[Recommendations for the Protocol of functional examination of the anorectal zone and disorders classification: the International Anorectal Physiology Working Group consensus and Russian real-world practice].

This manuscript summarizes consensus reached by the International Anorectal Physiology Working Group (IAPWG) for the performance, terminology used, and interpretation of anorectal function testing including anorectal manometry (focused on high-resolution manometry), the rectal sensory test, and the balloon expulsion test. Based on these measurements, a classification system for disorders of anorectal function is proposed. Aim to provide information about methods of diagnosis and new classification of functional anorectal disorders to a wide range of specialists general practitioners, therapists, gastroenterologists, coloproctologists all who face the manifestations of these diseases in everyday practice and determine the diagnostic and therapeutic algorithm. Current paper provides agreed statements of IAPWG Consensus and comments (in italics) of Russian experts on real-world practice, mainly on methodology of examination. These comments in no way intended to detract from the provisions agreed by the international group of experts. We hope that these comments will help to improve the quality of examination based on the systematization of local experience with the use of the methods discussed and the results obtained. Key recommendations: the International Anorectal Physiology Working Group protocol for the performance of anorectal function testing recommends a standardized sequence of maneuvers to test rectoanal reflexes, anal tone and contractility, rectoanal coordination, and rectal sensation. Major findings not seen in healthy controls defined by the classification are as follows: rectoanal areflexia, anal hypotension and hypocontractility, rectal hyposensitivity, and hypersensitivity. Minor and inconclusive findings that can be present in health and require additional information prior to diagnosis include anal hypertension and dyssynergia.

Angiopoietin-like 4 promotes osteosarcoma cell proliferation and migration and stimulates osteoclastogenesis.

BACKGROUND: Osteosarcoma is the most common primary bone cancer in children and young adults. It is highly aggressive and patients that present with metastasis have a poor prognosis. Angiopoietin-like 4 (ANGPTL4) drives the progression and metastasis of many solid tumours, but has not been described in osteosarcoma tissue. ANGPTL4 also enhances osteoclast activity, which is required for osteosarcoma growth in bone. We therefore investigated the expression and function of ANGPTL4 in human osteosarcoma tissue and cell lines. METHODS: Expression of ANGPTL4 in osteosarcoma tissue microarrays was determined by immunohistochemistry. Hypoxic secretion of ANGPTL4 was tested by ELISA and Western blot. Regulation of ANGPTL4 by hypoxia-inducible factor (HIF) was investigated using isoform specific HIF siRNA (HIF-1α, HIF-2α). Effects of ANGPTL4 on cell proliferation, migration (scratch wound assay), colony formation and osteoblastogenesis were assessed using exogenous ANGPTL4 or cells stably transfected with ANGPTL4. Osteoclastogenic differentiation of CD14+ monocytes was assessed by staining for tartrate-resistant acid phosphatase (TRAP), bone resorption was assessed by lacunar resorption of dentine. RESULTS: ANGPTL4 was immunohistochemically detectable in 76/109 cases. ANGPTL4 was induced by hypoxia in 6 osteosarcoma cell lines, under the control of the HIF-1α transcription factor. MG-63 cells transfected with an ANGPTL4 over-expression plasmid exhibited increased proliferation and migration capacity and promoted osteoclastogenesis and osteoclast-mediated bone resorption. Individually the full-length form of ANGPTL4 could increase MG-63 cell proliferation, whereas N-terminal ANGPTL4 mediated the other pro-tumourigenic phenotypes. CONCLUSIONS: This study describes a role(s) for ANGPTL4 in osteosarcoma and identifies ANGPTL4 as a treatment target that could potentially reduce tumour progression, inhibit angiogenesis, reduce bone destruction and prevent metastatic events.

Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure.

