Refractory coeliac sprue is a diffuse gastrointestinal disease.

BACKGROUND: Refractory coeliac sprue (RCS) with an immunophenotypically aberrant clonal intraepithelial lymphocyte (IEL) population is considered a cryptic form of intestinal T cell lymphoma. AIMS: To investigate the distribution of the abnormal and monoclonal IEL population in the digestive tract of RCS patients. PATIENTS AND METHODS: We compared the frequency of lymphocytic gastritis (LG) and lymphocytic colitis (LC), together with IEL phenotype and T cell clonality, in gastric and colonic samples from 15 adults with RCS (all with aberrant CD3 intracytoplasmic(+) surface(-) CD8(-) clonal IELs on duodenojejunal biopsies), 18 patients with active coeliac disease (ACD), and 10 patients with coeliac disease (CD) on a gluten free diet (GFD-CD) by means of immunohistochemistry and multiplex polymerase chain reaction amplification of the T cell receptor gamma gene (TCR-gamma) rearrangement. Blood samples of nine RCS patients were also tested for clonality. RESULTS: LG was found in 9/14 (64%), 11/18 (61%), and 3/10 (30%) patients with RCS, ACD, and GFD-CD, respectively, while LC was found in 6/11 (55%), 3/4 (75%), and 2/3 (66%) patients. Contrary to CD, all samples from patients with LG and LC showed an aberrant IEL phenotype. Monoclonal TCR-gamma rearrangements were detected in 8/13 (62%), 8/10 (80%), and 4/9 (44%) of gastric, colonic, and blood samples, respectively, from RCS patients, while in CD patients such rearrangements were only found in 2/25 (8%) gastric samples. CONCLUSION: The immunophenotypically aberrant monoclonal IEL population present in the small intestine of patients with RCS frequently disseminates to the blood and the entire gastrointestinal epithelium, suggesting that this is a diffuse gastrointestinal disease.

Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin.

The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.

Differentiation of Langerhans cells in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) consists of lesions composed of cells with a dendritic Langerhans cell (LC) phenotype. The clinical course of LCH ranges from spontaneous resolution to a chronic and sometimes lethal disease. We studied 25 patients with various clinical forms of the disease. In bone and chronic lesions, LCH cells had immature phenotype and function. They coexpressed LC antigens CD1a and Langerin together with monocyte antigens CD68 and CD14. Class II antigens were intracellular and LCH cells almost never expressed CD83 or CD86 or dendritic cell (DC)-Lamp, despite their CD40 expression. Consistently, LCH cells sorted from bone lesions (eosinophilic granuloma) poorly stimulated allogeneic T-cell proliferation in vitro. Strikingly, however, in vitro treatment with CD40L induced the expression of membrane class II and CD86 and strongly increased LCH cell allostimulatory activity to a level similar to that of mature DCs. Numerous interleukin-10-positive (IL-10(+)), Langerin(-), and CD68(+) macrophages were found within bone and lymph node lesions. In patients with self-healing and/or isolated cutaneous disease, LCH cells had a more mature phenotype. LCH cells were frequently CD14(-) and CD86(+), and macrophages were rare or absent, as were IL-10-expressing cells. We conclude that LCH cells in the bone and/or chronic forms of the disease accumulate within the tissues in an immature state and that most probably result from extrinsic signals and may be induced to differentiate toward mature DCs after CD40 triggering. Drugs that enhance the in vivo maturation of these immature DCs, or that induce their death, may be of therapeutic benefit.

A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes.

Immature dendritic cells (DC) sample Ags within nonlymphoid tissues and acquire exogenous proteins/pathogens via scavenger receptors or Ig FcR such as Fc gamma R and Fc epsilon R. IgA is present in a significant proportion among serum Ig and is the main isotype in mucosae, where DC are numerous. We found that a functional Fc alpha R (CD89) was expressed in situ and in vitro on interstitial-type DC but not on Langerhans cell-type DC. Interstitial-type DC expressed CD89 as a 50- to 75-kDa glycoprotein with a 32-kDa protein core, which was down-regulated upon addition of TGF-beta 1. DC, Fc alpha R specifically, bound IgA1 and IgA2. Cross-linking of CD89 on DC triggered endocytosis in time-dependent manner. In addition, internalization of polymeric IgA complexes induced the production of IL-10 and DC activation, as reflected by up-regulation of CD86 costimulatory molecules, class II MHC expression, and increased allostimulatory activity. Therefore, interstitial-type DC may use Fc alpha R-mediated Ag sampling in the subepithelium to check tissue integrity while Langerhans cells inside epithelial layers may neglect IgA immune complexes.

