Spatiotemporal regulation of galectin-1-induced T-cell death in lamina propria from Crohn's disease and ulcerative colitis patients.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p < 0.05) as revealed by flow cytometry using peanut agglutinin (PNA) binding and assessing dysregulation of the core-2 ß 1-6-N-acetylglucosaminyltransferase 1 (C2GNT1), an enzyme responsible for elongation of core 2 O-glycans. Consequently, Gal-1 binding was attenuated in CD3+CD4+ and CD3+CD8+ LP T-cells isolated from inflamed sites (p < 0.05). Incubation with recombinant Gal-1 induced apoptosis of LP CD3+ T-cells isolated from control subjects and non-inflamed areas of IBD patients (p < 0.05), but not from inflamed areas. In conclusion, our findings showed that transient regulation of the O-glycan profile during inflammation modulates Gal-1 binding and LP T-cell survival in IBD patients.

Human Neutrophil Elastase Proteolytic Activity in Ulcerative Colitis Favors the Loss of Function of Therapeutic Monoclonal Antibodies.

PURPOSE: Proteases play an essential role in the pathophysiology of inflammatory bowel disease (IBD), contributing to the intestinal mucosal lesions through the degradation of the extracellular matrix and alteration of the barrier function. Ulcerative colitis (UC) is characterized by an extensive infiltrate of neutrophils into the mucosa and hence, increased proteolytic activity. Human neutrophil elastase (HNE) is a serine protease that has been reported to be increased in UC patients' intestinal mucosa. Based on our previous studies, we hypothesized that HNE might induce proteolytic degradation and loss of function of therapeutic monoclonal antibodies in IBD patients. PATIENTS AND METHODS: Elastase expression and elastinolytic activity were determined in mucosal explants from ulcerative colitis patients (n=6) and cultured ex vivo in the presence or absence of recombinant elafin. Enzymatic digestions of therapeutic monoclonal antibodies were performed using recombinant HNE and elafin. The integrity of the therapeutic antibodies was evaluated by immunoblotting and protein G binding assay, whereas their TNF-neutralizing activity was assessed with a reporter cell line. RESULTS: We found that HNE and its elastinolytic activity were increased in the gut mucosa of UC patients. We also demonstrated that HNE cleaved biological drugs, impairing the TNF-α neutralizing capacity of anti-TNF monoclonal antibodies. This proteolytic degradation was inhibited by the addition of the specific inhibitor, elafin. CONCLUSION: Our results suggest that the high level of proteolytic degradation by mucosal neutrophil elastase, along with a potential imbalance with elafin, contributes to the loss of function of biologic agents, which are currently used in patients with IBD. These findings might explain the non-responsiveness of UC patients to therapeutic monoclonal antibodies and suggest the potential beneficial concomitant use of elafin in this treatment.

A functional IL1RL1 variant regulates corticosteroid-induced sST2 expression in ulcerative colitis.

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.

The IL-33/ST2 axis: Role in health and disease.

IL-33, an IL-1 family member, is expressed by many cell types and can regulate gene transcription. IL-33 is released upon cell necrosis and the precursor form is enzymatically processed, and then drives inflammation as a damage-associated molecular pattern. The IL-33 receptor ST2, encoded by IL1RL1, is expressed as both a membrane-anchored receptor (ST2L) activated by IL-33, and as a soluble variant (sST2) that exhibits anti-inflammatory properties. The IL-33/ST2 axis is involved in the pathogenesis of atopic and autoimmune diseases, cancer, and central nervous system disorders. Here, we review recent findings on the role of the IL-33/ST2 axis in health and disease.

Trichuriasis: spontaneous and stimulated Th1/Th2 cytokine production by isolated T-cells - abstract

