Evaluating newly approved drugs in combination regimens for multidrug-resistant tuberculosis with fluoroquinolone resistance (endTB-Q): study protocol for a multi-country randomized controlled trial.

BACKGROUND: Treatment for fluoroquinolone-resistant multidrug-resistant/rifampicin-resistant tuberculosis (pre-XDR TB) often lasts longer than treatment for less resistant strains, yields worse efficacy results, and causes substantial toxicity. The newer anti-tuberculosis drugs, bedaquiline and delamanid, and repurposed drugs clofazimine and linezolid, show great promise for combination in shorter, less-toxic, and effective regimens. To date, there has been no randomized, internally and concurrently controlled trial of a shorter, all-oral regimen comprising these newer and repurposed drugs sufficiently powered to produce results for pre-XDR TB patients. METHODS: endTB-Q is a phase III, multi-country, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of a treatment strategy for patients with pre-XDR TB. Study participants are randomized 2:1 to experimental or control arms, respectively. The experimental arm contains bedaquiline, linezolid, clofazimine, and delamanid. The control comprises the contemporaneous WHO standard of care for pre-XDR TB. Experimental arm duration is determined by a composite of smear microscopy and chest radiographic imaging at baseline and re-evaluated at 6 months using sputum culture results: participants with less extensive disease receive 6 months and participants with more extensive disease receive 9 months of treatment. Randomization is stratified by country and by participant extent-of-TB-disease phenotype defined according to screening/baseline characteristics. Study participation lasts up to 104 weeks post randomization. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 324 participants across 2 arms affords at least 80% power to show the non-inferiority, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per-protocol populations. DISCUSSION: This internally controlled study of shortened treatment for pre-XDR TB will provide urgently needed data and evidence for clinical and policy decision-making around the treatment of pre-XDR TB with a four-drug, all-oral, shortened regimen. TRIAL REGISTRATION: ClinicalTrials.Gov NCT03896685. Registered on 1 April 2018; the record was last updated for study protocol version 4.3 on 17 March 2023.

Psychomotor semiology in depression: a standardized clinical psychomotor approach.

BACKGROUND: Although psychomotor symptoms are associated with the clinical symptomatology of depression, they are rarely assessed and standardized clinical evaluation tools are lacking. Psychomotor retardation is sometimes assessed through direct patient observations by clinicians or through a clinical observation grid, in the absence of a standardized psychomotor assessment. In this pilot study, we evaluated the feasibility of standardized psychomotor examination of patients with major depressive disorder (MDD) and detailed a psychomotor semiology in these patients. METHODS: We used a standardized psychomotor assessment to examine 25 patients with MDD and 25 age- and sex-matched healthy controls (HC) and compared their psychomotor profiles. Using standardized tests, we assessed muscle tone and posture, gross motor skills, perceptual-motor skills, and body image/organization. Clinical assessments of depressive symptoms (levels of psychomotor retardation, anxiety, and self-esteem) comprised this detailed psychomotor examination. RESULTS: All participants were examined using the standardized psychomotor assessment. The main results of the psychomotor examination highlighted low body image of MDD participants (p < 0.001). Significant differences between groups were found in passive muscle tone, posture, emotional control, jumping, manual dexterity, walking, and praxis. Among these psychomotor variables, body image, passivity, jumping and rhythm scores predicted an MDD diagnosis. CONCLUSIONS: Beyond the psychomotor retardation known to be present in MDD patients, this examination revealed an entire psychomotor symptomatology characterized by elevated muscle tone, poor body image associated with poor self-esteem, slowness in global motor skills and manual praxis, and poor rhythmic adaptation. In light of these results, we encourage clinicians to consider using a standardized tool to conduct detailed psychomotor examination of patients with depressive disorders. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04031937 , 24/07/2019.

Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders.

Background: Retinoid-based therapies are commonly used in the treatment of disorders of keratinization and other skin disorders but can result in non-specific effects and adverse reactions. Use of retinoic acid metabolism blocking agents (RAMBAs) such as DX308 may address these shortcomings. Objectives: Characterize the therapeutic potential of recently discovered, CYP26-selective RAMBA, DX308. Materials and Methods: Preliminary in vitro assessment of potential off-target activity, metabolic and toxicologic profiling. Studies to assess safety and efficacy of topical treatment in correcting abnormal skin morphology in rhino mice. Extensive gene expression profiling by RNA sequencing and qPCR in 3D epidermis grown with keratinocytes (KCs) from keratinization disorders and healthy controls, to investigate modulation of retinoid biopathways. Results: In vitro, DX308 does not interact with off-target nuclear receptors or CYP450s, is not genotoxic, and is stable in skin, despite vigorous hepatic metabolism. In vivo, topical DX308 induces comedolysis and epidermal thickening without apparent adverse effects. Gene expression profiling shows potent modulation of retinoid-responsive genes by DX308 in both healthy and keratinization disorder KCs. Pathway analysis suggests DX308 may inhibit inflammatory and immune responses in KCs. Conclusions: These preliminary studies suggest that DX308 is an efficacious topical therapeutic with a favourable metabolic and safety profiles. DX308 may present an improved therapeutic alternative for the treatment of keratinization disorders and other retinoid-responsive skin ailments.

