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Background: Hirschsprung's disease (HSCR) is a congenital enteric nervous system (ENS) disorder. Genetics cannot explain most sporadic cases. To explore the relationship between pathogen infection, autoantibodies, innate immune, and HSCR. Methods: Pathogen microarray was conducted in the serum of the prospective neonatal abdominal distension (NAD) cohort, consisting of 56 children followed for at least 6 months until the final diagnosis of HSCR was determined or excluded. We conducted an autoantibody microarray in an HSCR cohort, which is comprised of diagnosed HSCR patients (HSCR) and healthy control subjects (HC). RNA-seq of colon tissues from aganglionic and ganglionic segments of HSCR patients was performed. Results: Experimental results show that the serum lgM and lgG of enterovirus 71 (EV71) were significantly higher in HSCR than in the gastrointestinal dysfunction (GI) group, with a prediagnose value reaching area under the curve (AUC) over 0.76. We discovered that a group of autoantibodies were significantly higher in HSCR including neuronal pentraxin 1 (NPTX1), amyloid, neuron lysate, and myelin-associated oligodendrocytic basic protein (MOBP) than that in the HC group. These four autoantibodies could distinguish HSCR from the HC group, with a combined AUC of over 0.90 using both serum IgG and IgM. Further analysis showed that wide activation of innate immune pathways, including toll-like receptor (TLR) signaling pathway, neutrophil-to-lymphocyte ratio (NLR) signaling pathway, red cell distribution width to lymphocyte ratio (RLR) signaling pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway in aganglionic compared to ganglionic segments of HSCR. Conclusion: This study suggested that virus-triggered autoimmunity may contribute to HSCR through activation of innate immunity, which facilitates the diagnosis and prevention of HSCR., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Weiyong Zhong et al.)

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat-Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients.

Cartilage-hair hypoplasia (CHH) is an autosomal recessive syndromic immunodeficiency with skeletal dysplasia, short stature, hypotrichosis, variable degree of immune dysfunction and increased incidence of anaemia, Hirschsprung disease and malignancy. CHH is caused by variants in the RMRP gene, encoding the untranslated RNA molecule of the mitochondrial RNA-processing endoribonuclease, which participates in for example cell cycle regulation and telomere maintenance. Recent studies have expanded our understanding of the complex pathogenesis of CHH. Immune dysfunction has a major impact on clinical course and prognosis. Clinical features of immune dysfunction are highly variable, progressive and include infections, lung disease, immune dysregulation and malignancy. Mortality is increased compared with the general population, due to infections, malignancy and pulmonary disease. Several risk factors for early mortality have been reported in the Finnish CHH cohort and can be used to guide management. Newborn screening for severe combined immunodeficiency can possibly be of prognostic value in CHH. Regular follow-up by a multidisciplinary team should be implemented to address immune dysfunction in all patients with CHH, also in asymptomatic cases. Haematopoietic stem cell transplantation can cure immune dysfunction, but its benefits in mildly symptomatic patients with CHH remain debatable. Further research is needed to understand the mechanisms behind the variability of clinical features, to search for potential molecular treatment targets, to examine and validate risk factors for early mortality outside the Finnish CHH cohort and to develop management guidelines. This review focuses on the pathogenesis, clinical course and management of CHH., (© 2020 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

Background: Live viral vaccines are generally contraindicated in patients with combined immunodeficiency including cartilage-hair hypoplasia (CHH); however, they may be tolerated in milder syndromes. We evaluated the safety and efficacy of live viral vaccines in patients with CHH., Methods: We analyzed hospital and immunization records of 104 patients with CHH and measured serum antibodies to measles, mumps, rubella, and varicella zoster virus (VZV) in all patients who agreed to blood sampling ( n = 50). We conducted a clinical trial (ClinicalTrials.gov identifier: NCT02383797) of live VZV vaccine on five subjects with CHH who lacked varicella history, had no clinical symptoms of immunodeficiency, and were seronegative for VZV; humoral and cellular immunologic responses were assessed post-immunization., Results: A large proportion of patients have been immunized with live viral vaccines, including measles-mumps-rubella (MMR) ( n = 40, 38%) and VZV ( n = 10, 10%) vaccines, with no serious adverse events. Of the 50 patients tested for antibodies, previous immunization has been documented with MMR ( n = 22), rubella ( n = 2) and measles ( n = 1) vaccines. Patients with CHH demonstrated seropositivity rates of 96%/75%/91% to measles, mumps and rubella, respectively, measured at a medium of 24 years post-immunization. Clinical trial participants developed humoral and cellular responses to VZV vaccine. One trial participant developed post-immunization rash and knee swelling, both resolved without treatment., Conclusion: No serious adverse events have been recorded after immunization with live viral vaccines in Finnish patients with CHH. Patients generate humoral and cellular immune response to live viral vaccines. Immunization with live vaccines may be considered in selected CHH patients with no or clinically mild immunodeficiency., (Copyright © 2020 Vakkilainen, Kleino, Honkanen, Salo, Kainulainen, Gräsbeck, Kekäläinen, Mäkitie and Klemetti.)