Cells, particularly mechano-sensitive musculoskeletal cells such as tenocytes, routinely encounter oxidative stress. Oxidative stress can not only stimulate tissue repair, but also cause damage leading to tissue degeneration. As diabetes is associated with increased oxidative damage as well as increased risk of tendon degeneration, the aim of this study was to determine if extracellular glucose levels alter the response of tendon cells to oxidative stress. Primary human tenocytes were cultured in either high (17.5 mM) or low (5 mM) glucose and treated with 100 µM hydrogen peroxide. In low glucose, peroxide-treated cells remained fully viable and collagen synthesis was increased, suggesting an anabolic response. In high glucose, however, peroxide treatment led to increased bim-mediated apoptosis. The activities of both forkhead box O (FOXO1) and p53 were required for upregulation of bim RNA expression in high glucose. We found that both p53-mediated inhibition of the bim repressor micro RNA (miR17-92) and FOXO1-mediated upregulation of bim transcription were required to permit accumulation of bim RNA. High glucose coupled with oxidative stress resulted in upregulation of miR28-5p, which directly inhibited expression of the p53 deacetylase sirtuin 3, resulting in increased levels of acetylated p53. In peroxide-treated cells in both high and low glucose, protein levels of acetylated FOXO1 as well as HIF1α (hypoxia-inducible factor 1α) were increased. However, under low-glucose conditions, peroxide treatment resulted in activation of p38, which inhibited FOXO1-mediated but promoted HIF1α-mediated transcriptional activity. In low glucose, HIF1α upregulated expression of sox9 and scleraxis, two critical transcription factors involved in establishing the tenocyte phenotype, and increased collagen synthesis. The switch from FOXO1-mediated (proapoptosis) to HIF1α-mediated (prodifferentiation) transcription occurred at an extracellular glucose concentration of 7 mM, a concentration equivalent to the maximum normal blood glucose concentration. Extracellular glucose has a profound effect on the cellular response to oxidative stress. A level of oxidative stress normally anabolic may be pathological in high glucose.

Investigation of osteoclastogenic signalling of the RANKL substitute LIGHT.

LIGHT (TNFSF14) is a member of the TNF superfamily and is known to substitute for RANKL to induce osteoclast differentiation. LIGHT binds HVEM and LTßR, but it is not known whether these receptors play a role in osteoclast formation or whether LIGHT acts via RANKL signalling pathways. We found that both RANKL and LIGHT strongly induced phosphorylation of Akt and NFκB but not JNK in mouse osteoclast precursor cells. The addition of an Akt inhibitor showed decreased osteoclast differentiation and resorption mediated by both RANKL and LIGHT. RT-PCR and FACS analysis showed that CD14(+) human osteoclast precursors expressed HVEM and LTßR; expression levels of HVEM increased in the course of osteoclastogenesis and a decrease in LIGHT expression was associated with an increase in HVEM suggesting that there is a feedback loop related to this receptor. Our findings show that LIGHT is not inhibited by the soluble RANKL receptor OPG and that LIGHT is a potent osteoclastogenesis factor that activates the Akt, NFκB and JNK pathways.

Chondroclasts are mature osteoclasts which are capable of cartilage matrix resorption.

Multinucleated cells termed chondroclasts have been observed on the deep surface of resorbed hyaline cartilage but the relationship of these cells to macrophages and osteoclasts and their role in rheumatoid arthritis (RA) and other arthritic conditions is uncertain. Multinucleated cells in RA and other arthritic conditions showing evidence of cartilage resorption were characterised immunohistochemically for expression of macrophage/osteoclast markers. Mature human osteoclasts formed from circulating monocytes and tissue macrophages were cultured for up to 4 days on slices of human cartilage and glycosaminoglycan (GAG) release was measured. Multinucleated cells resorbing unmineralised cartilage were seen in osteoarthritis, RA, septic arthritis, avascular necrosis and in four cases of giant cell tumour of bone that had extended through the subchondral bone plate. Chondroclasts expressed an osteoclast-like phenotype (TRAP+, cathepsin K+, MMP9+, CD14-, HLA-DR-, CD45+, CD51+ and CD68+). Both macrophages and osteoclasts cultured on cartilage released GAG. These findings indicate that chondroclasts have an osteoclast-like phenotype and that mature human osteoclasts are capable of cartilage matrix resorption. Resorption of unmineralised subchondral cartilage by chondroclasts and macrophages can be a feature of joint destruction in inflammatory and non-inflammatory arthropathies as well as inflammatory and neoplastic subchondral bone lesions.

