Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

BACKGROUND: Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function. METHODS: Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) colonic epithelial cells. Protein expression was measured by western blotting and intracellular Ca(2+) levels by Fura-2 fluorescence. KEY RESULTS: While acute (15 min) treatment of T(84) cells with a cholinergic G(q) PCR agonist, carbachol (CCh), rapidly stimulated Cl(-) secretion, subsequent CCh-induced responses were attenuated in a biphasic manner. The first phase was transient and resolved within 6 h but this was followed by a chronic phase of attenuated responsiveness that was sustained up to 48 h. CCh-pretreatment did not chronically alter responses to another G(q)PCR agonist, histamine, or to thapsigargin or forskolin which elevate intracellular Ca(2+) and cAMP, respectively. This chronically acting antisecretory mechanism is not shared by neurotransmitters that activate G(s)PCRs. Conditioned medium from CCh-pretreated cells mimicked its chronic antisecretory actions, suggesting involvement of an epithelial-derived soluble factor but further experimentation ruled out the involvement of epidermal growth factor receptor ligands. Acute CCh exposure did not chronically alter surface expression of muscarinic M(3) receptors but inhibited intracellular Ca(2+) mobilization upon subsequent agonist challenge. CONCLUSIONS & INFERENCES: These data reveal a novel, chronically acting, antisecretory mechanism that downregulates epithelial secretory capacity upon repeated G(q)PCR agonist exposure. This mechanism involves release of a soluble factor that uncouples receptor activation from downstream prosecretory signals.

Human herpesvirus 6B induces phenotypic maturation without IL-10 and IL-12p70 production in dendritic cells.

Human herpesvirus 6B (HHV-6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T-cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV-6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV-6B infection of DC. The addition of HHV-6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV-6B infection. Nevertheless, HHV-6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV-6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA-DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV-6B infection did not induce IL-10 and IL-12p70 production in immature DC. However, infected DC were still able to react to bacteria-derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL-10 and IL-12p70 when compared to that of uninfected DC.

Risk for contralateral breast cancer among carriers of the CHEK2*1100delC mutation in the WECARE Study.

The protein encoded by the CHEK2 gene is involved in cellular repair of DNA damage. The truncating mutation, CHEK2*1100delC, seems to increase the risk for breast cancer. We investigated whether the CHEK2*1100delC mutation carrier status increases the risk for asynchronous contralateral breast cancer (CBC) and whether it interacts with radiation therapy (RT) or chemotherapy in regard to CBC risk. The germline mutation frequency was assessed in 708 women with CBC and 1395 women with unilateral breast cancer (UBC) in the Women's Environment, Cancer and Radiation Epidemiology (WECARE) Study whose first primary breast cancer was diagnosed before age 55 years and during 1985--1999. Seven women with CBC (1.0%) and 10 women with UBC (0.7%) were CHEK2*1100delC variant carriers (rate ratio (RR)=1.8, 95% confidence interval (CI)=0.6-5.4 for CBC vs UBC). Carriers who received RT for their first breast cancer, compared with non-carriers not treated with RT, had an RR of developing CBC of 2.6 (95% CI=0.8-8.7). We found no significant associations between the CHEK2*1100delC mutation and CBC overall or among those treated with RT. However, the sampling variability was such that modest increases in risk could not be excluded. Nonetheless, because this is a rare mutation, it is unlikely to explain a major fraction of CBC in the population.

Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA.

BACKGROUND: Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO(+)T cell compartment. OBJECTIVE: To develop an ARMS assay for plasma virions and to investigate the expression of resistance mutations (103N and 184V) and dynamic interactions between proviral DNA and plasma virions. STUDY DESIGN: A clinical cross-sectional study, including 11 patients on lamivudine efavirenz and/or nevirapine therapy. The viral populations were determined by an assay based on real-time PCR and amplification refractory mutation system (ARMS). RESULTS: The 103N and 184V mutations were not detected in patients with stable low viremia. Patients previously exposed to mono or dual therapy often carried minor viral populations of either one or both mutations in plasma. The viral population with linked mutations (103N and 184V) was detected in two patients after more than 2 years of non-NNRTI HAART. CONCLUSION: The ARMS assay is useful for detecting viral quasi-species containing efavirenz and lamivudine resistant mutations in plasma virions and in proviral DNA. Data suggest an unequal distribution of linked-mutation populations in plasma and CD45RO(+)T cells. Furthermore, the linked 103N-184V mutation may be more fit than the single 184V mutation and this linked population emerges rapidly under inadequate drug pressure.