Background: Mutations in RMRP , encoding a non-coding RNA molecule, underlie cartilage-hair hypoplasia (CHH), a syndromic immunodeficiency with multiple pathogenetic mechanisms and variable phenotype. Allergy and asthma have been reported in the CHH population and some patients suffer from autoimmune (AI) diseases. Objective: We explored AI and allergic manifestations in a large cohort of Finnish patients with CHH and correlated clinical features with laboratory parameters and autoantibodies. Methods: We collected clinical and laboratory data from patient interviews and hospital records. Serum samples were tested for a range of autoantibodies including celiac, anti-cytokine, and anti-21-hydroxylase antibodies. Nasal cytology samples were analyzed with microscopy. Results: The study cohort included 104 patients with genetically confirmed CHH; their median age was 39.2 years (range 0.6-73.6). Clinical autoimmunity was common (11/104, 10.6%) and included conditions previously undescribed in subjects with CHH (narcolepsy, psoriasis, idiopathic thrombocytopenic purpura, and multifocal motor axonal neuropathy). Patients with autoimmunity more often had recurrent pneumonia, sepsis, high immunoglobulin (Ig) E and/or undetectable IgA levels. The mortality rates were higher in subjects with AI diseases ( χ ( 2 ) 2 = 14.056, p = 0.0002). Several patients demonstrated serum autoantibody positivity without compatible symptoms. We confirmed the high prevalence of asthma (23%) and allergic rhinoconjunctivitis (39%). Gastrointestinal complaints, mostly persistent diarrhea, were also frequently reported (32/104, 31%). Despite the history of allergic rhinitis, no eosinophils were observed in nasal cytology in five tested patients. Conclusions: AI diseases are common in Finnish patients with CHH and are associated with higher mortality, recurrent pneumonia, sepsis, high IgE and/or undetectable IgA levels. Serum positivity for some autoantibodies was not associated with clinical autoimmunity. The high prevalence of persistent diarrhea, asthma, and symptoms of inflammation of nasal mucosa may indicate common pathways of immune dysregulation.

Background: Mutations in the long noncoding RNA RNase component of the mitochondrial RNA processing endoribonuclease (RMRP) give rise to the autosomal recessive condition cartilage-hair hypoplasia (CHH). The CHH disease phenotype has some overlap with dyskeratosis congenita, a well-known "telomere disorder." RMRP binds the telomerase reverse transcriptase (catalytic subunit) in some cell lines, raising the possibility that RMRP might play a role in telomere biology., Objective: We sought to determine whether a telomere phenotype is present in immune cells from patients with CHH and explore mechanisms underlying these observations., Methods: We assessed proliferative capacity and telomere length using flow-fluorescence in situ hybridization (in situ hybridization and flow cytometry) of primary lymphocytes from patients with CHH, carrier relatives, and control subjects. The role of telomerase holoenzyme components in gene expression and activity were assessed by using quantitative PCR and the telomere repeat amplification protocol from PBMCs and enriched lymphocyte cultures., Results: Lymphocyte cultures from patients with CHH display growth defects in vitro, which is consistent with an immune deficiency cellular phenotype. Here we show that telomere length and telomerase activity are impaired in primary lymphocyte subsets from patients with CHH. Notably, telomerase activity is affected in a gene dose-dependent manner when comparing heterozygote RMRP carriers with patients with CHH. Telomerase deficiency in patients with CHH is not mediated by abnormal telomerase gene transcript levels relative to those of endogenous genes., Conclusion: These findings suggest that telomere deficiency is implicated in the CHH disease phenotype through an as yet unidentified mechanism., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21-24, prior to histological signs of enterocolitis. We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity. EdnrBNCC-/- Peyer's Patches demonstrate decreased B-lymphocytes, specifically IgM+IgDhi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrBNCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrBNCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.

Purpose of Review: Hirschsprung's disease (HSCR) is characterized by an absence of ganglion cells in the distal hindgut, extending from the rectum to a variable distance proximally, and results from a failure of cranial-caudal neural crest cell migration. Hirschsprung's-associated enterocolitis (HAEC) is a condition with classic manifestations that include abdominal distention, fever and foul-smelling stools, and is a significant and life-threatening complication of HSCR. The purpose of this review was to critically evaluate recent findings regarding the pathophysiology of HAEC., Recent Findings: Several recent studies have investigated the cause of HAEC in humans and mouse models. These studies suggest that alterations in the intestinal barrier, including goblet cell number and function, and Paneth cell function, impaired gastrointestinal mucosal immunity, including B-lymphocyte trafficking or function and secretory immunoglobulin A production, and dysbiosis of the intestinal microbiota may contribute to the development of HAEC., Summary: Recent studies add to the body of literature, suggesting that the intestinal defects observed in HSCR are not restricted to the aganglionic segment but extend to the mucosal immune system within and beyond the gastrointestinal tract. Future studies further dissecting the mechanisms of HAEC and validating these findings in humans will allow for the development of directed therapeutic interventions.