Although trichuriasis is the most prevalent known infection in the CARICOM countries, only a small proportion of infected children acquire intense infection, i.e., Trichuris Dysentry Syndrome (TDS). Hypotheses to be investigated are that there is a specific T-Cell anergy to the parasite in such hosts or that they have general predisposition to the "TH2" T-helper-cell response, which would be in keeping with the TDS local anaphylactic response we have shown by various techniques in our other reports. We investigated 3 groups of children: controls from a surgical private practice unlikely to have ever been significantly infected TDS cases. We used the ELISPOT technique with primary and secondary monoclonal antibodies to show cytokine production by cells separated from peripheral blood or colonic mucosal biopsies, expressing the final result in numbers of positive cells per 100,000 CD3+ cells (T-cells). Interferon-gamma detection was taken as indicative of the TH1 phenotype and Interleukin 4 (IL4) as indicative of TH2. Spontaneous production in short-term culture was compared with that after stimulation ionophore, staphylococcal enterotoxin B (superantigen) or T. trichiura excretory-secretory (ES) antigen. There was great variability in proportions of cytokine positive cells after culture, both spontaneously and after stimulation. There was also variability in the rank of potency of a given stimulant to "the T-cells of different children. ES antigen provoked the greatest IL4 response for one active TDS case, refuting the anergy hypothesis. The active cases had two highest productions (spontaneous) of interferon-gamma and (stimulated) of IL4. No significant difference in Th1/Th2 phenotypic profile emerged between the control and ex-TDS groups. We conclude that it is not innate character in T-helper-cell response to mitogen or antigen that accounts for predisposition to intense trichuriasis (AU)

Immediate hypersensitivity in colon of children with chronic Trichuris trichiura dysentery

There are few data on mucosal immune responses to intestinal helminths in human beings, especially those involving the IgE system, which is thought to be important in parasite expulsion. We sought evidence of an immediate hypersensitivity reaction in the colon of children with chronic dysentery due to Trichuris trichiura. 28 children with Trichuris dysentery syndrome (TDS) were compared with 16 control children (with no TDS or worms visible on colonoscopy). All children were aged 1-11 years. Rectal biopsy samples were taken before and after expulsion of the worms by means of mebendazole treatment. Children wtih TDS had significantly greater numbers than controls of mast cells (mean [SD] 10.9 [1.3] vs 3.9 [0.6] percent of all cells; p<0.0003) and of cells with surface IgE (median [range] 11.1 [7.5-11.6] vs 1.0 [0-1.5] percent; p<0.001) in the subepithelial region of the mucosa. On electronmicroscopy, degranulating mast cells were prominent in parasitised children. In culture, rectal biopsy samples from parasitised children showed high rates of spontaneous histamine release, but only low rates of antigen-specific release. After treatment, spontaneous histamine release was significantly reduced and antigen-specific histamine release could be provoked. Thus, an IgE-mediated immune mucosal response to a helminth infection does occur in human beings but is not sufficient to cause appreciable parasite expulsion. (Summary)

Tissue histamine release in rectal mucosa from trichuris dysentery syndrome children: infected, after worm expulsion, and during the recovery growth spurt - abstract

There is clearly some form of inflammatory response to the heavy load of epithelially invasive whipworms in the colonic and rectal mucosa in the fully developed trichuris dysentery syndrome (TDS). We have shown by extensive immunohistochemical studies of caecal biopsies that there is no evidence that this is "immunologically medicated" (essentially, T-cell-controlled) inflammation with mucosal structural damage. One major difference between infected and control children shown by immunohistochemistry, however, was a 10-fold increase in lamina propria cells with surface IgE, presumably mast-cells (MacDonald et al, CCMRC, 1989). Children with TDS were studied before treatment, 3 days after the completion of worm-expulsive chemotherapy, and 3 or 6 weeks later when they had shown a height gain of >0.7 percent over a 3-week interval. On each of these 3 occasions rectal mucosa was placed in ice-cold Tyrode's buffer for in vitro histamine release studies, either spontaneous (at 37§C) or following challenge with rabbit anti-human IgE or T.trichiura excretory-secretory (ES) product. Residual tissue histamine concentration was also assayed. Spontaneous histamine release tended to be high on the first biopsy (maximum 81 percent of total in 20 mn), and was not then capable of being boosted by anti-IgE or Trichuris ES. However, following worm expulsion (max 16 percent) and on the third biopsy (max 6 percent) there was a clear trend for spontaneous histamine release to be diminishing. In some specimens at the 2nd and 3rd stages Trichuris ES antigen could then provoke a massive discharge of intracellular histamine into the supernatant. These studies of cell histamine release have advanced our understanding of the inflammatory mechanism of this disease (Type I hypersensitivity) and have shown in some children a persisting worm-sensitive state of the rectal mucosa (AU)

Histopathology and immunohistochemistry of the caecum in children with the Trichuris dysentery syndrome