Distinction between coeliac disease and refractory sprue: a simple immunohistochemical method.

AIMS: We recently showed that refractory sprue is distinct from coeliac disease, the former being characterized by abnormal intraepithelial T-lymphocytes expressing a cytoplasmic CD3 chain (CD3c), lacking CD3 and CD8 surface expression, and showing TCRgamma gene rearrangements. To take advantage of the abnormal phenotype of CD3c + CD8 - intraepithelial lymphocytes (IEL) in refractory sprue we developed a simple method to distinguish coeliac disease from refractory sprue. METHODS AND RESULTS: Comparative immunohistochemical studies using anti-CD3 and anti-CD8 antibodies were applied on paraffin-embedded and frozen biopsy specimens in refractory sprue (n = 6), coeliac disease (n = 10), healthy controls (n = 5) and suspected refractory sprue (n = 6). Comparable results were obtained on fixed and frozen biopsy specimens. In four of the six patients with suspected refractory sprue, abnormal CD3c + CD8 - IEL and TCRgamma gene rearrangements were found, as in refractory sprue; the remaining two patients had normal (CD3 + CD8 +) IEL and no TCRgamma gene rearrangements. Both patients had coeliac disease, as one failed to comply with a gluten-free diet, while the other was a slow responder. CONCLUSION: This simplified immunostaining method using anti-CD3 and anti-CD8 antibodies on paraffin sections can distinguish active coeliac disease from refractory sprue and should prove useful in clinical practice.

Immunohistologic and morphometric analysis of cytotoxic T lymphocytes in gingivitis.

BACKGROUND: Gingivitis is an inflammatory phenomenon localized in gingival tissues and histologically characterized by an infiltration of several inflammatory cell populations. The purpose of this study was to characterize, localize, and quantify in situ inflammatory and cytotoxic T lymphocytes using immunolabeled gingival tissue sections in order to specify their implication during human gingivitis, since it is well known that such cells play an important role in the defense against bacterial elements. METHODS: Paraffin gingival tissue sections from 7 patients with gingivitis (G) and from 7 clinically and histologically healthy controls (C) were immunohistochemically stained by specific antibodies (anti-CD45, anti-CD3, anti-CD8, anti-CD20, anti-TIA-1, anti-GrB, and anti-CD68), allowing the quantification of inflammatory cells in upper gingival epithelium (Ep), in the basal epithelium layer (BEp), and in upper connective tissue (CT). Collagen fibers were stained by sirius red F3Ba in order to evaluate, by morphometric and automated image analysis, the surface occupied by collagen bundles and to histologically confirm the absence of pathology of the clinically selected healthy controls. RESULTS: In the gingivitis group, CD45+, CD3+, CD8+, TIA-1+, and GrB+ lymphocyte numbers were significantly increased in Ep (P<0.05); and CD45+, CD3+, and TIA-I+ lymphocyte numbers were significantly increased in BEp (P <0.05) compared respectively to Ep and BEp of group C. In Ep of group G, mean CD8+/CD3+ cell ratio was significantly increased (P<0.05) compared to BEp and CT, and 25% of TIA-1+ cytotoxic cells were activated GrB+ cells. CONCLUSIONS: The present study suggests that intraepithelial cytotoxic T lymphocytes play an important role during gingivitis and CD8 expression and that activation of TIA-1+ cytotoxic cells could be induced in Ep in response to epithelial environment. Thus, gingival epithelial tissue, which is generally only considered as a physical barrier, in fact contains numerous immune cell populations preventing the infiltration of pathogenic elements into the connective tissue. Particular clinical attention must be taken for the preservation of the epithelial tissue integrity.

ICAM-3 and E-selectin endothelial cell expression differentiate two phases of angiogenesis in infantile hemangiomas.