Potentially life-threatening enterocolitis is the most frequent complication in children with colonic aganglionosis (Hirschsprung disease, HSCR), and little is known about the mechanisms leading to enterocolitis. Splenic lymphopenia has been reported in the Endothelin Receptor B (Ednrb)-null mouse model of HSCR that develops enterocolitis. In this study, we sought to identify molecular mechanisms underlying this immune phenotype. We employed the Ednrb(-/-) mouse, and the knockout of its ligand, Edn3 (Edn3(-/-)). The major finding is that enterocolitis in the Ednrb(-/-) and Edn3(-/-) mice lead to thymic involution, splenic lymphopenia, and suppression of B lymphopoiesis as a consequence of colonic aganglionosis, not an intrinsic Edn3-Ednrb signaling defect directly affecting the lymphoid organs. We showed that adoptive transfer of Ednrb(-/-) marrow repopulated the RAG2-null mice marrow, thymus and spleen without development of enterocolitis. We identified the glucocorticoid corticosterone, as a potential mediator of the immune phenotype. This previously unrecognized pattern of immune abnormalities in mouse is nearly identical to lymphoid depletion in neonatal sepsis during severe physiological stress, suggesting that the mouse model used here could be also used for sepsis studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The most common congenital gut motility disorder is the Hirschsprung disease (HSCR). This anomaly is characterized by absence of neural crest-derived enteric neuronal ganglia. The aim of our study was to analyze the relationship between HSCR and other congenital anomalies or malfunctions. We examined 130 patients with Hirschsprung disease from Slovakia for last 10 years. During patients examination we focused not only on morphological abnormalities, but also functional anomalies. The incidence of associated congenital anomalies in our patients with HSCR was 26.1 %. But if we add functional defects (hypothyroidism, malfunction in cellular immunity, neurological deficit) to the morphological congenital abnormalities, the rate of the patients with HSCR with additional defects achieves 50.1 %. Nine of our patients (6.9 %) had syndromic HSCR. The most frequent disorder (13.6 % of patients) was primary deficiency in cellular immunity. More than 12.3 % of patients with HSCR had genitourinary abnormalities, in 10.0 % of patients variable degree of psychomotor retardation was observed, and skeletal, muscle and limb anomalies involved 7.7 % of patients. In 7.6 % cases of patients we found congenital hypothyroidism (including 2 cases of agenesis of thyroid gland). More than 6.1 % of patients presented with an associated anomaly in gastrointestinal tract (mostly anorectal malformations). Up to 5.5 % patients had congenital anomaly of heart, 3.8 % had ophthalmic and 3.1 % had craniofacial anomalies. Down syndrome was the main diagnosis in 3.8 % patients. We discussed  the relationship between HSCR and other anomalies, which are probably caused by abnormal migration, proliferation, or differentiation, of neural crest cells during embryogenesis (Tab. 1, Fig. 2, Ref. 75).

Background/purpose: Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model., Methods: Mice with neural crest conditional deletion of Endothelin receptor B (EdnrB) and their littermate controls were used (EdnrB-null and EdnrB-het). Bacterial DNA was prepared from cecal contents of P16-18 and P21-24 animals and pyrosequencing employed for microbiome analysis. Ileal tissue was isolated and secretory phospholipase A2 (sPLA2) expression and activity determined. Enteroinvasion of Escherichia coli into ileal explants was measured using an ex vivo organ culture system., Results: EdnrB-het and EdnrB-nulls displayed similar flora, sPLA2 expression and activity at P16-18. However, by P21-24, EdnrB-hets demonstrated increased Lactobacillus and decreased Bacteroides and Clostridium, while EdnrB-nulls exhibited reciprocal changes. EdnrB-nulls also showed reduced sPLA2 expression and luminal activity at this stage. Functionally, EdnrB-nulls were more susceptible to enteroinvasion with E. coli ex vivo and released less sPLA2 than EdnrB-hets., Conclusions: Initially, EdnrB-het and EdnrB-nulls contain similar cecal flora but then undergo reciprocal changes. EdnrB-nulls display dysbiosis, demonstrate impaired mucosal defense, decreased luminal sPLA2 and increased enteroinvasion of E. coli just prior to robust colonic inflammation and death. These findings suggest a role for the intestinal microbiome in the development of HAEC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To study the changes in the immunological status in 2 children with cartilage hair hypoplasia (CHH)., Methods: A 4-6 year immunological follow-up from infancy., Results: In infancy the children presented a combined T cell and B cell immunodeficiency which partly resolved in time. Mitogen-induced T cell proliferation values fluctuated but lymphopenia has remained constant. Both patients had no recent thymic emigrants (TREC). Both children have suffered from a prolonged viral infection. Hypogammaglobulinemia normalized during the first years of life but both children have a specific antibody deficiency (SAD)., Conclusions: The changes in the immunological status in CHH patients emphasize the importance of a regular follow-up. SAD should be searched for in CHH. The absence of TRECs supports combined immunodeficiency and possible need of hematopoietic stem cell transplantation.