Leptin acts as a mitogenic and antiapoptotic factor for colonic cancer cells.

BACKGROUND: Obesity is associated with increased levels of leptin. The mitogenic actions of leptin have been identified in various cell types. Because obesity may be a risk factor for colonic cancer, the proliferative and antiapoptotic effects of leptin on colonic cancer cells and the role of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) signalling were investigated. METHODS: Three human colonic cancer cell lines (T(84), HT29/Cl.19A and Caco-2) were treated with leptin. Cell proliferation was measured using the XTT colorimetric assay and apoptosis by a cell death enzyme-linked immunosorbent assay. Inhibitors of MAPK and PI3-K were used to evaluate the role of these signalling pathways. Phosphorylation of the downstream components extracellular signal-regulated kinase (ERK) 1/2 and Akt was detected by western blotting. RESULTS: Leptin increased cell number in all cell lines in a dose-dependent manner and reduced the number of apoptotic cells in a cell line-dependent manner. Leptin also caused ERK1/2 and Akt phosphorylation. Pretreatment with inhibitors of MAPK and PI3-K inhibited these responses, attenuated the mitogenic action of leptin and abolished its antiapoptotic effects. CONCLUSION: Chronic increases in leptin concentration may enhance the growth of colonic cancers via MAPK and PI3-K pathways. These effects of leptin could provide a link between obesity and colonic cancer, and may represent a target for anticancer drug development.

Salivary gland and temporomandibular joint involvement in rheumatoid arthritis: relation to disease activity.

OBJECTIVES: To study temporomandibular joint (TMJ) involvement, salivary gland dysfunction and oral mucosal lesions in rheumatoid arthritis (RA), and to investigate the relationship to general disease activity. SUBJECTS AND METHODS: The TMJ dysfunction index (D(i)), mean salivary flow and disease activity score (DAS28), were calculated for 50 RA-patients, and 23 non-RA patients (controls). RESULTS: Median D(i) was 5.5 (range: 0-21) for the RA-patients compared with 2.0 (range: 0-9) for the controls (P < 0.0001). Pain on movement of the TMJ (P = 0.015), muscular pain (P = 0.006), TMJ pain (P = 0.019) and D(i) as a total (P = 0.009), significantly correlated with DAS28. Mean resting whole saliva (RWS) flow was 2.6 (s.d. 2.4) ml per 15 min for the RA-patients and 4.5 (s.d. 3.0) for the controls (P = 0.003). RWS correlated positively with haemoglobin (P = 0.021) and negatively with Westergren erythrocyte sedimentation rate (ESR) (P = 0.029). No major differences in frequency of oral mucosal lesions were seen between RA-patients and controls. CONCLUSIONS: Higher frequency of TMJ and salivary gland dysfunction in RA-patients compared with controls has been demonstrated. RA disease activity is associated with hyposalivation and TMJ dysfunction.

Characterization of ribosomal P autoantibodies in relation to cell destruction and autoimmune disease.