Automatic depletion with Spectra Optia allows a safe 16% reduction of red blood cell pack consumption in exchanged sickle cell anemia patients.

BACKGROUND: Chronic red blood cell exchanges (RBCXs) are frequently used to prevent complications in patients with sickle cell anemia, but the scarcity of matched red blood cell packs (RBCPs) is a serious concern. The main goal of this study was to compare the number of RBCPs used during RBCXs between the Spectra Optia (SO) device (with the automatic depletion step) and the former Cobe Spectra (CSP) device. STUDY DESIGN AND METHODS: The performances and safety of 300 SO sessions using the automatic depletion step (SO/DE) in 50 patients with sickle cell anemia under a chronic transfusion program over a 1-year period were prospectively analyzed. The numbers of RBCPs saved using this protocol compared to the SO device without depletion and to the CSP device were determined. RESULTS: The SO/DE protocol appeared to be safe, as only 5% and 17% of the sessions were characterized by a significant decrease in blood pressure and increase in heart rate (grade 2 adverse events), respectively. Postapheresis hematocrit and fraction of cells remaining reached expected values. The SO/DE protocol required 16% fewer RBCPs compared to SO without depletion, allowing a mean saving of 12 RBCPs per patient and per year and 13% fewer compared to CSP device. Interestingly, the saving was more important for patients with high total blood volume and/or high preapheresis hematocrit. CONCLUSION: The SO/DE protocol is an efficient, safe and cost-effective procedure for patients with sickle cell anemia under a chronic transfusion program.

Laterality and Lateralization in Autism Spectrum Disorder, Using a Standardized Neuro-Psychomotor Assessment.

A detailed assessment of laterality in children with Autism Spectrum Disorder (ASD) was realized, including handedness and other measures (muscle tone, manual performance, dominant eye), using a standardized battery for the developmental assessment of neuro-psychomotor functions. The results of the laterality tests relating to cerebral hemisphere organization (spontaneous gestural laterality and tonic laterality) were different in ASD children, and indicate that the cerebral organization could be disrupted. These assessments, added to the observations of usual laterality most often reported in the literature, provide better understanding of the developmental organization from the pathophysiological point of view in children with ASD.

Copy-number analysis identified new prognostic marker in acute myeloid leukemia.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.

[Formula: see text]Current knowledge on motor disorders in children with autism spectrum disorder (ASD).

Motor symptomatology in autism is currently poorly understood, and still not included in the autism spectrum disorder (ASD) diagnostic criteria, although some studies suggest the presence of motor disturbances in this syndrome. We provide here a literature review on early motor symptoms in autism, focusing on studies on psychomotor issues (tone, postural control, manual dexterity, handedness, praxis). The approach adopted in research to study altered motor behaviors is generally global and there is no detailed semiology of the motor or neuromotor disorders observed in people with ASD. This global approach does not enable understanding of the neuro-developmental mechanisms involved in ASD. Identification of clinical neuro-psychomotor profiles in reference to a standard would help to better understand the origin and the nature of the disorders encountered in ASD, and would thus give new directions for treatment.

Very high quantitative tumor HER2 content and outcome in early breast cancer.