In vitro generation of mature human osteoclasts.

Mononuclear precursors of human osteoclasts are found in the CD14(+) monocyte fraction of circulating peripheral blood mononuclear cells (PBMCs). It is possible to generate osteoclasts in vitro from PBMCs cultured with macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand. In these cultures, however, it is not possible to distinguish the effect of a specific agent on osteoclast resorption activity as opposed to osteoclast differentiation. To produce a population of mature human osteoclasts to study osteoclast lacunar resorption specifically, we cultured CD14(+) human monocytes on hydrophobic dishes in order to generate and maintain osteoclasts in suspension prior to culturing them on coverslips and dentine slices. Multinucleated cells formed in these cultures expressed vitronectin receptor, tartrate-resistant acid phosphatase, and cathepsin K. These cells also produced F-actin rings and were capable of extensive lacunar resorption on dentine slices after 24 h in culture. Lacunar resorption was inhibited by calcitonin and zoledronate but not by osteoprotegerin. This method of generating a highly enriched population of mature human osteoclasts should provide a valuable means of specifically assessing the effect of molecular factors (e.g., cytokines, growth factors, hormones) and therapeutic agents on osteoclast resorption activity.

RANKL-independent human osteoclast formation with APRIL, BAFF, NGF, IGF I and IGF II.

Non-canonical pathways of osteoclastogenesis have been described in which several cytokines are able to substitute for RANKL. These cytokines are few in number and their role(s) in pathological bone resorption has not been ascertained. We have identified five additional cytokines, APRIL, BAFF, NGF, IGF I and IGF II, that can induce RANKL-independent osteoclastogenesis. All five cytokines induced both osteoclast differentiation and activation with respect to the formation of significant numbers of TRAP(+) and VNR(+) multinucleated cells that were capable of resorbing bone. The number of TRAP(+) multinucleated cells that formed was in the range of 40-75% of that supported by MCSF plus RANKL. Resorption was at a similar level to that induced by the other known RANKL substitutes TNFα, IL-6 and TGF-ß. The addition of osteoprotegrin, the endogenous decoy receptor of RANKL, revealed that this resorption was independent of RANKL. APRIL, BAFF, IGF I and IGF II were found to be expressed in giant cell tumour of bone. IGF I and IGF II demonstrated very strong expression in the stromal cell population of all tumour samples. This data suggests that non-canonical osteoclastogenesis plays a role in both normal and pathological bone resorption.

Tendinopathy and tears of the rotator cuff are associated with hypoxia and apoptosis.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff. We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1alpha (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1alpha regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis). The HIF-1alpha expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears. These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

Internal iliac artery angioplasty and stenting: an underutilized therapy.

BACKGROUND: Internal iliac artery (IIA) stenosis is a common finding in patients undergoing angiography. In patients with localized thigh and buttock claudication, endovascular treatment of an isolated IIA stenosis may lead to symptomatic improvement. METHODS: We retrospectively reviewed the records of nine patients who underwent IIA intervention for symptomatic thigh/buttock claudication. Patient demographics, angiographic status of both IIAs, and technical success were assessed by chart and angiogram review. Symptom relief was considered a successful outcome. RESULTS: Nine patients underwent unilateral or bilateral IIA angioplasty and/or stenting. There was a 100% technical success rate, and there were no complications. Six patients underwent a bilateral intervention and three underwent unilateral intervention. Fifteen arteries were treated. Seven arteries were treated with angioplasty, two with angioplasty and stenting, and six with primary stenting. Of the nine patients treated, seven had symptomatic relief from their claudication. Mean follow-up was 1 month. CONCLUSION: Percutaneous angioplasty and stenting of the IIA is technically feasible and safe. In patients who present with isolated proximal thigh and buttock claudication, IIA occlusive disease should be considered as an etiology. A majority of patients undergoing intervention report symptomatic improvement. Percutaneous intervention of the IIA has not been reported previously and should be an endovascular treatment option given its low morbidity and success rate. Also, there may be a beneficial role for IIA intervention in those patients undergoing unilateral IIA embolization during the course of endovascular aneurysmorrhaphy.

