Functional interaction between the tumor necrosis factor and interleukin 2 during induction of lymphokine activated killer cytotoxic activity

TNF has a differential effect on the induction of LAK activity which is dependent on the IL-2 concentrations used for LGL stimulation. TNF was found to synergize with low concentrations of IL-2 which are normally ineffective in inducing LAK activity. When IL-2 was used at concentrations optimal for LAK generation, TNF did not further enhance the differentiation of LGL into LAK effectors. When anti-TNF was included in the 51Cr-release assay, no effect on the cytolytic activity was observed. We also demonstrate that IL-2 induced an increase of specific TNF binding to LGL.     

The role of caspase activation and mitochondrial depolarisation in cultured human apoptotic eosinophils

Caspases are key intracellular molecules in the control of apoptosis, but little is known concerning their relative contribution to the cascade of events leading to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation dependent apoptosis induction in the cultured eosinophils (CE). CE cultured alone for 48 hours exhibited constitutive apoptosis (12% ± 1.2). Significant (P < 0.05) enhancement of eosinophil apoptosis was observed following monoclonal antibody (Mab) treatment with CD45 (40% ± 0.7), CD95 (36% ± 1.6), or CD69 (34% ± 0.2). Caspase activity was analysed using the novel CaspaTagTM technique and flow cytometry. CE ligated with CD45 (Bra55), CD95 (Fas) and CD69 Mab resulted in caspase-3 and -9 activation after 16 hours post-ligation. This trend in caspase-3 and -9 activation continued to increase significantly through to the 20 and 24 hours time points when compared to isotype control. Activated up-stream caspase-8 was detected 16 and 20 hours after treatment with CD45, CD95 and CD69 Mab followed by a trend toward basal levels at 24 hours. Ligation of CD95 was followed by mitochondrial permeabilization, as demonstrated by marked increase in mitochondrial transmembrane potential (ΔΨ_m) at all time points. However, ligation with CD45 and CD69 failed to induce a change in ΔΨ_m at 16 hours post-treatment compared to isotype control even though there was an alteration in mitochondrial downstream-caspase activity following ligation with these Mab(s) at this time point. At 20 and 24 hours post-ligation, CD45 or CD69 induce significantly altered levels of ΔΨ_m. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti inflammatory therapy for asthma.     

Green sensitive spectrofluorimetric determination of pemetrexed as antineoplastic drug: Application in pharmaceutical dosage forms and spiked human plasma

A recent antineoplastic medication is pemetrexed, this medicine is now being developed and produced on a large scale, thus approaches for quality control are urgently needed. Spectrofluorimetric guidelines for the simple estimation of pemetrexed were validated. Pemetrexed's assay depends on observations of its native fluorescence at wavelengths 275/450 nm and pH 4. The proposed approach was also used to identify the examined drug in both its formulation and in human plasma that had been spiked.     

Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer.Impact statementThese data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.     

Sensitivity of ruminal bacteria isolates of sheep,cattle and buffalo to some heavy metals

This study was conducted to investigate the toxicity of 12 heavy metals (i.e., HM), namely Hg, Cd, Co, Ni, Cr, Cu, Ag, Mo, Zn, Pb, Li and Na on bacterial isolates from the rumen of sheep, cattle and buffaloes. Rumen samples were collected immediately after slaughter during the dry season. Ruminal bacterial isolates, 59, were obtained from two sheep, five cows and nine buffaloes. Sensitivity of ruminal bacterial isolates to each HM was determined by the clearance zone (CZ) using the Kirby-Bauer disc diffusion susceptibility test. Bacterial populations isolated from the rumens of sheep and buffalo had a lower resistance (P<0.001) to HM toxicity than did cattle. Regardless of ruminant species, bacterial isolates revealed a higher tolerance (P<0.001) to Li and Na, whereas Hg and Cd were the most toxic HM for all isolates. We conclude that inhibition of these HM to the isolated microbial population of sheep, cattle and buffalo is ranked: Hg (most toxic) > Cd > Co ～ Ag ～ Cu ～ Cr ～ Ni > Mo ～ Zn ～ Pb > Li ～ Na. Further research is required to explain the mechanism of toxicity of these HM, and also to explore the variation among ruminant species.