BACKGROUND: It is unknown how a very high tumor total HER2 (human epidermal growth factor receptor-2) content (H2T) influences outcome in early breast cancer treated with adjuvant trastuzumab plus chemotherapy. PATIENTS AND METHODS: H2T was measured using a novel quantitative assay (HERmark(®)) from formalin-fixed tumor tissue of 899 women who participated in the FinHer trial (ISRCTN76560285). In a chromogenic in situ hybridization (CISH) test, 197 (21.9%) patients had HER2-positive cancer and were randomly assigned to receive trastuzumab or control. RESULTS: Cancer H2T levels varied 1808-fold. High H2T levels were correlated with a positive HER2 status by CISH (P < 0.0001). A nonlinear association was present between H2T and the hazard of distant recurrence in a subpopulation treatment effect pattern plot analysis in CISH-positive disease. Patients with very high H2T (defined by ≥22-fold the median of HER2-negative cancers; 13% of CISH-positive cancers) did not benefit from adjuvant trastuzumab [hazard ratio (HR) 1.23; 95% confidence interval (CI) 0.33-4.62; P = 0.75], whereas the rest of the patients with HER2-positive disease by CISH (87%) did benefit (HR 0.52; 95% CI 0.28-1.00; P = 0.050). CONCLUSION: Patients with HER2-positive breast cancer with very high tumor HER2 content may benefit less from adjuvant trastuzumab compared with those whose cancer has more moderate HER2 content.

Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab.

BACKGROUND: Patients with metastatic breast cancer (MBC) overexpressing HER2 (human epidermal growth factor receptor 2) are currently selected for treatment with trastuzumab, but not all patients respond. PATIENTS AND METHODS: Using a novel assay, HER2 protein expression (H2T) was measured in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for MBC. Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T < 13.8), H2T high (H2T ≥ 68.5), and H2T intermediate (13.8 ≤ H2T < 68.5) subgroups. Kaplan-Meier (KM) analyses were carried out comparing the groups for time to progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results. RESULTS: TTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors [median TTP 4.2 months, hazard ratio (HR) = 3.7, P < 0.0001] or H2T high tumors (median TTP 4.6 months, HR = 2.7, P = 0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP 12 months). OS analyses yielded similar results. CONCLUSIONS: MBC patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab. These results are preliminary and require confirmation in larger controlled clinical cohorts.

Toward Automatic Label-Free Whispering Gallery Modes Biodetection with a Quantum Dot-Coated Microsphere Population.

We explore a new calibration-free approach to biodetection based on whispering gallery modes (WGMs) without a reference measure and relative shifts. Thus, the requirement to keep track of the sensor position is removed, and a freely moving population of fluorophore-doped polystyrene microspheres can now fulfill this role of sensing resonator. Breaking free from fixed surface-based biosensing promotes adhesion between the microsphere sensors and the analytes since both can now be thoroughly mixed. The 70-nm-wide spectrum of green fluorescent microbeads allows us to monitor over 20 WGMs simultaneously without needing evanescent light coupling into the microspheres, hence enabling remote sensing. Since the exact radius of each microsphere is unknown a priori, it requires algorithmic analyses to obtain a reliable result for the refractive index of a solution. We first test our approach with different solutions of alcohol in water obtaining 3 x 10(-4) precision on the refractive index at lower concentrations. Then, the solutions of bacterial spores in water yield clear evidence of biodetection in the statistical analysis of WGMs from 50 microspheres. To extend the fluorescence spectral range of our WGM sensors, we present preliminary results on coating microspheres with CdSe/ZnS quantum dots.

Maternal deprivation alters epithelial secretory cell lineages in rat duodenum: role of CRF-related peptides.

OBJECTIVE: Chronic psychological stress is associated with development of intestinal barrier dysfunction and impairs host defence mechanisms. The intestinal epithelium, consisting of enterocytes, endocrine cells, goblet cells and Paneth cells, is an important component of this barrier. In the present study, the impact of maternal deprivation (MD) on secretory lineages of duodenal epithelium and the involvement of the peripheral corticotropin-releasing factor (CRF) pathway were investigated. METHODS: Rat pups were deprived of their dam for 3 h/day (days 5-20). Non-deprived pups served as controls. On days 8, 13, 20, 24, 34, 44 and 84, duodenal tissues were collected for quantitative real-time PCR and immunohistochemistry studies. RESULTS: MD induced a sustained decrease in the number of Paneth and goblet cells but hyperplasia of endocrine cells. These alterations were associated with a duodenal increase of CRF, urocortin 2 and CRF receptor subtype 2 (CRFR(2)) mRNA, whereas CRFR(1) expression was decreased. The effects of MD on intestinal epithelium were inhibited by the CRFR(1)/R(2) antagonist astressin injected daily before MD. Studies using specific receptor antagonists in rats subjected to MD revealed that CRFR(1) was involved in the hyperplasia of endocrine cells and CRFR(2) in the depletion of Paneth cells. Conversely, daily injection of CRF and of the CRFR(2) agonist urocortin 2 in control rats resulted in changes in epithelial differentiation similar to MD. CONCLUSIONS: The activation of CRFR(1) and CRFR(2) induced by MD markedly altered the quantitative distribution of secretory cells of the intestinal epithelium. These alterations, in particular the depletion of Paneth and goblet cells, may create conditions leading to the development of an epithelial barrier defect.