Canonical and non-canonical pathways of osteoclast formation.

Physiological and pathological bone resorption is mediated by osteoclasts, multinucleated cells which are formed by the fusion of monocyte / macrophage precursors. The canonical pathway of osteoclast formation requires the presence of the receptor activator for NFkappaB ligand (RANKL) and macrophage colony stimulating factor (M-CSF). Non-canonical pathways of osteoclast formation have been described in which cytokines / growth factors can substitute for RANKL or M-CSF to induce osteoclast formation. Substitutes for RANKL include LIGHT, TNFalpha and interleukins 6, 11 and 8. M-CSF substitutes include vascular endothelial growth factor (VEGF), placental growth factor (PlGF), FLt-3 ligand and hepatocyte growth factor (HGF). These growth factors can also influence canonical (RANKL / M-CSF-induced) osteoclast formation. Both canonical and non-canonical pathways of osteoclast formation play a role in the formation of osteolytic lesions where there is increased osteoclast formation and activity, such as in giant cell tumour of bone.

Hypoxia-inducible factor is expressed in giant cell tumour of bone and mediates paracrine effects of hypoxia on monocyte-osteoclast differentiation via induction of VEGF.

Hypoxia is an important regulator of bone biology and stimulates osteoclast differentiation from monocytic precursors. Hypoxia-inducible factor (HIF) is a key pro-tumourigenic transcription factor mediating pathways of hypoxia-inducible gene expression. We have described expression of HIF-1alpha and HIF-2alpha in the multi-nucleated, osteoclast-like giant cells and the mononuclear stromal component of giant cell tumour of bone (GCTB), a locally osteolytic primary bone tumour. HIF induction was observed in culture in the osteoblastic MG-63 cell line, primary GCTB stromal cells, and monocyte-derived osteoclasts following stimulation with hypoxia (0.1% O2) or the osteoclastogenic cytokines hepatocyte growth factor (HGF) and macrophage colony-stimulating factor (M-CSF). This was accompanied by increased expression of the downstream target genes Bcl-2/adenovirus E1B 19 kD-interacting protein 3 (BNIP3), Glut-1, and vascular endothelial growth factor (VEGF). As VEGF can substitute for M-CSF to support osteoclastogenesis in the presence of receptor activator for nuclear factor kappaB ligand (RANKL), we assessed the effect of MG-63 hypoxic conditioned media on osteoclast differentiation. In the presence of RANKL, hypoxic conditioned media induced the formation of active osteoclasts, as assessed from the numbers of TRAP-positive multi-nucleated cells and the area of lacunar bone resorption, which was inhibited by co-incubation with a neutralizing anti-VEGF antibody. Targeted siRNA ablated HIF-1alpha and/or HIF-2alpha expression in MG-63 cells and reduced hypoxic secretion of VEGF. Hypoxic conditioned media from cells treated with siRNA for (HIF-1alpha + HIF-2alpha) produced a significant decrease in osteoclast number (p < 0.005) and activity (p < 0.05) in comparison with the scrambled siRNA control. These results suggest that local hypoxia could indirectly influence osteoclastogenesis via autocrine and paracrine secretion of VEGF under the control of HIF. This is potentially an important mechanism of pathogenesis for GCTB and other osteolytic lesions.

Novel aspects of signaling and ion-homeostasis regulation in immunocytes. The TRPM ion channels and their potential role in modulating the immune response.

In just a few years, the discovery and subsequent characterization of several members of the TRPM family of cation channels have provided us with surprising new insights into unknown aspects of cellular ion-homeostasis regulation. This includes reports about ADP-ribose functioning as a novel intracellular second messenger and gating molecule of the Ca(2+)-permeable TRPM2 channel, studies demonstrating the central role of mouse TRPM5 in taste signaling, as well as the unexpected involvement of TRPM6 and TRPM7 in regulating Mg(2+)-homeostasis, or the cool properties of TRPM8 acting as a cold and menthol sensor in sensory neurons. At least four of the eight known TRPM proteins have been shown to be present in the immune context: TRPM1 (melastatin), TRPM2, TRPM4 and TRPM7. Although we currently lack animal models allowing a detailed assessment of the potential involvement of TRPM family members in modulating the immune response, the powerful combination of molecular and cellular biology, biochemistry, and electrophysiology have provided the first clues as to how these molecules could contribute to immunity.