Purpose: To investigate the impact of cow's milk allergy (CMA) on infants with Hirschsprung's disease (HD)., Methods: Twenty-four patients, who developed gastrointestinal symptoms before the age of 60 days and underwent surgery for HD in the period between January 2003 and December 2012, were enrolled in this study. They were divided into two groups based on CMA-related findings: stimulation index of lymphocyte stimulation test >300 % and the presence of eosinophilic infiltration in the resected colon. Ten patients were determined specimen as not having CMA (Group A), because they did not satisfy any of the criteria. The remaining 14 were determined as having possible CMA (Group B), because they satisfied either or both findings. Patient background characteristics, pre- and postoperative clinical history, and laboratory data were compared between Groups A and B., Results: Pre- and postoperative enterocolitis did not occur in Group A patients. Postoperative enterocolitis was more frequent in Group B than in Group A (p = 0.04). Other clinical and laboratory data did not show significant difference between the two groups., Conclusion: CMA is a possible risk factor for postoperative enterocolitis in patients with HD.

Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling.

Background: Cartilage-hair hypoplasia (CHH) is a rare autosomal recessive disorder characterized by short-limbed skeletal dysplasia. Some patients also have defects in cell-mediated immunity and antibody production. Granulomatous inflammation has been described in patients with various forms of primary immunodeficiencies but has not been reported in patients with CHH., Objective: We sought to describe granulomatous inflammation as a novel feature in patients with CHH, assess associated immunodeficiency, and evaluate treatment options., Methods: In a retrospective observational study we collected clinical data on 21 patients with CHH to identify and further characterize patients with granulomatous inflammation., Results: Four unrelated patients with CHH (with variable degrees of combined immunodeficiency) had epithelioid cell granulomatous inflammation in the skin and visceral organs. Anti-TNF-α mAb therapy in 3 of these patients led to significant regression of granulomas. However, 1 treated patient had fatal progressive multifocal leukoencephalopathy caused by the JC polyomavirus. In 2 patients immune reconstitution after allogeneic hematopoietic stem cell transplantation led to the complete disappearance of granulomas., Conclusion: To the best of our knowledge, this is the first report of granulomatous inflammation in patients with CHH. Although TNF-α antagonists can effectively suppress granulomas, the risk of severe infectious complications limits their use in immunodeficient patients., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Cartilage-hair hypoplasia (CHH) is characterized by metaphyseal dysplasia, bone marrow failure, increased risk of malignancies, and a variable degree of immunodeficiency. CHH is caused by mutations in the RNA component of the mitochondrial RNA processing (RMRP) endoribonuclease gene, which is involved in ribosomal assembly, telomere function, and cell cycle control., Objectives: We aimed to define thymic output and characterize immune function in a cohort of patients with molecularly defined CHH with and without associated clinical immunodeficiency., Methods: We studied the distribution of B and T lymphocytes (including recent thymic emigrants), in vitro lymphocyte proliferation, cell cycle, and apoptosis in 18 patients with CHH compared with controls., Results: Patients with CHH have a markedly reduced number of recent thymic emigrants, and their peripheral T cells show defects in cell cycle control and display increased apoptosis, resulting in poor proliferation on activation., Conclusion: These data confirm that RMRP mutations result in significant defects of cell-mediated immunity and provide a link between the cellular phenotype and the immunodeficiency in CHH., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Cartilage hair hypoplasia (CHH) is an autosomal recessive disorder caused by mutations in the ribonuclease mitochondrial RNA-processing (RMRP) gene. Although its most constant feature is metaphyseal dysplasia with short stature, CHH is associated with extraskeletal defects such as thin hair, anemia, immunodeficiency, and increased incidence of lymphomas. The spectrum of immunologic phenotypes in CHH translates into clinical severity. Whereas T-cell deficiency may remain subclinical or may result in severe combined immunodeficiency or Omenn syndrome, humoral immunodeficiency has only rarely been noted in these patients. Here we report the diagnosis of CHH in a woman who presented with severe short stature and a full-blown antibody deficiency, clinically resembling common variable immunodeficiency. Sequencing of the RMRP gene revealed compound heterozygosity for two novel mutations (g.68_69delinsTT and g.76C>T). Despite the late onset of immunodeficiency in the patient, its clinical course was severe, and the patient died 3 years after the first diagnosis., (Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Pediatric surgeons often encounter neonates who present with Hirschsprung's disease (HD)-like symptoms and plain x-ray findings, but respond well to conservative treatment. During our investigation of the etiology of this condition, which we named "benign transient nonorganic ileus of neonates" (BTNIN), we noticed that BTNIN included cases of cow's milk allergy (CMA). Therefore, a prospective study of the identity of BTNIN and CMA was conducted., Methods: Cow's milk allergy was diagnosed when a baby showed HD-like symptoms after oral feeding, and a drug-induced lymphocyte stimulation test was positive for cow's milk with a titer of more than 300%., Materials: Of 38 neonates with suspected HD, a surgical disorder was excluded by plain x-ray in 9, intestinal atresia was diagnosed in 3, and the remaining 26 were enrolled in this study., Results: Of 26 cases, 9 were diagnosed as HD by manometric studies and 17 as CMA. Thirteen of 17 CMA cases had been fed with breast milk and 4 with formula milk., Conclusion: The proportion of CMA in the cases presenting with HD-like symptoms in the neonatal period is much higher than what we expected, and most cases of BTNIN are caused by CMA. If HD is ruled out, CMA should be considered.

