Transcriptome analysis of ankylosing spondylitis patients before and after TNF-α inhibitor therapy reveals the pathways affected.

Tumor necrosis factor-α (TNF-α) inhibitors are highly effective in suppressing inflammation in ankylosing spondylitis (AS) patients, and operate by suppression of TFN-α and downstream immunological pathways. To determine the mechanisms of action of TNF-α inhibitors in AS patients, we used transcriptomic and bioinformatic approaches on peripheral blood mononuclear cells from AS patients pre and post treatment. We found 656 differentially expressed genes, including the genome-wide significant AS-associated genes, IL6R, NOTCH1, IL10, CXCR2 and TNFRSF1A. A distinctive gene expression profile was found between male and female patients, mainly because of sex chromosome-linked genes and interleukin 17 receptor C, potentially accounting for the differences in clinical manifestation and treatment response between the genders. In addition to immune and inflammation regulatory pathways, like intestinal immune network for IgA production, cytokine-cytokine receptor interaction, Ras signaling pathway, allograft rejection and hematopoietic cell lineage, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that infection-associated pathways (influenza A and toxoplasmosis) and metabolism-associated pathways were involved in response to TNF-α inhibitor treatment, providing insight into the mechanism of TNF-α inhibitors.

Epigenetic and gene expression analysis of ankylosing spondylitis-associated loci implicate immune cells and the gut in the disease pathogenesis.

Ankylosing spondylitis (AS) is a common immune-mediated arthropathy primarily affecting the spine and pelvis. Most AS patients have subclinical intestinal inflammation, suggesting the gut microbiome and the immune response play a role in pathogenesis. Susceptibility to AS is primarily genetic, and at least 114 susceptibility variants have been identified to date. We applied bioinformatic methods utilizing epigenetic and gene and protein expression data to identify the cell types through which AS-associated variants operate. Variants were enriched in transcriptionally regulated regions in monocytes, CD4+ and CD8+ T cells, natural killer cells, regulatory T cells and B cells and mucosa from the small intestine, sigmoid colon and rectum. Weak signals were detected in bone cells, consistent with bone disease being a secondary manifestation. RNA sequencing of blood cells from AS patients and controls identified differentially expressed genes. Interrogation of expression databases showed that the upregulated genes were enriched in monocytes and downregulated genes were enriched in CD8+ T cells and natural killer cells. Gene Ontology term enrichment analysis identified microbes and the gut in the aetiology of AS. These findings identify the key immune cell types that drive the disease, and further highlight the involvement of the gut microbiome in the pathogenesis of AS.

Does a more extensive mucosal excision prevent haemorrhoidal recurrence after stapled haemorrhoidopexy? Long-term outcome of a randomized controlled trial.

AIM: The study aimed in a multicentric randomized controlled trial to define the role of a more extensive mucosal resection on recurrence of mucosal prolapse in patients with Stage III haemorrhoids undergoing stapled haemorrhoidopexy. METHOD: In all, 135 patients were randomized to treatment with a PPH-01/03 (Ethicon EndoSurgery) or an EEA (Covidien) stapler. They were reviewed after a minimum follow-up of 4 years to determine the rate of recurrent mucosal prolapse and general condition (wellness evaluation score). Postoperative bowel dysfunction was assessed using the Rome III criteria. RESULTS: Eighty-seven (65%) of the 135 patients (48 in the EEA stapler group and 37 in the PPH group) were available for long-term follow-up. The two groups were comparable for age, gender and duration of follow-up (mean 49.3 ± 5.4 months and 49.0 ± 5.3 months respectively). In the EEA group, 11 (23%) patients had some degree of recurrent prolapse compared with 12 (32%) in the PPH group (P = 0.409). Persistence of anal bleeding was significantly higher in the PPH group (P = 0.04) while the postoperative Haemorrhoid Symptom Score was significantly better in the EEA group (1.73 ± 1.65 vs 3.17 ± 1.94, P < 0.001). The wellness evaluation score was significantly better in the EEA group (1.2 ± 1.27 vs 0.6 ± 1.0, P = 0.028). Furthermore, 7 (15%) of the patients in the EEA group complained of some evacuation disturbance compared with 13 (36%) in the PPH group (P = 0.021). CONCLUSION: The study failed to demonstrate any significant difference in the long-term recurrence rate of Stage III haemorrhoids using EEA or PPH. Nevertheless, use of the larger volume EEA provides better symptom resolution compared with PPH.