Caecal biopsy specimens from Jamaican children with the Trichuris dysentery syndrome (TDS) and age matched Jamaican controls were investigated by immunohistochemistry and by light microscopy. Biopsy specimens from all children (with TDS and controls) showed a mild to moderate increase in inflamatory cells. Except in the vicinity of the worm, where the epithlium was flattened, there was no other epithelial abnormality. Compared with controls, children with TDS had increased IgM lamina propria plasma cells and decreased intaepithelial T cells. There was also an increase in crypt epithelial cells proliferation. Lamina propia T cells (both activated and non-activated) were no more common in children with the Trichuris syndrome than controls. Epithelial cell HLA-DR and VLA-1 expression (which are increased in other colitides) were the same in both groups. Despite the presence of large worm burdens and chronic dysentery, therefore, only minor changes were seen in the caecal mucosa of children with TDS. (AU)

Tumour necrosis factor/cachectin may mediate growth suppression in children with Trichuris colitis - abstract

As we have argued before (Cooper et al, CCMRC, 1989) the association between chronic Trichuris colitis and linear growth retardation is specific: the variability in the degree of wasting, the pattern of post-treatment catch-up growth in which weight-for-height often remains constant or actually falls and the accelerated height velocity without a change in environment, all point to the existence of some endogenous growth-suppressing factor. A candidate for this factor is the cytokine and mediator of systemic inflammation tumour necrosis factor alpha (TNF), also known as cachectin. Thirteen children with the Trichuris dysentery syndrome had a mean height-for-age of -2.3 (SD 1.34) NCHS Z-scores. Plasma TNF was assayed by ELISA. 9/13 were positive (>20 pg ml-1). Values ranged from 0 to 99 pg/ml,-1, similar to those reported in acute but uncomplicated attacks of malaria. Controls: 10 paediatric surgery follow-up patients (height Z-score + 0.1) had plasma TNF < 20 pg ml-1; in 9 stunted but parasite-free children from the community (study of Grantham-Mcgregor) TNF was undetectable. Culture of colonic mucosal mononuclear cells from four of the Trichuris colitis children yielded higher concentrations of TNF in the supernatants than those of four non-Trichuris mild, non-specific colitis patients. This suggests that the source of the increased plasma TNF may be the lamina propria macrophages, increased in number in trichuriasis as we have already described (MacDonald et al, CCMRC. 1989) (AU)

Growth suppression in the Trichuris syndrome

The Trichuris Dysentery Syndrome (Ramsey, 1962) is an insidious, chronic condition which has clinical features similar to Chron's ileocolitis and ulcerative colitis, disease similarly associated with growth retardation. The attained heights and weights of 19 children at the time of diagnosis of intense, symptomatic Trichuris (whipworm) infection showed and average height of -2,4 Standard Deviation (Z) scores from the Tanner-Whitehouse median with weight, adjusted for height-age, -1.3 Z. We present data on the growth velocities of 11 of the children, in the half-year following worm explusion by mebendazole. These children returned to their home environment without food supplementation or close follow-up, but showed and average height velocity of + 5.5 Z and weight velocity (for height-age) of + 2.4 Z. Of 8 children with unequivocal height spurts only 3 had any weight spurt. We suggest that the pattern of catch-up growth points to the existence of some specific link between allergy or inflamation in the lower intestinal tract and suppression of linear growth, rather than to stunting due to general deprivation and undernutrition. (AU)