Hirschsprung disease (HD), a developmental disorder, is associated with failure of enteric ganglia formation. Signaling molecules, including secreted basement membrane molecules, derived from the mesenchyme of the gut wall play an important role in the colonization and/or differentiation of the enteric nervous system. The current study aims to define the possible alterations of laminins involved in the pathogenesis of HD. Expression of the various laminin alpha, beta, and gamma chains, was assessed in the aganglionic, transitional, and ganglionic bowel segments of patients with HD or with other motor disorders. Cytoskeletal, neuronal, and glial markers were also included in this study. The major finding highlighted by the present work concerns the clear identification and location of myenteric aganglionic plexuses in HD with some of the laminin antibodies, which reveal a peripheral nerve type of differentiation. Furthermore, we could show an increase of laminin alpha5 chain immunostaining in the dilated muscle of the ganglionic bowel upstream the distal aganglionic region in a subgroup of patients with HD, as well as a relocalization of laminin alpha2 chain in the subepithelial basement membrane. Overall, these basement membrane molecules could provide useful markers for diagnosis of aganglionosis or hypoganglionosis.

Hirschsprung's disease is of multifactorial origin including genetic as well as other region-specific neuroenvironmental factors as well as focal contact with the extracellular matrix. Immunological activity is elevated in patients with enterocolitis associated with Hirschsprung's disease suggesting an immunological role in HD. Increased expression of major histocompatibility complexes (MHC) class II antigens and ICAM-1 in the aganglionic segments of bowel further strengthen this association and suggest early immunologic involvement. In this study, immunoglobulin activity was biochemically assessed in surgically resected specimens of 23 patients with Hirschsprung's disease in whom there was no clinical or histological evidence of enterocolitis. IgG and IgM were significantly increased in segments of bowel histologically identified as aganglionic or transitional zone, suggesting an area of maximal immunological activity in the transitional zone. The lack of an increase in IgA immunoglobulin activity in the same bowel segments suggests that IgA response may be related to associated enterocolitis in HD. Elevated tissue IgG and IgM levels were observed in 5 neonates presenting within the first week of life and older patients did not have significantly higher immunoglobulin levels, suggesting that the immunological response was not part of an ongoing infection or progressive condition. Increased IgM in the neonatal period suggests an early immune response in Hirschsprung's disease and strengthens the hypothesis that the immune system may be involved in its pathogenesis. Possible causes include the effects of an antenatal infection or local microenvironmental influences on the differentiation or maturation of migrating neuroblasts of the enteric nervous system.

The development of B-cells and immunoglobulin-isotype and IgA subclass-positive cells in the lamina propria of the large intestine during infancy was investigated. Biopsy specimens from 36 infants, taken for diagnostic purposes, were available. All samples showed normal morphology. Monoclonal antibodies to CD22, IgA, IgA1, IgA2, IgM and IgG and a peroxidase method were used to demonstrate positive cells in the cryostat sections. Cell densities were counted from a known area. T-cells had been measured in a previous study using the same specimens. The density of CD22+ cells was already high in infants below the age of 1 month and increased little with age. Four specimens from infants below the age of 40 days lacked IgA-, IgA1- and IgA2-positive cells. The densities of these cells increased with age; the correlation coefficient between the age and the density of the cells was for IgA: R=0.47, p=0.04; for IgA1: R=0.57, p=0.001, and for IgA2: R=0.34, p=0.04. The densities of IgG- and IgM-positive cells remained unchanged with age. The negative correlation between density of IgG+cells and CD8+ cells in the lamina propria (R= -0.43, p=0.01) was significant. Strong local stimulation results in early accumulation of CD22+, IgM+ and IgG+ cells in the large intestine, but little change takes place after the first few days of life. The terminal differentiation to IgA-positive cells is slowest, and this population showed significant developmental change.

In 1967, Okamoto et al suggested that the absence of ganglion cells in Hirschsprung's disease (HD) was attributable to failure of migration of neural crest cells. The earlier the arrest of migration, the longer the aganglionic segment. Since then, this hypothesis generally has been accepted. However, subsequent experiments using mouse models of intestinal aganglionosis indicate that nerve cells may reach the correct position but then fail to develop or survive. An alternative hypothesis has been proposed that the aganglionosis may be caused by failure of differentiation as a result of microenvironmental changes after the migration has occurred. Extracellular matrix proteins are recognized as important microenvironmental factors. It has been shown that enteric neurogenesis is dependent on extracellular matrices, which provide a migration pathway for neural crest-derived cells and promote the maturation of settled neural crest-derived cells. Altered distributions of extracellular matrices have been shown in human HD cases and murine HD models, suggesting the role of extracellular matrices in the pathogenesis of HD. Recent studies suggest that intestinal smooth muscle cells, target cells of enteric neurons, play an important role in guiding and influencing its own innervation. Normal maturation was inhibited in neurons cultured with smooth muscle cells of aganglionic colon in comparison to normal colon. Furthermore, it was demonstrated that levels of neurotrophic factors, crucial in the development and survival of enteric neurons, are decreased in circular muscle layers of aganglionic colon in comparison to normoganglionic colon. The smooth muscle cells of the aganglionic colon may represent an unfavorable microenvironment for neuronal development compared with the normally innervated region. Recently, markedly increased immunoreactivity of major histocompatibility complex (MHC) class II antigens and ICAM-1 was demonstrated in aganglionic bowel, suggesting the immunological mechanisms may be involved in the etiology of HD. Genetic factors have been implicated in the etiology of this condition because HD is known to occur in families and in association with some chromosomal abnormalities. Recent expansion of molecular genetics identified multiple susceptibility genes of HD, including the RET gene, the glial cell line-derived neurotrophic factor gene, the endothelin-B receptor gene, and endothelin-3 gene. Of these, inactivating mutations of the RET gene are the most frequent, occurring in 50% of familial and 15% to 20% of sporadic cases of HD. To date, despite extensive research, the exact etiology of this condition remains poorly understood. The present report describes the authors' current understanding of and recent progress in the etiology of HD.

