Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling

In stimulated neutrophils, the majority of tyrosine phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserved and solubilized tyrosine phosphorylated proteins but also retained their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilisation of a significant fraction of tyrosine phosphorylated proteins. Furthermore, we observed that in monosodium urate crystals-stimulated neutrophils, Lyn activity was enhanced in the soluble fraction recovered from the hypertonic fraction, but not from that of the first hypotonic lysis. The distribution of tyrosine phosphorylated proteins between the NP-40 soluble and insoluble fractions was both substrate- and agonist-dependent. In neutrophils stimulated with fMet-Leu-Phe, MSU crystals or by CD32 ligation, the tyrosine phosphorylated proteins were mostly insoluble. On the other hand, in GM-CSF-treated cells, the phosphoproteins were more equally distributed between the two fractions. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.     

Autocrine IL-10 impairs dendritic cell (DC)-derived immune responses to mycobacterial infection by suppressing DC trafficking to draining lymph nodes and local IL-12 production

The production of IL-12 by dendritic cells (DC) early in an immune response is considered critical for the polarization of CD4(+) T lymphocyte response towards a Th1 pattern, a key process in the clearance of intracellular pathogens. Infection of bone marrow-derived DC with Mycobacterium bovis Bacillus Calmette Guérin (BCG) induced a concurrent and dose-dependent releaseof IL-10 and IL-12. Here we examined whether the production of IL-10 by DC affected their IL-12 response to mycobacterial infection and the generation of protective immune responses in vivo. Compared to wild-type (WT) DC, DC deficient for IL-10 synthesis (IL-10(-/-)) showed increased IL-12 production in response to BCG infection and CD40 stimuli in vitro. Moreover, when transferred into mice, infected IL-10(-/-) DC were more efficient than WT DC at inducing IFN-gamma production to mycobacterial antigens in the draining lymph nodes (DLN).This effect was associated with increased trafficking of IL-10(-/-) DC to the DLN and enhanced IL-12 production by DC within the DLN. These data show that autocrine IL-10 exerts a dual inhibitory effect on the induction of primary immune responses by DC: first, by down-regulating the migration of infected DC to the DLN and second, by modulating the IL-12 production by DC in the DLN.     

Accessibility, clinical effectiveness, and practice costs of providing a telephone option for routine asthma reviews: phase IV controlled implementation study.

BACKGROUND: Attendance for routine asthma reviews is poor. A recent randomised controlled trial found that telephone consultations can cost-effectively and safely enhance asthma review rates; however, concerns have been expressed about the generalisability and implementation of the trial's findings. AIM: To evaluate the effectiveness of a telephone option as part of a routine structured asthma review service. DESIGN OF STUDY: Phase IV controlled before-and-after implementation study. SETTING: A large UK general practice. METHOD: Using existing administrative groups, all patients with active asthma (n = 1809) received one of three asthma review services: structured recall with a telephone-option for reviews versus structured recall with face-to-face-only reviews, or usual-care (to assess secular trends). Main outcome measures were: proportion of patients with active asthma reviewed within the previous 15 months (Quality and Outcomes Framework target), mode of review, enablement, morbidity, and costs to the practice. RESULTS: A routine asthma review was provided for 397/598 (66.4%) patients in the telephone-option group compared with 352/654 (53.8%) in the face-to-face-only review group: risk difference 12.6% (95% confidence interval [CI] = 7.2 to 17.9, P<0.001). The usual-care group achieved a review rate of 282/557 (50.6%). Morbidity was equivalent in the three groups; however, enablement (P = 0.03) and confidence (P = 0.007) in asthma management were greater in the telephone-option versus face-to-face-only group. The cost per review achieved by providing the telephone-option service was lower than the face-to-face-only service (10.03 pounds versus 12.74 pounds, mean difference 2.71 pounds; 95% CI = 1.92 to 3.50, P<0.001); usual-care costs were 11.85 pounds per review achieved. CONCLUSION: Routinely offering telephone reviews cost-effectively increased asthma review rates, enhancing patient enablement and confidence with management, with no detriment to asthma morbidity. Practices should consider a telephone option for their asthma review service.     

Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies for inflammatory bowel disease: high prevalence in patients with celiac disease

Both celiac disease and inflammatory bowel disease (IBD) are characterized by chronic diarrhea and the presence of distinct (auto)antibodies. In the present study we wanted to determine the prevalence of serological markers for inflammatory bowel disease, i.e., perinuclear antineutrophil cytoplasmic antibodies (pANCA) and/or anti-Saccharomyces cerevisiae antibodies (ASCA), in 37 patients with biopsy-confirmed celiac disease (Marsh IIIb/c). The majority of the patients was positive for IgA (auto)antibodies typically associated with celiac disease, i.e., antiendomysium antibodies (EMA) (86.5%), antigliadin antibodies (AGA) (73%), and antirecombinant human tissue transglutaminase antibodies (rh-tTGA) (86.5%). Four patients with selective IgA deficiency could be identified by analyzing EMA, AGA, and rh-tTGA for the IgG isotype. The prevalence of pANCA and ASCA, markers that are used for IBD, was unexpectedly high in our cohort of patients with celiac disease: 8 patients were positive for pANCA (IgG) and 16 patients were positive for ASCA (IgG and/or IgA). These results indicate that the presence of pANCA or ASCA in the serum of patients with chronic diarrhea does not exclude celiac disease. A prospective study is required to determine whether pANCA and/or ASCA identify patients at risk for developing secondary autoimmune disease.     

Null mutation in human ciliary neurotrophic factor gene confers higher body mass index in males

Ciliary neurotrophic factor (CNTF) administration reduces weight in leptin-resistant mice via the signalling pathway normally activated by leptin. A G>A null mutation in the CNTF gene results in complete absence of protein. We hypothesised that absence of CNTF could lead to diminished initiation of anorectic pathways, with consequent increase in body mass. In 575 Caucasian men aged 59-73 years, the A/A genotype (frequency 1.9%) was associated with a 10 kg increase in weight (P=0.03, 2 df) and 3 kg/m(2) greater BMI (P=0.02, 2 df). There was no effect in women. The CNTF G>A null mutation therefore confers a moderate effect on obesity in males of A/A genotype, who represent 1% of the general population.     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region

Anophthalmia or microphthalmia occur in approximately one in 10 children who have severe visual impairment. These eye malformations are often of unknown aetiology, but can be inherited in autosomal dominant, recessive or X-linked forms, and can also occur in association with specific chromosome abnormalities. Four children are described in the medical literature with microphthalmia or anophthalmia in association with chromosome rearrangements involving distal 3q, suggesting the presence of a micro/anophthalmia gene in this region. We have identified two further patients with micro/anophthalmia and chromosome rearrangements involving 3q26-->3q27 and identified a 6.7 MB common deleted region. Patient 1 had multiple abnormalities including bilateral anophthalmia, abnormalities of the first and second cranial nerves and partial absence of the corpus callosum. His karyotype was 46,XY,del(3)(q26.33q28). Patient 2 had right anophthalmia and left extreme microphthalmia. Her karyotype was 46,XX,del(3)(q26.33q28)t(3;7)(q28;q21.1). Both patients had intrauterine growth retardation (IUGR) and strikingly similar dysmorphic facies consisting of bossed forehead, downward-slanting palpebral fissures, grooved bridge of the nose, prominent low-set ears, small down-turned mouth and small mandible. We identified BAC clones mapping to distal 3q from the ENSEMBL and NCBI Entrez databases. These BAC clones were used as fluorescence in situ hybridisation (FISH) probes to identify the minimum deleted region common to both patients. This interval, between clones RPC11-134F2 and RPC11-132N15, was estimated to be 6.7 MB. We conclude that there is an anophthalmia locus within this interval. Candidate genes mapping to this region include Chordin and DVL3, a homologue of the Drosophila Dishevelled gene.     

