Chemokine expression and leucocyte infiltration in Sjogren's syndrome

OBJECTIVE: To investigate the expression and source of chemokines in minor salivary gland biopsies (MSGs) in patients with Sjogren's syndrome (SS). METHODS: Immunohistochemical analysis was used to determine the pattern of chemokine expression in MSGs from patients with (n=6) and without (n=5) SS, as well as to examine the phenotype of both resident and infiltrating cells expressing chemokines. RESULTS: Significant differences in the number of infiltrating mononuclear (MN) cells in patients with and without SS were noted. Ductal epithelial cells of SS biopsies expressed significantly increased levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted). Biopsies from patients with SS showed that MIP-1beta was expressed by 51% of infiltrating cells, while 41% expressed MIP-1alpha, whereas 22 and 7% expressed RANTES and IL-8, respectively. CONCLUSION: Chemokines expressed by ductal epithelial cells may attract circulating leucocytes, in particular CD4+ T cells, towards the site of inflammation, thereby orchestrating the influx of MN cells characteristically seen in MSGs in SS. Chemokines may be induced directly by a putative triggering agent for SS, or secondary to the release of pro-inflammatory cytokines produced by epithelial cells. These findings further implicate epithelial cells as playing a major role in the pathogenesis of SS and implicate chemokines in the leucocyte recruitment in this setting.      

Spinning top urethra: not a normal variant

Spinning top urethra (STU) is a term used to describe a widened posterior urethra seen mainly in girls. It is commonly regarded as a normal variant. The authors studied 30 girls with STU using videourodynamics. Twenty-eight showed bladder instability; 21, a congenital wide bladder neck anomaly; and 20, both instability and a wide bladder neck. One patient had a sensitive bladder. All patients had a urodynamic abnormality. The authors believe that the STU is nearly always an indication of bladder instability or wide bladder neck anomaly. The most common mechanism for the dilatation of the posterior urethra is that unstable contractions are resisted by a voluntary increase in distal sphincter tension so as to prevent leakage of urine. The resulting pressure rise produces distention of the posterior urethra, which will be maximal in subjects with a weak bladder neck mechanism as in the congenital wide bladder neck anomaly. The authors believe that STU is seldom if ever a normal variant.     

Thrombin-induced secretion of serotonin from platelets can occur in seconds

The platelet release reaction was studied by a new quenched-flow approach. Platelets labeled with 14C-serotonin were reacted for short times (up to 5 sec) with thrombin and then quenched with glutaraldehyde or paraformaldehyde. Serotonin secretion began within 1 sec and was nearly complete by 4 sec. Aggregation recorded by a resistive-particle counter was similarly fast. Therefore, the quenched-flow system reveals that serotonin secretion can occur more rapidly than estimated in earlier studies.     

Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia

AIM： To identify the distribution of hepatitis B virus （HBV） subgenotype and basal core promoter （BCP） mutations among patients with HBV-associated liver disease in Indonesia.
METHODS： Patients with chronic hepatitis （CH, n =61）, liver cirrhosis （LC, n = 62）, and hepatocellular carcinoma （HCC, n = 48） were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing.
RESULTS： HBV genotype B （subgenotypes B2, B3, B4, 85 and B7） the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C （subgenotypes C1, C2 and C3） was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 （84.9%） and C1 （82.2%） were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 （84.9%） and adrq＋ （89.4%） were the most prevalent in HBV genotype B and C, respectively. Double mutation （A1762T/G1764A） in the BCP was significantly higher in LC （59.7%） and HCC （54.2%） than in CH （19.7%）, suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC （46.8%）, but lower in HCC （22.9%） and CH （18.0%）, suggesting that this mutation may be an indicator of cirrhosis.
CONCLUSION： HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.     

Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels

Background and purpose:

ClC-K kidney Cl⁻ channels are important for renal and inner ear transepithelial Cl⁻ transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above ∼1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.

Experimental approach:

We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects.

Key results:

Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation.

Conclusions and implications:

The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.     

Manipulation of liver regeneration with macrophages to influence the hepatic progenitor cell niche

Insufficient regeneration of the adult liver is believed to cause failure to recover from severe liver disease. An undifferentiated cell population with stem-cell-like qualities known as hepatic progenitor cells (HPCs) is hypothesised to have a central role in regeneration of the adult liver during massive or chronic liver disease. Stem cells in other organ systems are believed to reside in a specialised microenvironment or niche that supports their maintenance and function. The existence of a hepatic stem cell niche might provide a means of therapeutically manipulating endogenous HPCs in vivo as a regenerative therapy.To investigate the physiological potential of HPCs to regenerate the mammalian liver, we have established a novel model of hepatocellular injury and HPC activation using genetic manipulation of hepatocytes. After hepatocyte senescence and death in this model (AhCre Mdm2^flox), HPCs expand and bring about the complete regeneration of the liver parenchyma.We demonstrate that a stereotypical niche, consisting partly of macrophages, exists in both animal models and correlating human disease. Using cell tracking, we show active recruitment of extrahepatic macrophages into this niche during injury. In health, intravenous injection of macrophages results in macrophage engraftment to the liver niche, with subsequent HPC activation and changes to liver structure and function.Within the niche, macrophages use paracrine signalling to control both HPC proliferation and cell fate via TWEAK (tumour-necrosis-factor-like weak inducer of apoptosis) and the Wnt signalling pathway, respectively. After hepatocellular injury, macrophages ingest hepatocyte debris, and release Wnt which promotes HPC differentiation into hepatocytes. TWEAK is vital for HPC proliferation in the AhCre Mdm2^flox model of regeneration. Here, the absence of TWEAK signalling results in liver failure and mortality.This work has demonstrated for the first time the ability of a solid organ to fully regenerate in the adult mammal from progenitor cells, and additionally highlights mechanisms by which this process can be modulated by either small molecule or cell therapy.FundingUniversity of Edinburgh.     

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

Background and Purpose

The Ca²⁺-permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (–/–) mice, and the increases in [Ca²⁺]_i produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF.

Experimental Approach

Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca²⁺ concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists.

Key Results

Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and –/– mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca²⁺]_i in neurons from +/+ and –/– mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca²⁺ in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF.

Conclusions and Implications

TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist.     

Distress due to unwanted side-effects of prostate cancer treatment is related to impaired well-being (quality of life)

Based on traditional quality of life scales, it has been suggested that known side-effects of prostate cancer treatment do not influence the quality of life. The present authors have developed an alternative approach to quality of life assessment applying epidemiological methods. Using a self administered questionnaire, we investigated 431 prostate cancer patients and an age stratified sample of 435 randomly selected men. Patients reporting any level of distress due to waning sexual functions (66%) or urine or bowel symptoms (38%), reported a lower psychological well-being compared to patients not reporting these symptoms or patients not distressed by their symptoms. Our results stress that an intact sexual and urinary and bowel functions are important for the quality of life among elderly men with or without prostate cancer.