Background: Subpopulations of c-Kit immunopositive cells in the muscle coat of the gastrointestinal tract are considered pacemaker cells and have been investigated in human tissue relating to motility disorder. However, the morphology of c-kit immunopositive cells in intact human tissue is still unclear., Methods: The authors studied the distribution of c-Kit immunopositive cells in the normal human colon and their cellular configuration by confocal microscopy on whole-mount preparations. The authors then compared them with six cases of Hirschsprung's disease (HD; two of short segment aganglionosis, three of extensive aganglionosis, and one of total aganglionosis)., Results: In the normal colon regional differences were found in the distribution of c-Kit immunopositive cells. The population in the muscle layers and at the submucosal border was larger in the anal part than in the oral part. Accumulation of positive cells at the myenteric plexus level was prominent only at the descending colon. In the descending colon of HD the authors could not demonstrate any differences in c-Kit immunopositive cells on aganglionic segments compared with the corresponding area of intact tissue., Conclusion: More attention must be paid to these regional differences of distribution, and identical regions of affected and unaffected bowels must be compared when discussing the relation between the abnormality of c-Kit-positive cells and motility disorders including HD.

Background: There is only limited knowledge of the development of the immune responses of the gut in very young infants after exposure to bacterial and food antigens at birth., Methods: In this study, 49 large intestinal biopsy specimens, which were judged to have normal morphology, were taken from 49 young infants. Eleven patients had Hirschsprung's disease (group 1) and 38 had miscellaneous conditions (group 2). The densities of T cells, their subsets expressing surface antigens CD8 and CD4, and T-cell receptors alpha/beta or gamma/beta were measured, as well as densities of mononuclear and epithelial cells expressing HLA-class II antigens., Results: T-cell densities in groups 1 and 2 were similar. Patients with Hirschsprung's disease had significantly more HLA-DR (p = 0.006) and HLA-DP-expressing cells (p = 0.003) in the lamina propria than did the patients in group 2. In group 1, HLA-DR- (r = 0.58; p = 0.46) and HLA-DP-expressing cells (r = 0.66; p = 0.03) showed a significant positive regression with age in the lamina propria, whereas in group 2, HLA-DR+ cells in the lamina propria showed marked (r = -0.9; p = 0.006) negative regression during the first 1.5 months of life. In contrast to results in previous reports, in the current results, HLA-D region antigens were present in the epithelium in a considerable proportion (up to one fourth) of specimens from the large intestine in both groups. CD3+ (r = -0.59; p = 0.006) and CD4+ (r = -0.64; p = 0.002) cells showed a strong negative regression with age in the lamina propria during the first 2.5 months; and thereafter, there was a weak, insignificant rise in the numbers of these cells. The distribution of CD4+, CD8+, and TCR alpha/beta or gamma/beta T cells of the epithelium of the young infants did not differ significantly from that in the epithelium of adults., Conclusions: These results show that several significant changes occur in the mucosal immune system during the first few weeks of life.

Inflammatory infiltrations of the enteric plexuses are uncommon and are usually lymphoplasmacytic. Within the past 15 years, nine pediatric cases in which a predominantly eosinophilic infiltrate of the gastrointestinal wall with a predilection for the myenteric (Auerbach's) and deep submucosal (Henle's) plexuses were seen at our institution. Two were neonates without gastrointestinal abnormalities who expired shortly after birth. Seven were patients with short-segment Hirsch-sprung's disease. There was a mild increase in mucosal eosinophils in the overlying mucosa and only one patient had peripheral eosinophilia. Follow-up data obtained 1 month to 7 1/2 years after biopsy revealed no development of inflammatory bowel disease, connective tissue disease, malignancy, allergic disorder, or intestinal dysmotility. The proximal location of the infiltrate suggests that it may represent a secondary finding rather than a primary cause of aganglionosis.

