for clinical trials. Herein we summarize the precision and ruggedness of the testing procedure and the main findings with respect to 'coverage of amino acid sequence' and limits-of-detection for various hypothetical structural variants. We also describe, in more detail, two unanticipated insights into the method gained from the validation study. The first of these is a potentially troublesome side-product arising during the reduction/alkylation step. Once the cause of this side-product was identified, it was easily prevented. We also report on subtle changes to the peptide map upon extended storage of the digest in the autosampler. These findings helped us to develop a 'robust' method for implementation in a quality control laboratory.

A comparative field trial of cephalonium and cloxacillin for dry cow therapy for mastitis in Australian dairy cows.

OBJECTIVE: To investigate and compare the therapeutic efficacy of dry cow agents containing either cephalonium or cloxacillin within Australian dairy herds. DESIGN: A treatment-control trial. METHODS: Milk from infected quarters of cows with high somatic cell counts in milk on eight Australian dairy farms was cultured to identify bacterial pathogens. Cows were randomly assigned to treatment groups and one group was treated with cephalonium at drying off and the other group was treated with cloxacillin at drying off. Milk samples from infected quarters were collected immediately after calving and were cultured for pathogens. The effect of treatment on bacteriological cure was examined and somatic cell counts from infected cows from the first two herd tests after calving were examined for a treatment effect. On four farms, milk samples were collected for culture from all cases of clinical mastitis identified within the first 7 days after calving. The effect of treatment upon incidence of clinical mastitis after calving was examined. RESULTS: There was no significant difference between treatments on quarter cure rates for new infections, for chronic infections and for infections with Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis. Infected quarters treated with cephalonium had a significantly higher cure rate than quarters treated with cloxacillin when Corynebacterium bovis and Staphylococcus epidermids were included as pathogens combined (80.3% versus 70.7%). There was no significant difference between the treatments on somatic cell counts of infected cows at the first two herd tests after calving. There was no difference between treatments on the incidence of clinical mastitis in the first 7 days after calving.

Pyruvate kinase deficiency anaemia in a Basenji dog.

Pyruvate kinase deficiency anaemia was suspected in an 18-month-old male Basenji dog after other known causes of canine haemolytic anaemia had been excluded. Anaemia of moderate severity (packed cell volumes 0.20 to 0.26 1/1) and reticulocytosis (uncorrected reticulocyte counts 8 to 43%) persisted during 5 months' observation, and biopsies showed development of bone marrow fibrosis and sclerosis. The diagnosis of pyruvate kinase deficiency anaemia was presumptive because erythrocyte pyruvate kinase concentrations in the affected dog were inconclusive and related animals were not available for enzyme assay. However, the gene for pyruvate kinase deficiency is known to occur among Basenji dogs in Australia.