Mucosal-associated invariant T (MAIT) cells are activated in the gastrointestinal tissue of patients with combination ipilimumab and nivolumab therapy-related colitis in a pathology distinct from ulcerative colitis.

The aim of this study was to investigate the pathogenesis of combination ipilimumab and nivolumab-associated colitis (IN-COL) by measuring gut-derived and peripheral blood mononuclear cell (GMNC; PBMC) profiles. We studied GMNC and PBMC from patients with IN-COL, IN-treated with no adverse-events (IN-NAE), ulcerative colitis (UC) and healthy volunteers using flow cytometry. In the gastrointestinal-derived cells we found high levels of activated CD8+ T cells and mucosal-associated invariant T (MAIT) cells in IN-COL, changes that were not evident in IN-NAE or UC. UC, but not IN-C, was associated with a high proportion of regulatory T cells (Treg ). We sought to determine if local tissue responses could be measured in peripheral blood. Peripherally, checkpoint inhibition instigated a rise in activated memory CD4+ and CD8+ T cells, regardless of colitis. Low circulating MAIT cells at baseline was associated with IN-COL patients compared with IN-NAE in one of two cohorts. UC, but not IN-COL, was associated with high levels of circulating plasmablasts. In summary, the alterations in T cell subsets measured in IN-COL-affected tissue, characterized by high levels of activated CD8+ T cells and MAIT cells and a low proportion of Treg , reflected a pathology distinct from UC. These tissue changes differed from the periphery, where T cell activation was a widespread on-treatment effect, and circulating MAIT cell count was low but not reliably predictive of colitis.

Complete spontaneous regression of a metastatic melanoma of the mandible: a case report and follow-up recommendations.

Regression of metastatic melanoma is very rare and occurs in only 0.23% of cases. Metastasis to the oral cavity is particularly uncommon and accounts for only 1-3% of all oral malignancies. This report presents a case of spontaneous and complete regression of a metastatic melanoma in the mandibular ramus. The patient remains asymptomatic more than 2 years after diagnosis. The patient was followed up regularly. It is recommended that further surveillance imaging be performed in asymptomatic patients following discussion with the surgical and oncological teams. This type of surveillance, together with new systemic treatments, is advocated due to its potential to increase long-term survival even after relapse.

Trabectedin in Advanced High-Grade Uterine Leiomyosarcoma: A Case Report Illustrating the Value of (18)FDG-PET-CT in Assessing Treatment Response.

We report the case of a 60-year-old woman with metastatic high-grade uterine leiomyosarcoma who achieved a delayed response to second-line therapy with the marine-derived drug trabectedin (Yondelis(®), PharmaMar). We used 2-deoxy-2-[(18)F] fluorodeoxyglucose (FDG)-positron emission tomography (PET-CT) imaging as a tool for response monitoring in parallel with conventional re-staging according to Response Evaluation Criteria in Solid Tumours (RECIST) using computed tomography (CT). We illustrate the role of serial (18)FDG-PET-CT imaging in the functional assessment of tumour response. Three cycles after commencement of trabectedin treatment, a reduction of the maximum standardized uptake value (SUVmax) of the solid component of the pelvic mass was observed, indicating a cystic or necrotic response in the tumour to trabectedin. After 7 cycles of treatment, on (18)FDG-PET-CT there was clear evidence of ongoing disease improvement: the solid pelvic components were at worst stable, with an unchanged SUVmax, and possibly marginally reduced in size, while the pulmonary metastases had further reduced in size and become FDG negative; the bony metastases were stable. After a total of 13 cycles of treatment, administered over 13 months, the patient showed signs of progression on an (18)FDG-PET-CT scan. The safety profile of trabectedin remained manageable, showing no evidence of cumulative toxicity and being associated with a preserved quality of life. This report illustrates potential limitations of RECIST in response assessments and the critical role of serial (18)FDG-PET-CT imaging in assessing response to trabectedin treatment. Therefore, we propose that (18)FDG-PET-CT may improve the assessment of response to trabectedin in selected patients.

Development and certification of green tea-containing standard reference materials.

A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

Continuous low-dose cyclophosphamide and methotrexate combined with celecoxib for patients with advanced cancer.

BACKGROUND: Combined therapy of metronomic cyclophosphamide, methotrexate and high-dose celecoxib targeting angiogenesis was used in a phase II trial. METHODS: Patients with advanced cancer received oral cyclophosphamide 50 mg o.d., celecoxib 400 mg b.d. and methotrexate 2.5 mg b.d. for two consecutive days each week. Response was determined every 8 weeks; toxicity was evaluated according to CTC version 2.0. Plasma markers of inflammation, coagulation and angiogenesis were measured. RESULTS: Sixty-seven of 69 patients were evaluable for response. Twenty-three patients had stable disease (SD) after 8 weeks, but there were no objective responses to therapy. Median time to progression was 57 days. There was a low incidence of toxicities. Among plasma markers, levels of tissue factor were higher in the SD group of patients at baseline, and levels of both angiopoietin-1 and matrix metalloproteinase-9 increased in the progressive disease group only. There were no changes in other plasma markers. CONCLUSION: This metronomic approach has negligible activity in advanced cancer albeit with minimal toxicity. Analysis of plasma markers indicates minimal effects on endothelium in this trial. These data for this particular regimen do not support basic tenets of metronomic chemotherapy, such as the ability to overcome resistant tumours by targeting the endothelium.