The clinical course, diagnosis, and treatment of 2 patients with acquired intestinal aganglionosis without other neurological involvement or neoplasia are described. They initially presented with constipation and abdominal pain in late childhood. They were found to have enteric ganglionitis with a loss of neurons together with vacuolated nerve cells surrounded by CD3- and CD4-positive T lymphocytes. This process initially affected only the colon but later the entire gastrointestinal tract was involved in 1 patient. Associated with this process there were circulating immunoglobulin G class enteric neuronal antibodies in high titer (1:5000-8000). The staining of central nervous system neuronal nuclei and Western blotting indicated the presence of antineuronal nuclear protein antibodies of the ANNA-1 (anti-Hu) type usually associated with paraneoplastic sensory neuropathy. However, the reaction pattern in enteric neurons was quite different with strong reaction to perikarya and only weak staining of nuclear antigens.

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.

We describe a case of semi-lethal chondrodysplasia with skeletal manifestations, detectable at birth, typical of the round femoral inferior epiphysis dysplasia (RFIED) or 'Glasgow' variant. Immunological abnormalities and Hirschsprung disease, so far described only in cartilage hair hypoplasia (CHH), were documented during the first months of life in our patient. These findings confirm the hypothesis that RFIED is a form of CHH with early infantile onset.

The aim of this study was twofold. First, to establish quantitatively the distribution of the immunoglobulin-containing (plasma) cells, T and B lymphocytes in the lamina propria of the rectal mucosa of normal neonates and neonates with Hirschsprung's disease (HD). Second, to review the neonates with HD to determine any differences in these cell populations between those who subsequently developed Hirschsprung's enterocolitis (HEC) and those who did not. Two conclusions can be drawn from the results of our study of rectal mucosal immune defenses. First, neonates with HD have no deficiencies in these defenses when compared with normal neonates. Second, neonates with HD who subsequently develop HEC have no premorbid deficiency in these defenses. It was noted that the pan-T cell count in the infants who went on to develop HEC appeared to be increased, although this did not reach statistical significance. The use of fresh or frozen material would permit a more detailed analysis of the separate T cell subsets.

Although the etiology of Hirschsprung's disease and neuronal intestinal dysplasia remains obscure, both have histological abnormalities involving ganglion cells and neuronal elements. Searching for a common pathway that may inhibit normal maturation of neurogenic precursors, we examined the possible role of an immune mechanism in the maldevelopment of the enteric neural network. Six patients with Hirschsprung's disease were studied by comparing biopsy specimens from diseased colon with ones taken from proximal ganglionic colon in the same patients. These were similarly compared with colonic biopsy specimens from patients studied with chronic constipation or bowel removed at the time of operation for other disorders. Biopsies were taken from four other patients with neuronal intestinal dysplasia. Each was examined by hematoxylin & eosin staining, acetylcholinesterase histochemistry, and immunohistochemistry of major histocompatibility complex (MHC) class I and class II antigens. All rectal samples from Hirschsprung's disease patients exhibited elevated acetylcholinesterase histochemistry and absent ganglia to confirm the diagnosis. These findings were correlated with marked elevation of class II MHC in the aganglionic area, whereas the proximal normal ganglionic segments showed no elevation. Rectal biopsy specimens from patients with chronic constipation exhibited no such elevation. A similar elevation of class II MHC was detected in the mucosa and submucosa of all four patients with the rare neuronal intestinal dysplasia disorder whose diagnosis was confirmed by giant ganglia in Auerbach's plexuses, aberrant Meissner's ganglia in the lamina propria mucosa, and giant neurofibrils in the mucosa and submucosa. The correlation of elevated class II MHC in these two neuronal dysfunction disorders may indicate an underlying autoimmune mechanism as is seen in thyroiditis and insulin dependent diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)

The major histocompatibility complex (MHC) Class I and II cell surface antigens responsible for the recognition of self vs non-self were studied in patients with documented Hirschsprung's disease. Monoclonal antibodies reactive with monomorphic determinants of human lymphocyte antigen (HLA)-A,B,C (Class I) and HLA-DR (Class II) were used to demonstrate immunohistochemically the expression of MHC antigens in 27 biopsy specimens from a variety of colorectal disorders. The rectal specimens examined from patients with Hirschsprung's disease showed an unexpected, marked elevation of Class II antigens with abnormal localization in the mucosa and lamina propria. This ectopic expression was not seen in any portion of the small or large bowel of patients who did not have Hirschsprung's disease. Furthermore, proximal normal colon of children with Hirschsprung's disease failed to show increased expression of Class II antigen. In an attempt to better define the effector arm at a cellular level, the distribution of helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and natural killer cells (NK; CD16+) was examined in 5 cases. In Hirschsprung's disease, rectal infiltration of CD8+ and CD16+ cells was found, but not CD4+ cells. Ectopic expression of Class II antigen with increased numbers of rectal T cells and NK cells suggested that an early immunologic event may be causal in Hirschsprung's disease.