Autoantibodies against the ribosomal P proteins are related to cell death and tissue destruction and are frequently exhibited in patients with systemic lupus erythematosus (SLE). In an attempt to explore the effect of tissue destruction on the induction of anti-P autoantibodies, we searched for anti-P autoantibodies by enzyme-linked immunosorbent assay in 201 antinuclear antibody (ANA)-positive individuals, in 10 patients with treated kidney SLE and in 45 acute leukaemia patients undergoing intensive chemotherapy. The autoantibody reactivity was further characterized using one- and two-dimensional immunoblot analysis and immunofluorescence. Anti-P were detected in 5.5% (11/201) of ANA-positive individuals, but not in kidney-affected SLE patients or in patients with leukaemia. Seven of 11 anti-P-positive patients had SLE (3/11), primary Sjögrens's syndrome (1/11) and other autoimmune diseases (3/11). A relation between disease activity and anti-P was suggested by follow-up examinations in one SLE patient, supported by the absence of anti-P autoantibodies in the 10 treated kidney SLE patients. Anti-P autoantibodies were detected by immunoblot in one patient with SLE indicating anti-P2 predominance and in the patient with Sjögrens's syndrome indicating anti-P1 predominance. Diverging humoral responses in these ANA- and anti-P-positive patients were further illustrated by immunofluorescence, elucidating varying nuclear reactivity and anti-P pattern. The observation of anti-P in individuals with active autoimmune disease, but not in patients with chemotherapy-induced cell damage, suggests that anti-P antibodies are part of a specific disease process, and not elicited as a response to cell destruction per se.

Sjögren's syndrome in an out-patient clinic: classification of patients according to the preliminary European criteria and the proposed modified European criteria.

OBJECTIVES: To investigate whether out-patients with a clinical diagnosis of Sjögren's syndrome (SS) satisfied current preliminary European criteria for SS, and to determine the proportion of patients who satisfied the proposed modified European criteria for SS and thus had indication of an autoimmune process. METHODS: Out-patients with a clinical diagnosis of SS registered between 1 January 1999 and 1 November 2000 were included in the study. RESULTS: Of 203 patients with a clinical diagnosis of SS, 116 (57.1%) satisfied the current European criteria and 83 (40.9%) satisfied the proposed modified criteria. CONCLUSIONS: Sicca symptoms and signs may have a variety of causes. In our study only 40.9% of the patients with a clinical diagnosis of SS satisfied the proposed modified European criteria and had evidence of SS as an autoimmune condition. Our findings indicate that for patient populations with an established diagnosis of SS according to the preliminary European criteria, approximately one-third will lose the diagnosis according to the modified criteria.

Comparison of the effects of fish oil and olive oil on blood lipids and aortic atherosclerosis in Watanabe heritable hyperlipidaemic rabbits.

To compare the effects of fish oil and olive oil on the development of atherosclerosis in Watanabe heritable hyperlipidaemic (WHHL) rabbits, 6-week-old animals were given a daily dose (1.5 ml/kg body weight) of fish oil (n 10) or olive oil (n 10) by oral administration for 16 weeks. Plasma cholesterol and triacylglycerols were measured once monthly, and their concentrations in lipoproteins, together with susceptibility of LDL to oxidation were measured in vitro at the termination of the experiment. Aortic atherosclerosis was quantified biochemically and microscopically. After 4 weeks of treatment, and throughout the study thereafter, blood lipids were significantly (P < 0.05) lower in the fish-oil group than in the olive-oil group (cholesterol: 17.0 v. 30.3 mmol/l, triacylglycerols 2.97 v. 6.25 mmol/l, at termination). In the fish-oil group cholesterol was significantly lower in intermediate-density lipoproteins (2.69 v. 6.76 mmol/l) and VLDL (3.36 v. 11.51 mmol/l). Triacylglycerol levels of intermediate-density lipoproteins and VLDL in the fish-oil group were also significantly lower when compared with the olive-oil group (0.54 v 1.36 mmol/l and 0.92 v. 2.87 mmol/l respectively). No group differences were recorded for LDL- and HDL-cholesterol or triacylglycerol levels. A significantly higher oxidation of LDL was recorded 1 h after exposure to CuSO4 in the fish-oil group when compared with the olive-oil group (0.465 v. 0.202, arbitrary units). The following indicators of atherosclerosis development were significantly lower in the fish-oil group than in the olive-oil group: the cholesterol content (mg/g tissue) in the ascending aorta (29.8 v. 48.9), the intima:media value (4.81 v. 18.24) and the area of intima (0.10 v. 0.57 mm2) in the thoracic aorta. It was concluded that fish-oil treatment decreased blood lipids and the development of aortic atherosclerosis in WHHL rabbits when compared with olive-oil treatment.