Temozolomide in the treatment of solid tumours: current results and rationale for dosing/scheduling.

This review examines the current evidence for the use of temozolomide in the treatment of solid tumours. The possible molecular and clinical advantages of temozolomide are identified and the molecular mechanism of temozolomide resistance is explored. Attempts to maximise efficacy have led to manipulation of both dosage and drug scheduling and the evidence for the various strategies is reviewed. Finally, the potential role of combination therapy is considered.

Changes in amylin and amylin-like peptide concentrations and beta-cell function in response to sulfonylurea or insulin therapy in NIDDM.

OBJECTIVE: Amylin, a secretory peptide of beta-cells, is the constituent peptide of islet amyloid, which is characteristic of NIDDM, and changes in amylin secretion in response to therapies may influence the rate of production of islet amyloid. The primary objective of this study was to determine whether therapy with sulfonylurea or basal insulin in NIDDM would alter amylin secretion in a way that might affect the formation of islet amyloid. RESEARCH DESIGN AND METHODS: In a randomized crossover design, eight subjects with NIDDM underwent three 8-week periods of therapy with diet alone, sulfonylurea, or exogenous basal insulin, with evaluation of amylin, amylin-like peptide (ALP), and glucose and C-peptide concentrations, both during fasting and after a standard breakfast. Changes in beta-cell function (% beta) were assessed, in the basal state by homeostasis model assessment (HOMA) and in the stimulated state by hyperglycemic clamps. Seven nondiabetic control subjects each underwent a meal profile and hyperglycemic clamp. RESULTS: Both sulfonylurea and insulin therapy reduced basal glucose concentrations compared with diet alone, but neither reduced the increased postprandial glucose increments. Both sulfonylurea and insulin therapy increased basal % beta, assessed by HOMA, but only sulfonylurea increased the second-phase C-peptide responses to the hyperglycemic clamp. Sulfonylurea increased time-averaged mean postprandial amylin and ALP concentrations compared with diet alone (geometric mean [1-SD range] for amylin, 4.9 [2.0-11.8] vs. 3.0 [1.4-6.2] pmol/l, P = 0.003; for ALP, 16.4 [8.5-31.7] vs. 10.1 [4.9-20.8] pmol/l, P = 0.001). Insulin therapy reduced basal ALP concentrations compared with diet alone (2.9 [1.5-5.6] vs. 6.0 [2.6-13.6] pmol/l, P = 0.03), but had no effect on postprandial concentrations of amylin (3.0 [1.3-6.5] pmol/l) or ALP (10.0 [5.5-18.1] pmol/l). CONCLUSIONS: By increasing postprandial concentrations of the constituent peptides of islet amyloid, sulfonylurea therapy might increase the rate of deposition of islet amyloid and thereby accelerate the decline of % beta in NIDDM, compared with diet therapy alone.

Rapid physicochemical detachment, separation and concentration of bacteria from beef surfaces.

A simple, rapid, physicochemical treatment for the removal of viable bacteria from the surface of raw and cooked beef is described. The detachment method was linked to a differential centrifugation step which removed large amounts of particulate food matter and concentrated the detached bacteria. The method increased the numbers of bacteria released from beef surfaces and increased the numbers detected by at least one and a half orders of magnitude, when compared to the traditional 'stomaching' technique. This 1-h separation and concentration method produced cleaner suspensions of bacteria and improved the sensitivity of detection by DEFT and direct plate count.

Resistance of melanoma cell lines to interferons correlates with reduction of IFN-induced tyrosine phosphorylation. Induction of the anti-viral state by IFN is prevented by tyrosine kinase inhibitors.

Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN-induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN-resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN.

An evaluation of the local reaction and biodegradation of calcium sodium alginate (Kaltostat) following subcutaneous implantation in the rat.

Kaltostat swabs were implanted subcutaneously in rats to evaluate their biodegradability and ability to evoke local tissue reactions. Implant sites were evaluated after 24 h and after 7 days, 28 days and 12 weeks. Histological sections showed no noticeable degradation of the Kaltostat within the 3 month observation period, contrary to some published reports. Following subsidence of a modest foreign body reaction, implants became embedded in thin fibrous sheaths which were infiltrated with vascular channels and fibroblasts. This study demonstrates that Kaltostat fibres are well tolerated following subcutaneous implantation in the rat model and present no obvious toxic risk or contraindication to their use as wound dressings or as haemostatic agents in general surgery.

cDNA sequence identity for the type I interferon receptor subunit from cell lines of widely differing responsiveness to interferon.