A breeding colony of piebald lethal mice was established in order to study the natural history of congenital megacolon in a mouse model of Hirschsprung's disease and to investigate mucosal defense mechanisms and secretory functions in enterocolitis complicating congenital megacolon. In experiment 1, 214 mice were studied, 53 of which had congenital megacolon. All piebald lethal mice with congenital megacolon (S1/S1) died at 3 to 11 weeks of age. Two distinct patterns of mortality were identified. A majority of mice (64%) became acutely ill at 3 to 4 weeks of age and died, whereas the remainder died between 9 and 11 weeks of age. The former group of mice exhibited clinical and histologic evidence of severe enterocolitis while the latter group had massive abdominal distension and classical megacolon. In experiment 2, piebald mice with congenital megacolon were killed at the time of acute illness. Significant histologic and immunohistochemical differences were seen in the ganglionic colon between piebald mice with early clinical onset of acute illness and piebald mice with the classical clinical picture of congenital megacolon. In the former group of mice the number of immunocytes in the lamina propria was significantly higher than in control mice (P less than .001), immunoglobulin-producing cells were equally distributed throughout the lamina propria and IgA-containing cells were by far the most abundant cell type identified in the colon. In the latter group of mice, immunocyte responses were significantly low and the distribution of immunocytes markedly different, with the immunoglobulin-producing cells being located only at the deep layer of lamina propria.(ABSTRACT TRUNCATED AT 250 WORDS)

Twelve patients with Hirschsprung's disease were studied to investigate why some children develop enterocolitis. Previous studies have failed to explain this adequately; they have also failed to explain why enterocolitis can occur many years after definitive surgery. Six of the 12 patients had enterocolitis. By assessing immunological mucosal defense, it was shown that these patients had a marked deficiency in transfer of secretory IgA across the gastrointestinal mucosal cell, and thus were prone to mucosal invasion of both pathogenic and commensal organisms. The abnormalities were detected upon initial investigation (at presentation) prior to the onset of enterocolitis, and persisted into later life. The significance of these findings with reference to predicting which children are prone to develop enterocolitis and their long-term susceptibility to recurrence are discussed.

The distributions of gut hormones in the colon of Hirschsprung's disease were investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments (ganglionic, oligoganglionic, and aganglionic) were stained by the unlabeled antibody enzyme method. The immunoreactivity of vasoactive intestinal polypeptide (VIP) was found to be reduced in the oligoganglionic and aganglionic segments. Antisera to substance P and met-enkephalin demonstrated immunoreactive cells and fibers in the ganglionic segment, whereas these cells and fibers were almost completely absent in the oligoganglionic and aganglionic segments. A similar distribution was seen for the mucosal endocrine cells with somatostatin immunoreactivity. Antisera to neurotensin, motilin, bombesin, and cholecystokinin revealed no immunoreactivity in the normal colon or the three segments. The differences in these peptides between normal and impaired colonal segments may be one of the causes of colon constriction in Hirschsprung's disease.

The pathophysiology of Hirschsprung's disease has not been fully elucidated but is known to have a neurogenic basis. In recent years, new neural proteins and peptides have been discovered and our aim in this study was to use immunocytochemistry to investigate their involvement in the neuronal abnormalities associated with this condition. Large bowel samples from 9 children undergoing surgery for Hirschsprung's disease were compared with those taken from 8 children with other gastrointestinal diseases but no aganglionosis. Immunocytochemistry was carried out using antibodies to a wide range of neuron specific proteins and peptides. Examination of sections immunostained for the general neuronal markers, protein gene product 9.5, neuron specific enolase and neurofilament triplet proteins, allowed rapid identification of aganglionic segments. Nerves containing vasoactive intestinal polypeptide/peptide histidine methionine (VIP/PHM), galanin, substance P, somatostatin, met-enkephalin or calcitonin gene-related peptide (CGRP) showed a marked reduction in all layers of the aganglionic bowel. However, scattered VIP/PHM immunoreactive fibres were also found in the hypertrophied nerve bundles. In contrast with these reduced peptide-containing nerves, fibres displaying NPY immunoreactivity showed a marked increase in all aganglionic segments, particularly in the circular muscle where few are found normally. Our findings shed further light on the neurobiology of aganglionic bowel and suggest that immunostaining of neural proteins and the peptide NPY can aid rapid histopathological diagnosis of congenital aganglionosis.

In a retrospective study of Hirschsprung's disease two monoclonal antineurofilament antibodies were tested immunohistochemically. Biopsy samples of rectum and colon were fixed in formalin and embedded in paraffin. Tissue sections from normal controls and from unaffected colon of patients with Hirschsprung's disease showed partial staining of only some of the axon bundles in the myenteric and in the submucous plexus. In contrast, in all tissue sections from colon affected by Hirschsprung's disease there were heavily stained hyperplastic axon bundles. Sections of colon from patients with various other neurogenic disorders of the colon were studied by the same method; the characteristic Hirschsprung picture was never seen.

In 55 children with Hirschsprung's disease, 64 of their normal siblings and 120 other members of their families, HLA-A, B and C types were examined. The statistical significance of the raised incidence of A1, B14, B37 and Bw35 in the patients disappeared after correction for multiple testing. The levels of homozygosity were very similar in patients and normal controls. The results indicate no direct association of the disease with HLA type.

An immunological study of B cells in patients with colitis that was associated with Hirschsprung's disease was undertaken and compared with that in patients with ulcerative colitis and in normal controls. There was an appreciable increase in IgA and a decrease in IgG in both disease groups. Humoral reaction in colitis associated with Hirschsprung's disease seems to be indistinguishable from that in ulcerative colitis and possibly in other inflammatory bowel diseases.