Cellular adhesion molecules are newly identified mediators of angiogenesis. Infantile hemangiomas, characterized in the early stages by a proliferation of poorly differentiated vessels followed in the late stages by a vascular differentiation and regression of the tumor, represent an interesting model to study angiogenesis. We studied by immunohistochemistry the distribution of HLA-DR and three adhesion molecules ICAM-3, E-selectin and VCAM-1 on endothelial cells in different stages of vessel differentiation in infantile hemangiomas. We found high levels of ICAM-3 expression on proliferating vessels, while its expression was low or undetectable on well differentiated vessels. A different set of E-selectin antibodies showed a more heterogenous pattern of distribution and VCAM-1 antigens were found in both proliferating and differentiated vessels. HLA-DR expression on endothelial cells was inversely correlated to the vascular differentiation. Our results are consistent with the hypothesis that ICAM-3 plays a role in the early stages of vessel formation. Our results also suggest that variation of E-selectin and HLA-DR expression may be related either to vessel differentiation or may reflect the acquisition of an activated endothelial cell status.

Gluten-free diet induces regression of T-cell activation in the rectal mucosa of patients with celiac disease.

OBJECTIVE: An increase in the number of intraepithelial lymphocytes (IEL) in the rectal epithelium of patients with active celiac disease has been described. No data are available about how they vary during a gluten-free diet. The aim of the study was to assess the effect of a gluten-free diet on T-cell activation in the rectal mucosa of adult patients with celiac disease. METHODS: Frozen duodenal and rectal biopsies were available in four celiac patients (one male, three female, mean age 39 yr) both before and after 7 to 24 months on a gluten-free diet. Biopsy samples were stained using monoclonal antibodies directed against CD3, betaF1, TcRdelta1, CD25, and HLADR. Numbers of IEL were estimated by counting the peroxidase-stained cells per 100 epithelial cells. Four patients without histological abnormalities were used as control subjects. RESULTS: In the four patients with active celiac disease but in none of the controls, CD25 was expressed by both duodenal and rectal lamina propria cells and HLADR was expressed by duodenal (4/4) and rectal (2/4) epithelial cells. In addition, two patients with active celiac disease had features of lymphocytic colitis, i.e., >20 IEL per 100 epithelial cells. After a gluten-free diet, the mean number of rectal CD3+ betaF1+ IEL decreased (9% vs 21%) and the expression of CD25 and HLADR was no longer present. These changes mirrored those found in the small intestinal biopsies. CONCLUSION: These results suggest that in celiac disease, gluten-driven T-cell activation is not restricted to the proximal part of the intestine but is present on the whole intestinal length. Assessment of the effectiveness of a gluten-free diet through rectal biopsies warrants investigation, as it could lessen discomfort for patients and prove more cost-effective.

In-situ endothelial cell adhesion molecule expression in ulcerative colitis. E-selectin in-situ expression correlates with clinical, endoscopic and histological activity and outcome.

BACKGROUND AND OBJECTIVES: Little is known of the in-situ expression of adhesion molecules in ulcerative colitis (UC) according to disease activity. In the present study we investigate the vascular expression of endothelial leucocyte adhesion molecule 1 (ELAM-1/E-selectin), vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecules (ICAM-1 and ICAM-3) on the rectal mucosa of patients with UC in order to identify links between in-situ expression of these adhesion molecules and clinical, endoscopic and histological parameters. DESIGN AND METHODS: At inclusion, 16 untreated patients with UC at different stages of disease activity were assessed clinically and endoscopically and underwent rectal biopsy. Ten patients had similar assessments during follow-up. Quantitative histological and immunohistochemical scores were established with anti-E-selectin, VCAM-1, ICAM-1, ICAM-3 and HLA-DR monoclonal antibodies on frozen biopsy specimens. RESULTS: (1) At inclusion, E-selectin in-situ expression correlated with clinical activity (r = 0.7, P = 0.05), endoscopic severity (r = 0.74, P = 0.04), the histological score (r = 0.57, P = 0.02) and in-situ expression of HLA-DR on epithelial cells (r = 0.74, P = 0.01). (2) After remission, there was a significant decrease in ELAM-1 in-situ expression (P = 0.04). (3) In patients with clinical, endoscopic and histological remission the level of residual E-selectin expression appeared to be predictive of clinical relapse. (4) Vascular expression of VCAM-1 and ICAM-1 did not correlate with clinical, endoscopic or histological parameters, or with changes in disease activity. (5) ICAM-3 was never detected on endothelial cells of the colonic mucosa of controls or patients with UC. CONCLUSION: In ulcerative colitis, E-selectin, but not VCAM-1, ICAM-1 or ICAM-3, appears to play a central role in leucocyte migration into the colonic mucosa. Elevated vascular expression of E-selectin after remission may be involved in clinical recurrence.