Growth suppression in the trichuris dysentery syndrome - abstract

There is a well-established, although poorly understood, association between inflammatory bowel disease and growth retardation. The trichuris dysentery syndrome (Ramsey, 1962) is an insidious, chronic condition which has clinical features similar to Crohn's colitis and ulcerative colitis. We present data on the attained heights and weights of 19 children at the time of diagnosis of intense, symptomatic Trichuris (whipworm) infection. Their ages ranged from 2 to 10 years and their histories of bowel symptoms from 6 months to 7 years. We further present data on the growth velocities of 10 of them in the half-year following worm expulsion by mebendazole. These children returned to their home environments without food supplementation or close follow-up. All measurements are converted to Standard Deviation Scores for age (denoted Z) based upon the Tanner-Whitehouse distance and velocity growth charts. Mean height was minus 2.6 Z (SD 1.4) and weight-for-height minus 1.5 Z (SD 1.0). Where bone age was obtained, it corresponded closely to the child's height age. Only 4 of the 19 had greater than minus 2 Z: the syndrome was thus predominantly associated with stunting rather than wasting. Following worm expulsion, usually accompanied by a therapeutic course of ferrous sulphate, height velocities averaged +6.8 Z (SD 3.6) and weight velocities +2.5 Z, (SD 4.6). All height velocities were greater than the standard for age, with 9 out of 10 greater than +2 Z, whereas only 4 weight velocities were greater than +1 Z, 6 values being the same as, or less than, the standard for children of that age or that height. We conclude that the Trichuris Dysentery Syndrome is associated more with suppression of linear growth than with loss of weight-for-height, and that the suppression is reversible upon expulsion of the worms - the symptoms then having resolved. Catch-up height growth can then achieve velocities as great as any recorded, including those that follow growth hormone therapy in pituitary deficiency (AU)

An immuno-histochemical study of the inflammatory cell infiltrate in the large intestine of Jamaican children with trichuris trichuria infection - abstract

The symptomatology of children with Trichuris Dysentry Syndrome (Ramsey, 1962) has much in common with that of idiopathic inflammatory bowel disease (Crohn's and ulcerative colitis). However, the pathology is confined to the mucosa and includes relatively little disturbance of the crypt architecture or epithelial histology. We undertook an immuno-histochemical study of the faecal mucosa of five heavily infected children with non specific, bloody diarrhoea (in whom such gross inflammatory changes as did exist were confined to the rectum and distal colon). Tissue obtained by colonoscopic biopsy was fixed in formalin and embedded in paraffin wax. Routine histological sections revealed only a non-specific increase in inflammatory cells in the lamina propria and failed to differentiate between trichuriasis and non-specific bloody diarrhoea. Specimens of the fixed tissue were transported to London where a panel of peroxidase-labelled antibodies was applied. Expressing immuno-stained cells as a percentage of total lamina propria cells, we showed marked difference between the Trichuris dysentery and control patients, summarised below using group means: Igm plasma cell, IgE-coated mast cell, Macrophages; TRICHURIS - 12.4, 9.6 33.3 respectively; NON-TRICHURIS - 7.2, 0.5, 8.9 respectively; SIGNIF. LEVEL - 0.05, 0.0001, 0.005 respectively. Further immuno-histochemistry on snap-frozen tissue will allow specification of the type and activation state of the cells involved in the inflammatory response to T. trichiura. It is our hope in due course to relate these changes to the symptomatology, both intestinal and extra-intestinal, of this important disease (AU)

Colonoscopy and studies of cellular immune function in children with geophagia and mucoid dysentery - abstract

Many West Indian children eat earth, of whom a number have frequent unformed stools with mucus; of these children, some are heavily infected by the whipworm, Trichuris trichiura. We have begun a study of the mucosal pathology in these children, and of their cellular immune function, both systemic and local. We performed 8 complete (i.e. to the caecum) colonoscopies on 5 children aged 3.1 to 4.8 years, of whom two had trichuriasis. Bowel preparation with senna, bisacodyl and magnesium sulphate and sedation with chlorpromazine, diazepam and pethidine were satisfactory in all cases. Chronic inflammation with crypt hyperplasia and lymphoid hyperplasia were seen in the initial mucosal biopsies of 4 patients. Lymphocytes were separated both from colonic biopsies and from blood samples and maintained in culture for 48 hours. To some of the cultures were added lectin mitogens. The lymphokines, Interleukin-2 (IL-2) and Inteferon-gamma (IFN-t), which are released by activated T-cells, were bioassayed in London from the culture supernatants. All supernatants from the mitogen-stimulated blood lymphocytes contained IL-2 and IFN-t, diminishing as expected in serial dilution. In general, the concentrations of IL-2 released by activated colonic T-cells were similar, and similar to those in children with inflammatory bowel disease previously investigated. However, one child showed no evidence of mitogen-activated of either caecum or transverse colon T-cells. The two children with Trichuris showed less IL-2 in the 1:2 dilution from their caecal T-cells than from their transverse colon T-cells. This study shows the feasibility of paediatric colonoscopy and functional studies on blood and colonic lymphocytes in Jamaica. The findings on these patients with chronic dysentery suggest that some have a reduction in T-cell function specific to the colonic mucosa (AU)