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Alleviation of the effects of endotoxin exposure on behavior and hippocampal IL-1beta by a selective non-peptide antagonist of corticotropin-releasing factor receptors.

Previous research has shown that lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) administration produces learning/memory deficits in a variety of paradigms. In our laboratory, we have consistently observed LPS-induced behavioral alterations in a two-way active avoidance conditioning paradigm. Following LPS administration, one factor that affects cytokine production is corticotropin-releasing factor (CRF). CRF has well known anti-inflammatory effects, via stimulation of ACTH and corticosterone release. However, CRF acting directly on immune cells or within the CNS may potentiate proinflammatory effects. The current experiments explored the potential of antalarmin, a CRF-R1 non-peptide antagonist, to diminish or negate deficits observed with LPS administration. On the first day of testing, four-month-old male C57BL/6J mice received an intraperitoneal (i.p.) injection of antalarmin, followed 90min later by a second i.p. injection of LPS 4h prior to two-way active avoidance conditioning testing. As hypothesized, LPS administration altered performance. However, pretreatment with antalarmin attenuated the adverse effects of LPS administration. Moreover, evidence indicates that antalarmin attenuated hippocampal, but not peripheral, cytokine release. The behavioral results cannot be explained by alterations in the HPA axis, as antalarmin did not affect the LPS-induced rise in corticosterone. The current research contributes preliminary evidence that CRF may be an important factor in the development of LPS-induced behavioral effects, and that blocking the activity of CRF may be sufficient to alleviate some of the effects of endotoxin exposure, possibly due to diminished LPS-induced IL-1beta release in the dorsal hippocampus.

The ability of the rat to metabolize myristoyl-methionine: an acylamino acid with potentially useful antibacterial properties.

Two experiments with Sprague Dawley rats tested their ability to hydrolyse myristoyl-methionine (M-M) into myristic acid and L-methionine (M). In the first experiment, lasting for 3 days. male rats were orally administered [9,10-3H]myristoyl-L-[35S]methionine. The recovery of radioactivity was approximately 90% for both isotopes; 19% of the administered 3H was recovered in the urine and 16% in the faeces, while the recovered 35S activity was 13 and 12%, respectively. The balance of the radioactivity was found among the tissues, organs and blood. In the second experiment, male and female rats received soybean-based diets which were supplemented with either 0.305% M-M or 0.2% M (both diets contained equal amounts of M) for periods up to 4 weeks. The growth rate of the rats receiving the 0.305% M-M diets was slightly slower than that for the rats on the 0.2% M diet, but the difference was not statistically significant (P > 0.05). The M-M rats had a transitory decrease in feed consumption, suggesting that palatability may have contributed to the growth difference and that a somewhat greater amount of M-M was necessary for the rat to attain the same growth rate as that produced by 0.2% M. When the amount of dietary M-M was increased to 3.05% M-M, a greater reduction in feed consumption and body weight gain was observed. This latter diet was an initial attempt to study the potential toxicity of M-M. None of the haematological, clinical chemistry or organ weight data suggested that M-M was overtly toxic per se, but longer-term feeding studies are needed to evaluate the potential toxicity of M-M more fully.

Influence of carbohydrate status on immune responses before and after endurance exercise.

To determine the effect of carbohydrate (CHO) status on immune responses after long-duration exercise, on two occasions, 10 men completed a glycogen-depleting bout of cycle ergometry followed by 48 h of either a high-CHO diet (HiCHO; 8.0 g CHO/kg) or a low-CHO diet (LoCHO; 0.5 g CHO/kg). After the 48 h, subjects completed a 60-min ride at 75% maximal O2 uptake (EX). Blood samples were taken predepletion, pre-EX, post-EX, and 2 and 24 h post-EX and were assayed for leukocyte number and function, glucose, glutamine, and cortisol. The glucose responses were significantly higher in the HiCHO (4.62 +/- 0.26 mM) vs. the LoCHO (3.19 +/- 0.15 mM) condition post-EX, and glutamine was significantly higher in the HiCHO (0.472 +/- 0.036 mM) vs. the LoCHO (0.410 +/- 0.025 mM) condition throughout. Cortisol levels were significantly greater in the LoCHO (587 +/- 50 nM) vs. the HiCHO (515 +/- 62 nM) condition throughout the trial. Lymphocyte proliferation (phytohemagglutinin) was significantly depressed after exercise. However, there was no difference between conditions, and the depression was not correlated with elevations in cortisol. Circulating numbers of leukocytes, neutrophils, lymphocytes, and lymphocyte subsets were significantly greater in the LoCHO vs. the HiCHO condition at the post-EX and 2 h post-EX time points. These data indicate that the exercise and diet manipulation altered the number of circulating leukocytes but did not affect the decrease in lymphocyte proliferation that occurred after exercise.