Redesigning postnatal care: a randomised controlled trial of protocol-based midwifery-led care focused on individual women's physical and psychological health needs.

OBJECTIVES: To develop, implement and test the cost-effectiveness of redesigned postnatal care compared with current care on women's physical and psychological health. DESIGN: A cluster randomised controlled trial, with general practice as the unit of randomisation. Recruited women were followed up by postal questionnaire at 4 and 12 months postpartum and further data collected from midwife and general practice sources. SETTING: Thirty-six randomly selected general practice clusters in the West Midlands Health Region, UK. PARTICIPANTS: All women expected to be resident within recruited practices for postnatal care were eligible for inclusion. Attached midwives recruited 1087 women in the intervention and 977 in the control practice clusters. INTERVENTIONS: The systematic identification and management of women's health problems, led by midwives with general practitioner contact only when required. Symptom checklists and the Edinburgh Postnatal Depression Scale (EPDS) were used at various times to maximise the identification of problems, and individual care and visit plans based on needs. Evidence-based guidelines were used to manage needs. Care was delivered over a longer period. MAIN OUTCOME MEASURES: Women's health at 4 and 12 months, assessed by the Physical and Mental Component Scores (PCS and MCS) of the Short-Form 36 (SF-36) and the EPDS. Women's views about care, reported morbidity at 12 months, health service usage during the year, 'good practice' indicators and health professionals' views about care were secondary outcomes. RESULTS: At 4 and 12 months postpartum the mean MCS and EPDS scores were significantly better in the intervention group and the proportion of women with an EPDS score of 13+ (indicative of probable depression) was significantly lower relative to controls. The physical health score (PCS) did not differ. Health service usage was significantly less in the intervention group as well as reported psychological morbidity at 12 months. Women's views about care were either more positive or did not differ. Intervention midwives were more satisfied with redesigned care than control midwives were with standard care. Intervention care was cost-effective since outcomes were better and costs did not differ substantially. CONCLUSIONS: The redesigned community postnatal care led by midwives and delivered over a longer period, resulted in an improvement in women's mental health at 4 months postpartum, which persisted at 12 months and at equivalent overall cost. It is suggested that further research should focus on: the identification of postnatal depression through screening; whether fewer adverse longer term effects might be demonstrated among the children of the women who had the intervention care relative to the controls; testing interventions to reduce physical morbidity, including studies to validate measures of physical health in postpartum women. Further research is also required to investigate appropriate postnatal care for ethnic minority groups.

Effects of redesigned community postnatal care on womens' health 4 months after birth: a cluster randomised controlled trial.

BACKGROUND: Much postpartum physical and psychological morbidity is not addressed by present care, which tends to focus on routine examinations. We undertook a cluster randomised controlled trial to assess community postnatal care that has been redesigned to identify and manage individual needs. METHODS: We randomly allocated 36 general practice clusters from the West Midlands health region of the UK to intervention (n=17) or control (19) care. Midwives from the practices recruited women and provided care. 1087 (53%) of 2064 women were in practices randomly assigned to the intervention group, with 977 (47%) women in practices assigned to the control group. Care was led by midwives, with no routine contact with general practitioners, and was extended to 3 months. Midwives used symptom checklists and the Edinburgh postnatal depression scale (EPDS) to identify health needs and guidelines for the management of these needs. Primary outcomes at 4 months were obtained by postal questionnaire and included the women's short form 36 physical (PCS) and mental (MCS) component summary scores and the EPDS. Secondary outcomes were women's views about care. Multilevel analysis accounted for possible cluster effects. FINDINGS: 801 (77%) of 1087 women in the intervention group and 702 (76%) of 977 controls responded at 4 months. Women's mental health measures were significantly better in the intervention group (MCS, 3.03 [95% CI 1.53-4.52]; EPDS -1.92 [-2.55 to -1.29]; EPDS 13+ odds ratio 0.57 [0.43-0.76]) than in controls, but the physical health score did not differ. INTERPRETATION: Redesign of care so that it is midwife-led, flexible, and tailored to needs, could help to improve women's mental health and reduce probable depression at 4 months' postpartum.