Expression of GM-CSF receptor by Langerhans' cell histiocytosis cells.

Langerhans' cell histiocytosis (LCH) is characterized by the proliferation of large mononucleated cells containing Birbeck granules and expressing CD1a. Recent studies have demonstrated that LCH is a clonal proliferation; however, its aetiology is still unknown. Growth and differentiation of bone-marrow-derived cells are controlled by cytokines. The proliferation, differentiation and activation of normal Langerhans cells are controlled by granulocyte/macrophage colony-stimulating factor (GM-CSF) in vitro. Therefore, GM-CSF could be implicated in the pathogenesis of LCH. Indeed, LCH cells contain GM-CSF, and children with disseminated LCH have an elevated GM-CSF serum level. As a cytokine only acts on cells expressing a specific receptor, we investigated the presence of GM-CSF receptor on LCH cells. Fourteen frozen tissue samples from children with LCH were studied by in situ immunohistochemistry with two mouse monoclonal antibodies specific for the alpha chain of the GM-CSF receptor (CDw116). LCH cells of all the samples were positively stained with both antibodies. This study suggests that GM-CSF may be a growth factor for LCH cells.

Langerhans' cell histiocytosis cells are activated Langerhans' cells.

Langerhans' cell histiocytosis (LCH) is characterized by the presence of large mononucleated cells, associated with inflammatory cells. The Langerhans' cell (LC) lineage of the mononucleated cells is suggested by the presence of Birbeck granules and the expression of CD1a. We investigated the presence of 14 markers expressed by normal LCs in vitro. Nine skin and one lymph node frozen biopsies of LCH children were analysed by in situ immunohistochemistry. The data were compared with six skin and five lymph node frozen biopsies. LCH cells of the ten samples were positive for all 14 LC markers. We observed three different groups of markers, according to the respective staining of normal LCs and LCH cells. Group 1 included DR, DQ, CD1a, CD1c, and ICAM-3. Markers of group 1 were present on the majority of both normal LCs and LCH cells. Group 2 included CD1b, CD4, LFA-1, LFA-3, CD32, and CD68. Markers of group 2 were detected on the majority of LCH cells, but only on a fraction of normal LCs. Group 3 included CD11b, CD24, and B7/BB1. Markers of this group were detected on LCH cells, but not on normal LCs. This in situ immunohistochemical study confirms that LCH cells belong to the LC lineage. The different clinical LCH syndromes had the same immunohistochemical staining. The expression of some markers of groups 2 and 3 is known to be related to the activation of LCs in vitro. Our study suggests that LCH cells are activated LCs.

[Histopathological diagnosis of Hirschsprung's disease]. / Diagnostic histopathologique de la maladie de Hirschsprung.

The basis of the histological diagnosis of Hirschsprung's disease (HD) is the demonstration of ganglionic nerve cells in the myenteric plexus of the rectum. The deep rectal biopsy with inclusion of colonic muscle fibres can now be replaced by a quantitative study of the cholinergic fibres in the sub-mucosa, which can be stained histochemically by the acetylcholinesterase technique (ACE). This technique only requires an aspiration biopsy which is innocuous and able to be repeated. Provided the disease is limited to the rectum and in the absence of any malformation syndrome and any previous pelvic operation, this technique is reliable with a 3 to 4 p. cent false negative and false positive rate. This technique therefore has an important place in the diagnosis of HD, together with the clinical signs, radiology and recto-manometric findings. This technique also opens the way for promising histochemical studies of intestinal neuromediators.

[Acetylcholinesterase activity in suction rectal biopsies. An appraisal of its value in the diagnosis of Hirschsprung's disease (author's transl)]. / Etude de l;activité acétylcholinestérasique dans les biopsies rectales obtenues par aspiration. Son utilité et sa valeur dans le diagnostic de la maladie de Hirschsprung.

Demonstration of an increase in Acetylcholinesterase activity within the terminal nerves of the aganglionic segment in Hirschsprung's disease can provide a useful additional criterion to the diagnosis of this disease. The value as well as the limitations of this histochemical investigation, when performed on superficial suction biopsies, is evaluated. 135 rectal biopsies taken from 123 children were studied. In 28 biopsies performed on children with Hirschsprung's disease, the correct diagnosis was established in 19 cases suggested in 5 and missed in 2. Conversely in 107 control specimens, the results of the histochemical method were correctly negative on 97 occasions and falsely positive on 2.