Flavonoids mixture (diosmin, troxerutin, hesperidin) in the treatment of acute hemorrhoidal disease: a prospective, randomized, triple-blind, controlled trial.

BACKGROUND: The role of a mixture of phlebotonics in the treatment of acute hemorrhoid crisis is investigated to test their efficacy. METHODS: One hundred and thirty-four consecutive patients with an acute hemorrhoidal crisis recruited in five colorectal units entered the study. Sixty-six of them were randomized to receive a mixture of diosmin, troxerutin and hesperidin (group A), and 68 a placebo (group B). The main symptoms, the use of oral painkillers and the Bristol scale score were recorded at each scheduled visit and compared using both Student's t test for independent samples and the ANOVA models for repeated measures. The presence of edema, prolapse and thrombosis were also recorded and compared using the Chi-square test. Furthermore, the trend of proportions during the time of the evaluations was assessed by the Chi-square test for linear trend. RESULTS: Pain, bleeding and the proportion of patients who reported persistence of edema and thrombosis decreased significantly after 12 days of treatment in group A. After 6 days, the number of paracetamol tablets taken by patients in group A was significantly lower than the amount of flavonoid mixture. CONCLUSIONS: The use of a mixture of diosmin, troxerutin and hesperidin is a safe and effective mean of managing symptoms of acute hemorrhoidal disease. Furthermore, in patients receiving treatment, there was faster control and lower persistence of edema and thrombosis.

Prospective multicenter observational trial on the safety and efficacy of LEVORAG® Emulgel in the treatment of acute and chronic anal fissure.

BACKGROUND: Anal fissure (AF) is a common cause of anal pain with a tendency not to heal spontaneously because of ischemia of the anoderm caused by sphincter spasm. Lateral internal sphincterotomy, while very effective, can cause fecal incontinence and chemical sphincterotomy by application of cream may have discouraging side effects and/or low efficacy. The aim of this prospective multicenter study was to evaluate the safety and effectiveness of a new medical treatment based on Emulgel cream, with emollient, soothing and protective agents, on AF healing. METHODS: Consecutive patients with AF treated in nine coloproctology units during 6 months entered the study on topical treatment with Levorag(®) Emulgel (THD S.p.A Correggio (RE), Italy). Before treatment, they had a proctologic examination and pain was measured using a visual analog scale. THD Levorag(®) Emulgel was applied every 12 h for 40 days. Monitoring was scheduled at 10, 20 and 40 days. At time 0 and at the end of treatment, patients underwent anorectal manometry, if possible. RESULTS: Two hundred eighty-four AF patients were recruited (171 acute fissures). Complete healing was achieved in 47.9 % of the cases, an improvement in 31.0 % (global efficacy 78.9 %). In patients with acute fissure, the rate of efficacy was 89.4 % (complete healing: 64.3 %, improvement: 25.1 %), in those with chronic fissure the rate of efficacy was 62.8 % (complete healing: 23 %, improvement: 39.8 %), p < 0.001. Pain and resting anal pressure decreased significantly after treatment. CONCLUSIONS: Treatment with THD Levorag(®) Emulgel proved to be effective for the reepithelization of AF and the reduction of pain in the short term in about 80 % of patients.

Control of post-hemorrhoidectomy symptoms and wound healing by Triclosan: a randomized, double-blind, controlled trial.