Identification of differentially expressed genes in experimental models of the tumor microenvironment using differential display.

BACKGROUND: Genes upregulated within the tumour microenvironment represent potential targets for rational drug design. Most studies to date concentrate on the effects of hypoxia, although it is likely many genes are regulated by a more physiological combination of factors. MATERIALS & METHODS: Cells under conditions analogous to the normal and tumour microenvironments were isolated from the plateau-phase system and multicellular spheroids. Gene expression was analysed by differential display and confirmed by Northern blot or semiquantitative RT-PCR. RESULTS: p21-activated kinase (PAK1), a calmodulin-related mRNA, cytochrome oxidase subunit I and an H3.3 histone were upregulated within the in vitro tumour microenvironment, the last 3 within spheroids. CONCLUSIONS: Both models exhibit a range of microenvironmental parameters, although spheroids are more physiological with respect to the presence of extreme hypoxia and the formation of 3-dimensional interactions. We have shown that it is feasible to manipulate the spheroid system by serial trypsinisation to obtain reproducible cell populations for gene expression studies.

Hypoxia and oxidative stress in breast cancer. Hypoxia and tumourigenesis.

The microenvironmental hypoxia that arises as a consequence of the development of a solid tumour also acts to promote tumour growth. Hypoxia induces the expression of key components of the angiogenic and apoptotic signalling cascades, the glycolytic pathway and various cell-cycle control proteins. At the cellular level it mediates the infiltration and accumulation of tumour-associated macrophages within avascular tumour regions. Complex interactions between tumour cell and macrophage hypoxia-regulated gene products and their associated pathways form the basis for the hypoxic promotion of tumourigenesis and malignant progression.

Curcumin inhibits activation of Vgamma9Vdelta2 T cells by phosphoantigens and induces apoptosis involving apoptosis-inducing factor and large scale DNA fragmentation.

Curcumin, in addition to its role as a spice, has been used for centuries to treat inflammatory disorders. Although the mechanism of action remains unclear, it has been shown to inhibit the activation of NF-kappaB and AP-1, transcription factors required for induction of many proinflammatory mediators. Due to its low toxicity it is currently under consideration as a broad anti-inflammatory, anti-tumor cell agent. In this study we investigated whether curcumin inhibited the response of gammadelta T cells to protease-resistant phosphorylated derivatives found in the cell wall of many pathogens. The results showed that curcumin levels > or =30 microM profoundly inhibited isopentenyl pyrophosphate-induced release of the chemokines macrophage inflammatory protein-1alpha and -1beta and RANTES. Curcumin also blocked isopentenyl pyrophosphate-induced activation of NF-kappaB and AP-1. Commencing around 16 h, treatment with curcumin lead to the induction of cell death that could not be reversed by APC, IL-15, or IL-2. This cytotoxicity was associated with increased annexin V reactivity, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of apoptosis-inducing factor to the nucleus, and morphological evidence of nuclear disintegration. However, curcumin led to only large scale DNA chromatolysis, as determined by a combination of TUNEL staining and pulse-field and agarose gel electrophoresis, suggesting a predominantly apoptosis-inducing factor-mediated cell death process. We conclude that gammadelta T cells activated by these ubiquitous Ags are highly sensitive to curcumin, and that this effect may contribute to the anti-inflammatory properties of this compound.

An open-label pilot trial of cladibrine (2-cholordeoxyadenosine) in patients with primary sclerosing cholangitis.

