Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Expression and function of colonic epithelial K(v)LQT1 K+ channels

1. K(V)LQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential Defects in ion channels have been demonstrated in cardiac arrhythmia. This channel is inhibited potently by the chromanol 293B, The same compound has been shown to block cAMP-dependent electrolyte secretion in rat and human colon, Therefore, it was suggested that a K+ channel similar to K(V)LQT1 is expressed in the colonic epithelium. 2, In the present paper, expression of K(V)LQT1 and its function in colonic epithelial cells is described. Reverse transcription-polymerase chain reaction analysis of rat colonic mucosa demonstrated expression of K(V)LQT1 in both crypt cells and surface epithelium. When expressed in Xenopus oocytes, K(V)LQT1 induced a typical delayed activated K+ current. 3, As demonstrated, the channel activity could be further activated by increases in intracellular cAMP. These and other data support the concept that K(V)LQT1 is forming a component of the basolateral cAMP-activated Kf conductance in the colonic epithelium.

When state silence becomes persecution: The case of domestic violence refugees in minister for immigration and multicultural affairs v Khawar

In Minister for Immigration and Multicultural Affairs v Khawar, the Federal Court upheld a finding setting aside the refusal of the Refugee Review Tribunal (RRT) to grant a protection visa to a Pakistani woman - Tribunal's failure to consider the notion that state tolerance of violence for discriminatory reasons could amount to persecution under the definition of 'refugee' in the Convention Relating to the Status of Refugees.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4(+) V alpha 24 natural killer T cells expressing CD40L

Human V alpha 24 natural killer T (V alpha 24NKT) cells are activated by -glycosylceramide-pulsed dendritic cells (DCs) in a CDld-dependent and T-cell receptor-mediated manner. There are two major subpopulations of V alpha 24NKT cells, CD4(-) CD8(-) V alpha 24NKT and CD4(+) V alpha 24NKT cells. We have recently shown that activated CD4(-) CD8 V alpha 24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of V alpha 24NKT cells is currently limited. We aimed to investigate whether CD4(+) V alpha 24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4(+) V alpha 24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4(+) V alpha 24NKT cells, but not with resting CD4(+) V alpha 24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb, Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Va24NKT cells. The apoptosis of DCs from normal donors. triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4(+) V alpha 24NKT cells by virtue of apoptosis of DCs.

TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukema cells

Human V alpha 24NKT cells are activated by alpha -galactosylceramide (alpha -GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor-mediated manner. Here, we demonstrate that CD4(+)V alpha 24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) when the cells are activated by alpha -GalCer-pulsed dendritic cells but not prior to activation. We also show that myeloid that human activated CD4(+)V alpha 24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated V alpha 24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated V alpha 24NKT cells, or other strategies to increase activated V alpha 24NKT cells in vivo, may be of benefit to patients with AML M4.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.