AIM: Milligan-Morgan hemorrhoidectomy (MM) is still the most common treatment for grades III and IV hemorrhoids despite prolonged post-operative anal pain and wound healing. This multicenter, double blind, randomized, controlled trial was designed to assess the safety and the efficacy of anal wound cleansing with Triclosan (Proctocid®) in the control of symptoms and healing time after MM. METHODS: A total of 113 patients with grades III and IV hemorrhoids, undergoing open hemorroidectomy by diathermy or Ligasure vessel sealing device, were randomly assigned to Triclosan or sodium hypochlorite solution. All patients received analgesics and a fiber-rich diet after hemorrhoidectomy. Postoperative anal pain, bleeding and/or secretion and itch were assessed 7, 14 and 21 days after hemorrhoidectomy by a Visual Analogue Scale (VAS) and the day of complete re-epithelialization of anal wounds was recorded. RESULTS: Fifty-five patients were randomized for Triclosan treatment and 58 for the control drug. The two groups were comparable for demographics, severity of hemorrhoids and technique used for the hemorrhoidectomy. The comparison of days to get complete anal wound healing shows a trend of significance (P=0.05) for the Triclosan group. Bleeding and/or secretion, anal pain and itch were significantly better (P=0.003; P<0.0001 and P=0.01, respectively). CONCLUSION: Triclosan solution for the treatment of post-hemorrhoidectomy wounds is safe and improves the control of post-operative symptoms and wound healing time compared to sodium hypochlorite.

Crim1 has an essential role in glycogen trophoblast cell and sinusoidal-trophoblast giant cell development in the placenta.

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1(KST264), we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1(KST264/KST264) mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1(KST264) allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1(KST264/KST264) placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Audit of the care of the dying in a network of hospitals and institutions in Queensland.

BACKGROUND: Most Australians die in institutions and there is evidence to suggest that the care of these patients is not always optimal. Care pathways for the dying have been designed to transfer benchmarked hospice care to other settings (e.g. acute hospitals and residential age-care facilities) by defining goals of best care, providing guidelines to provide that care and documenting outcome. METHOD: A retrospective audit was undertaken across a network of health-care institutions in Queensland. The 18 goals considered essential for the care of the dying within the Liverpool Care Pathway were taken as a benchmark. Documentation of achievement of each of these goals was sought. RESULTS: The notes of 160 patients who had died in eight institutions (four hospitals, three hospices, one nursing home) were reviewed. Several areas for improvement were identified, particularly in those goals relating to communication, resuscitation orders and care after death. Few units documented the provision of written information to families. Most patients were prescribed medications in anticipation of pain and agitation but less were prescribed drugs for other common symptoms in the dying. Most of the goals were achieved in a higher percentage of cases in hospice units. Marked differences in practice were noted between different institutions. CONCLUSION: The audit identified several aspects in the care of the terminally ill that could be improved. End-stage pathways may provide a model for improving the care of patients dying in hospitals and institutions in Australia.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18.

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.

Mice null for sox18 are viable and display a mild coat defect.

We have previously shown that Sox18 is expressed in developing vascular endothelium and hair follicles during mouse embryogenesis and that point mutations in Sox18 are the underlying cause of cardiovascular and hair follicle defects in ragged (Ra) mice. Here we describe the analysis of Sox18(-/-) mice produced by gene targeting. Despite the profound defects seen in Ra mice, Sox18(-/-) mice have no obvious cardiovascular defects and only a mild coat defect with a reduced proportion of zigzag hairs. A reduction in the amount of pheomelanin pigmentation in hair shafts was also observed; later-forming hair follicles showed a reduced subapical pheomelanin band, giving Sox18(-/-) mice a slightly darker appearance than Sox18(+/+) and Sox18(+/-) siblings. Sox18(-/-) mice are viable and fertile and show no difference in the ability to thrive relative to littermates. Because of the mild effect of the mutation on the phenotype of Sox18(-/-) mice, we conclude that the semidominant nature of the Ra mutations is due to a trans-dominant negative effect mediated by the mutant SOX18 proteins rather than haploinsufficiency as has been observed for other SOX genes. Due to the similarity of SOX18 to other subgroup F SOX proteins, SOX7 and -17, and the overlap in expression of these genes, functional redundancy amongst these SOX proteins could also account for the mild phenotype of Sox18(-/-) mice.

Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice.

Analysis of classical mouse mutations has been useful in the identification and study of many genes. We previously mapped Sox18, encoding an SRY-related transcription factor, to distal mouse chromosome 2. This region contains a known mouse mutation, ragged (Ra), that affects the coat and vasculature. Here we have directly evaluated Sox18 as a candidate for Ra. We found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. Furthermore, we found no recombination between Sox18 and Ra in an interspecific backcross segregating for the Ra phenotype. We found point mutations in Sox18 in two different Ra alleles that result in missense translation and premature truncation of the encoded protein. Fusion proteins containing these mutations lack the ability to activate transcription relative to wild-type controls in an in vitro assay. Our observations implicate mutations in Sox18 as the underlying cause of the Ra phenotype, and identify Sox18 as a critical gene for cardiovascular and hair follicle formation.

The UTX gene escapes X inactivation in mice and humans.

We recently have identified a ubiquitously transcribed mouse Y chromosome gene, Uty , which encodes a tetratricopeptide repeat (TPR) protein. A peptide derived from the UTY protein confers H-Y antigenicity on male cells. Here we report the characterization of a widely transcribed X-linked homologue of Uty , called Utx , which maps to the proximal region of the mouse X chromosome and which detects a human X-linked homologue at Xp11.2. Given that Uty is ubiquitously transcribed, we assayed for Utx expression from the inactive X chromosome (Xi) in mice and found that Utx escapes X chromosome inactivation. Only Smcx and the pseudoautosomal Sts gene on the mouse X chromosome have been reported previously to escape inactivation. The human UTX gene was also found to be expressed from Xi. We discuss the significance of these data for our understanding of dosage compensation of X-Y homologous genes in humans and mice.

An H-YDb epitope is encoded by a novel mouse Y chromosome gene.

Rejection of male tissue grafts by genotypically identical female mice has been explained by the existence of a male-specific transplantation antigen, H-Y (ref. 1), but the molecular nature of H-Y antigen has remained obscure. Hya, the murine locus controlling H-Y expression, has been localized to delta Sxrb, a deletion interval of the short arm of the Y chromosome. In mice, H-Y antigen comprises at least four distinct epitopes, each recognized by a specific T lymphocyte clone. It has recently been shown that one of these epitopes, H-YKk, is a peptide encoded by the Y-linked Smcy gene, presented at the cell surface with the H-2Kk major histocompatibility complex (MHC) molecule. However, deletion mapping and the analysis of variable inactivation of H-Y epitopes has suggested that the Hya locus may be genetically complex. Here we describe a novel mouse Y chromosome gene which we call Uty (ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome). We identify the peptide WMHHNMDLI derived from the UTY protein as an H-Y epitope, H-YDb. Our data formally demonstrate that H-Y antigen is the product of more than one gene on the Y chromosome.

Targeted disruption of the Wnt2 gene results in placentation defects.

Wnt genes have been implicated in a range of developmental processes in the mouse including the patterning of the central nervous system and limbs. Reported here for the first time is the expression of Wnt2 in the early heart field of 7.5-8.5 dpc (days post-coitum) mouse embryos, making Wnt2 a potentially useful gene marker for the early stages of heart development. Expression was also detected in the allantois from 8.0 dpc and at later stages in the placenta and umbilicus. Mice deficient in Wnt2, generated by gene targeting, displayed runting and approximately 50% died perinatally. Histological analysis revealed alterations in the size and structure of placentas from these mice from 14.5 dpc. The placental defects were associated primarily with the labyrinthine zone and included oedema and tissue disruption and accumulation of maternal blood in large pools. There was also an apparent decrease in the number of foetal capillaries and an increase in the amount of fibrinoid material in the Wnt2 mutant placentas. These results suggest that Wnt2 is required for the proper vascularisation of the mouse placenta and the placental defects in Wnt2-deficient mice result in a reduction in birthweight and perinatal lethality.