Synthesis of phosphotyrosine-containing peptides using bis-(2,2,2-trichloro)ethyl groups for phosphate protection.

Suitability of bis-(2,2,2-trichloro)ethyl (Tc) groups for protection of phosphate moiety in Boc-mode synthesis of phosphotyrosine peptides is demonstrated Boc-Tyr(PO3Tc2)-OH and Fmoc-Tyr(PO3Tc2)-OH were prepared by acylating H-Tyr(PO3Tc2)-OH with (Boc)2O and Fmoc-ONSu, respectively. Phosphorus introduction was achieved by phosphorylating Boc-Tyr-OBzl with Tc phosphochloride. The Tc-phosphorus protector was found to be incompatible with the Fmoc group because the conditions of Fmoc removal (piperidine treatment) caused dephosphorylation. Complete NMR spectral assignments in the described compounds is presented. (Contribution No. 2398 from the Centre for Food and Animal Research).

The effect of moderate aerobic training on lymphocyte proliferation.

The purpose of this study was to determine the effect of 12 wks of aerobic training on resting lymphocyte number and proliferation, and immunoglobulin and cytokine levels. Eleven college-aged males (training group = EX) performed 30 min of cycling at 75% of VO2peak, 3 days/wk with VO2peak assessment and blood samples taken at 0,8 and 12 wks. A group of 10 sedentary controls (CT) underwent the same testing protocol. Lymphocyte proliferation response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was quantified as a stimulation index (SI) based on the ratio of stimulated versus control cultures, and as total counts per min (CPM). Immunoglobulin (Ig) levels (IgG, IgA, and IgM), and lymphocyte counts were also determined. There was a significant increase in VO2 in the EX group (41.0 +/- 1.8 vs. 46.3 +/- 1.4 ml.kg-1.min-1 pre and post training, respectively). Training had no effect on the PHA SI for the EX group (23.9 +/- 3.3, 27.7 +/- 4.1, and 26.3 +/- 4.0 at 0, 8 and 12 wks, respectively), or the responses of the CT group (28.8 +/- 6.0, 23.9 +/- 3.1, and 30.6 +/- 4.3). No changes were observed for the PWM SI. Significant increases were observed in the CPM for both groups. No differences in the Ig or lymphocyte levels were found during the study. These data indicate that 12 wks of moderate endurance training did not alter resting immune function as determined by mitogen stimulated lymphocyte proliferation, total circulating lymphocytes, or Ig levels.

Measurement of tumor necrosis factor activity by flow cytometry.

Tumor necrosis factor-alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity using flow cytometry. After an incubation with TNF, L929 cells were harvested and treated with a calcein-AM and ethidium homodimer-1 solution. Nonfluorescent calcein-AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer-1 is a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity.

Improved fluorescent bioassay for the detection of tumor necrosis factor activity.

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells were stained with a solution of ethidium homodimer-1, a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. The assay is sensitive, inexpensive and correlates with the already reported TNF assays while measuring the membrane alteration by TNF and not the cell detachment. It requires no rinsing before dye addition which may cause cell loss; there is no interference with culture medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.

Comparative metabolism of deltamethrin and 3-phenoxybenzoic acid in chickens.

A comparative metabolism of [benzyl 14C]deltamethrin and 3-phenoxybenzoic acid (3-PBacid) was carried out in chickens. The effect of oral and intravenous route of administration was also investigated. There was no difference in the metabolic profile of [14C] portion of deltamethrin and 3-PBacid. Similarly, no effect on the nature of metabolites excreted due to mode of administration was observed. Biotransformation of orally dosed compounds did not occur in the intestine, but were absorbed and metabolized by liver (deltamethrin) and kidney (3-PBacid). 3-PBacid, the primary metabolite of deltamethrin, was readily metabolized into 3-hydroxy benzoic acid (3-HO-Bacid) which is conjugated with a variety of endogenous substances to form sulphates, glucuronides, butyl ester and peptides. HPLC and LC-Ms methods were used to ascertain the structures of metabolites.