Melanoma cell lines exhibit strikingly different sensitivity to the antiproliferative effects of interferon. cDNAs encoding the Type I interferon receptor subunit were amplified by polymerase chain reaction, using as template RNA isolated from three melanoma cell lines displaying greater than 100 fold range in their sensitivity to the antiproliferative effects of IFN-beta. Comparison of the cDNA sequences obtained with the published cDNA sequence from the highly interferon-sensitive lymphoid cell line Daudi revealed only one base change that leads to a conservative amino acid substitution. It is concluded that the cellular differences in responsiveness to interferon, of the melanoma cell lines tested, do not arise from the expression of variants of the cloned Type I interferon receptor subunit.

Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae.

Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36 degrees C. The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript. In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature. A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized. This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9. The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence. Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb. None of the identified reading frames share significant homologies with entries of several data bases.

Separation of bacteria using agglutinins isolated from invertebrates.

The agglutination of a selection of Gram-positive and Gram-negative bacteria by the haemolymph and coelomic fluid from several invertebrates was studied. The haemolymph from Lumbricus terrestris and Limulus polyphemus caused the strongest agglutination of most of the bacteria studied. When the agglutinating fraction of Lim. Polyphemus was liganded to magnetic microspheres 53% of the cells in pure cultures of Listeria monocytogenes C200, 15% of Salmonella enteritidis 37782, 92% of Staphylococcus aureus NCDO 949, 19% of Escherichia coli E4936/76 and 65% of E. coli W2-2 were adsorbed to the beads. The immobilized haemolymph from Lumb. terrestris adsorbed 42% of Salm. enteritidis 37782, 64% of E. coli 4936/76 and 27% of Staph. aureus NCDO 1499 cells and the coelomic fluid from Haemopsis sanguisuga adsorbed 42, 48 and 50% of these cultures respectively. With immobilized Haem. sanguisuga agglutinins, 21-27% of Staph. aureus NCDO 2044 cells were recovered from full-fat pasteurized milk and 20-51% from braising steak. Immobilized Lim. polyphemus agglutinins recovered 17-34% of Staph. aureus cells from raw egg. The potential of agglutinins isolated from invertebrates for enhancing rapid microbiological assays of foods is discussed.

Use of the polymerase chain reaction and 16S rRNA sequences for the rapid detection of Brochothrix spp. in foods.

Oligonucleotide primers were designed against rRNA sequences to give a DNA-based PCR assay for the rapid identification/detection of Brochothrix spp. The PCR products could be confirmed by hybridization to an internal oligonucleotide probe. The method successfully and sensitively detected/identified these organisms in pure cultures but was of limited value as a detection method because the detection sensitivity, in relation to conventional plate counts, varied and the assay sensitivity was reduced in the presence of staphylococci. Furthermore, sensitivity was also lost when the assay was applied directly to meat samples. However, a separation step using a lectin (from Agaricus bisporus) immobilized on magnetic beads prior to the PCR assay, allowed the direct detection of low numbers (> 10(2) cfu g-1) of Brochothrix in meat samples within a working day.

Evidence for an [Fe]-type hydrogenase in the parasitic protozoan Trichomonas vaginalis.

The hydrogenase of the pathogenic protozoan Trichomonas vaginalis was extracted and partially purified. The catalytic and spectroscopic properties of the enzyme indicate that it belongs to the class of [Fe]-hydrogenases, rather than the [NiFe]-hydrogenases. The hydrogenase activity was highly sensitive to carbon monoxide, 50% inhibition being attained by 1 microM CO. The EPR spectrum of the most active fractions from chromatography, after reduction by hydrogen and partial reoxidation under argon, showed an EPR spectrum at g = 2.10, 2.04, 2.00. This unusual spectrum is characteristic of the 'H-cluster', as seen in [Fe]-hydrogenases of anaerobic bacteria such as Clostridium spp.

Immunoreactive prostaglandin G/H synthase content increases in ovine cotyledons during late gestation.

In this study, using gel electrophoresis and Western blotting, we have demonstrated that the content of irPGHS in ovine placenta increases during late gestation prior to the onset of labour. This increase in PGHS tissue content may contribute to the corresponding increased production of prostaglandins by the ovine placenta during this period of pregnancy. Although the mechanism by which PGHS tissue content is elevated at this time remains to be established, one possibility which is currently being investigated in our laboratory is that increased PGHS content reflects an increased level of PGHS gene expression.

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods.

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87-100% of cells from cultures of L. monocytogenes, 80-100% of Staph. aureus, 33-45% of Salmonella spp. and 42-77% of E. coli. The A. bisporus lectin bound 31-63% of cells in cultures of L. monocytogenes, 83% of Staph. aureus but only 3-5% of the salmonella cells. Similarly H. pomatia lectin bound greater than 92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31-54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10-47%) or ground beef (32-50%).