The effects of levetiracetam on objective and subjective sleep parameters in healthy volunteers and patients with partial epilepsy

Levetiracetam is a novel antiepileptic drug which has recently been released as an adjunctive treatment for partial epilepsy. In the two studies reported here we examined the objective and subjective effects of levetiracetam on sleep in 12 healthy volunteers and 17 patients [16 who could be evaluated for electroencephalogram (EEG) recordings] with a history of partial epilepsy on stable carbamazepine monotherapy. The studies were of a similar double-blind crossover placebo-controlled design with subjects' sleep being recorded in their own homes. The results from the two studies showed considerable similarities. In both, levetiracetam produced an increase in the time spent in stage 2 sleep, which in the patient study was accompanied by a decrease in the time spent in stage 4 sleep and in the volunteer study an increase in rapid eye movement (REM) latency. The subjective changes included reports that sleep was of a better quality with fewer awakenings and patients also reported that their sleep was more restful. Volunteers and patients did, however, feel less alert on waking in the morning. Therefore, both groups reported a decrease in awakenings after levetiracetam despite the finding from the EEG of no change in the actual number of awakenings. It may be concluded from both studies that levetiracetam does affect some indicators of subjective sleep perception, but does not influence objective sleep measures of sleep continuity. The results from the patient study during placebo add-on treatment also showed that patients on carbamazepine had a marked increase in SWS, an increase in stage 2 sleep and an increase in REM latency compared with healthy volunteers. Interestingly, levetiracetam also reduced bilateral epileptiform EEG activity, particularly in patients with more discharges.     

Myoblasts fail to stimulate T cells but induce tolerance

Recent interest in myoblast transfer and in the use of myoblastsas vehicles in gene therapy has made it important to understandthe potential immunogenicity of allogeneic or neoantlgen-expresslngmyoblasts. Given the problems of producing a pure populationof myoblasts, In this study we used a tumour-derived musclecell line (TE671), with phenotyplc features of myoblasts, whichwe transfected to express HLA-DR1. However, this cell line wasunable to stimulate either established HLA-DR1-specific alloreactlveT cell clones or a primary alloresponse. Nor could it presenthaemagglutlnln peptide HA 306–324 to DR1-restricted, HA306–324-speciflc T cell clones or lines. Indeed, prelncubatlonwith DR1-expressing TE671 and HA 306–324 rendered suchT cells tolerant as Judged by their subsequent inability toproliferate in response to a DR1⁺ B cell line plus peptide HA306–324. These results imply that myoblasts do not providecostlmulatory signals, and are therefore unlikely to stimulateallospeclfic T cells following myoblasts transplantation orto initiate neoantlgen-speclfic immune responses following Invivo transfection.     

Predictors of low CD4 count in resource-limited settings: based on an antiretroviral-naive heterosexual thai population

A barrier to the appropriate provision of antiretroviral therapy to treat immunosuppressed HIV-infected persons in resource-poor countries is identifying who requires treatment. The World Health Organization (WHO) has suggested using a clinical algorithm combined with a total lymphocyte count (TLC) < 1200 cells/mm as a surrogate for a CD4 count less than 200 cells/mm when it is not possible to measure the CD4 count. We evaluated various TLC levels, anemia, and body mass index and compared our data with the WHO criteria to develop a more sensitive algorithm to predict CD4 counts of < 200 cells/mm and < 350 cells/mm in 839 men and women from Thailand infected with HIV-1 subtype E (CRF01_AE). The December 2003 WHO guidelines had a sensitivity of 34.1% in men and 31.8% in women to detect persons with a CD4 count < 200 cells/mm in this HIV-infected population from Thailand. The use of a TLC < 1500 cells/mm or TLC < 2000 cells/mm combined with anemia or WHO stage II infection doubled the sensitivity to detect persons with a CD4 count < 200 (63.0% in men, 68.2% in women) with less than a 6% decrease in specificity.