Cladribine (2-chlorodeoxyadenosine) is a nucleoside analog with specific antilymphocytic activity that has been used in patients with a variety of lymphoid malignancies and autoimmune diseases. Primary sclerosing cholangitis (PSC) is a chronic hepatic autoimmune disorder of unknown etiology, thought to be mediated by biliary autoreactive cytotoxic lymphocytes. Because cladribine is an effective antilymphocytic drug, it may have potential disease-modifying activity in patients with PSC. We studied four patients with stages I and II PSC in an open-label pilot trial of 6 months' duration and 2 years' follow-up. Drugs were administered at 0.1 mg/kg/d subcutaneously for 5 days per monthly cycle for a total of 3 cycles. Patients evaluation included monthly liver panel test, cell count and lymphocytes subset, symptom severity score, posttreatment liver biopsy, and endoscopic retrograde cholangiopancreatography at 6 months and 2 years. All patients had a significant decrease in peripheral total lymphocyte (1,629 +/- 462 to 426 +/- 57; p < 0.01) and CD4 cell count (782 +/- 200 to 144 +/- 21; p < 0.05) with consequent decrease of CD4:CD8 ratio (3.82 +/- 1.96 to 1.84 +/- 0.69; p = 0.09). This was associated with a quantifiable decrease in the hepatic inflammatory infiltrate on liver biopsy. No significant changes were found in symptom scores, liver panel tests, or cholangiograms. The drug was well-tolerated and two of four patients reported remission of their inflammatory bowel disease symptoms. Cladribine decreases the hepatic lymphocytic inflammatory infiltrate in early-stage PSC, which did not translate into any short-term symptomatic, biochemical, or radiologic improvements. Further studies with long-term follow-up are needed to assess if this anti-inflammatory effect can modify the progression of disease.

Developmental expression of voltage-gated potassium channel beta subunits.

Expression of potassium channel beta subunits (Kvbeta) was determined in the developing mouse CNS using an antiserum against an amino acid sequence present in the C-terminus of Kvbeta1, Kvbeta2, and Kvbeta3. Using the anti-Kvbeta antiserum, we determined that Kvbeta expression is restricted to the spinal cord and dorsal root ganglia in the embryonic CNS. At birth, Kvbeta expression is detected in brainstem and midbrain nuclei, but was not detected in the hippocampus, cerebellum or cerebral cortex. During the first postnatal week, Kvbeta expression is present in hippocampal and cortical pyramidal cells and in cerebellar Purkinje cells. Expression of Kvbeta subunits reaches adult levels by the third postnatal week in all of the brain regions examined. A rabbit antiserum directed against a unique peptide sequence in the N-terminus of the Kvbeta1 protein demonstrates that this subunit displays a novel expression pattern in the developing mouse brain. Kvbeta1 expression is high at birth in all brain regions examined and decreases with age. In contrast, Kvbeta2 expression is low at birth and increases with age to reach adult levels by the third postnatal week. These findings support the notion that the differential regulation of distinct potassium channel beta subunits, in the developing mouse nervous system, may confer the functional diversity required to mediate both neuronal survival and maturation.

Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry.

Winged helix transcription factors play important roles in cellular differentiation and cell-specific gene expression. To define the role of the winged helix factor hepatocyte nuclear factor/forkhead homologue (HFH)-4, a targeted mutation was created in the mouse hfh-4 gene. No expression of HFH-4 was detected in hfh-4(-)/- mice by RNA blot analysis, in situ hybridization, or RT-PCR. hfh-4(-)/- mice were noted to have abnormalities of organ situs consistent with random determination of left-right asymmetry. In addition, a complete absence of cilia was noted in hfh-4(-)/- mice. The hfh-4 gene is thus essential for nonrandom determination of left-right asymmetry and development of ciliated cells. Homozygous mutant mice also exhibited prenatal and postnatal growth failure, perinatal lethality and, in some cases, hydrocephalus. RT-PCR revealed an absence of left-right dynein (lrd) expression in the embryonic lungs of hfh-4(-)/- mice, suggesting that HFH-4 may act by regulating expression of members of the dynein family of genes. The abnormalities in ciliary development and organ situs in hfh-4(-)/- mice are similar to those observed in human congenital syndromes such as Kartagener syndrome. Targeted mutation of hfh-4 thus provides a model for elucidating the mechanisms regulating ciliary development and determination of left-right asymmetry.