Immune function in patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited multisystem disorder characterized by exocrine pancreatic dysfunction and varying degrees of cytopenia. In addition, various immunological abnormalities have been noted. To clarify the issue of immunological competence or incompetence in SDS, we prospectively studied immune function in 11 patients with SDS. Seven suffered from recurrent bacterial infections and six from recurrent viral infections. Varying degrees of impairment were readily identified. All patients had neutropenia; total lymphocyte counts, however, were normal in all except one patient. Nine patients had B-cell defects comprising one or more of the following abnormalities: low IgG or IgG subclasses, low percentage of circulating B lymphocytes, decreased in vitro B-lymphocyte proliferation and a lack of specific antibody production. Seven out of nine patients studied had at least one T-cell abnormality comprising a low percentage of total circulating T lymphocytes or CD3+/CD4+ cell subpopulations or decreased in vitro T-lymphocyte proliferation. Five out of six patients studied had decreased percentages of circulating natural killer cells. Moreover, neutrophil chemotaxis was significantly low in all the patients studied. These data point to a major immunodeficiency component in SDS that places patients at heightened risk of infections, even if neutrophil numbers are protective. This finding broadens the definition of the syndrome substantially: it suggests that the SDS marrow defect occurs at the level of an early haematological-lymphocytic stem cell or that a combined marrow and thymic stromal defect accounts for the aberrant function of haematopoietic and lymphopoietic lineages.

Neutrophil-mediated epithelial injury during transmigration: role of elastase.

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, beta-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.

Shwachman-Diamond syndrome with exocrine pancreatic dysfunction and bone marrow failure maps to the centromeric region of chromosome 7.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. A genomewide scan of families with SDS was terminated at approximately 50% completion, with the identification of chromosome 7 markers that showed linkage with the disease. Finer mapping revealed significant linkage across a broad interval that included the centromere. The maximum two-point LOD score was 8.7, with D7S473, at a recombination fraction of 0. The maximum multipoint LOD score was 10, in the interval between D7S499 and D7S482 (5.4 cM on the female map and 0 cM on the male map), a region delimited by recombinant events detected in affected children. Evidence from all 15 of the multiplex families analyzed provided support for the linkage, consistent with a single locus for SDS. However, the presence of several different mutations is suggested by the heterogeneity of disease-associated haplotypes in the candidate region.

Deficiency of Src homology 2-containing phosphatase 1 results in abnormalities in murine neutrophil function: studies in motheaten mice.

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me(v)/me(v) mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. MOTHEATEN: neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum.

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1(-/-)) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen. Lsp1(-/-) mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5(-) macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in Lsp1(-/-) mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1(-/-) mice are similar to those of wt mice and Lsp1(-/-) mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the Lsp1(-/-) mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1(-) neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis. (Blood. 2000;96:1827-1835)

Neutrophil products and alterations in epithelial junctional proteins: prevention of artifactual degradation.

Recent reports of disruption of endothelial cell adherens junction proteins during neutrophil adhesion and transmigration have been challenged as being partly due to post-fixation artifactual release of neutrophil-derived proteases. In this study we examined alterations in the epithelial junctional complex during neutrophil adhesion. Using standard fixation protocols, neutrophil addition to epithelial monolayers resulted in gross disruption of apical junction protein immunofluorescence. However, the inclusion of a post fixation incubation step with formic acid resulted in epitope preservation. These observations indicate that neutrophil derived products, likely proteases, remain active despite prolonged exposure to conventional fixatives. This may result in diffuse and artifactual loss of epithelial junctional protein immunofluorescence. Formic acid prevents this loss of epitope staining and may be considered as an agent to preserve protease-sensitive endothelial or epithelial immunoreactivity.

Segregation analysis in Shwachman-Diamond syndrome: evidence for recessive inheritance.

Shwachman-Diamond syndrome is a rare disorder of unknown cause. Reports have indicated the occurrence of affected siblings, but formal segregation analysis has not been performed. In families collected for genetic studies, the mean paternal age and mean difference in parental ages were found to be consistent with the general population. We determined estimates of segregation proportion in a cohort of 84 patients with complete sibship data under the assumption of complete ascertainment, using the Li and Mantel estimator, and of single ascertainment with the Davie modification. A third estimate was also computed with the expectation-maximization (EM) algorithm. All three estimates supported an autosomal recessive mode of inheritance, but complete ascertainment was found to be unlikely. Although there are no overt signs of disease in adult carriers (parents), the use of serum trypsinogen levels to indicate exocrine pancreatic dysfunction was evaluated as a potential measure for heterozygote expression. No consistent differences were found in levels between parents and a normal control population. Although genetic heterogeneity cannot be excluded, our results indicate that simulation and genetic analyses of Shwachman-Diamond syndrome should consider a recessive model of inheritance.

Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar.

OBJECTIVES: With the use of clinical data from a large international cohort, we evaluated and compared affected siblings and isolated cases. STUDY DESIGN: Data from 116 families were collected, and patients conforming to our predetermined diagnostic criteria were analyzed. Phenotypic manifestations of affected siblings and singletons were compared with the use of t tests, Wilcoxon scores, and chi2 analysis. RESULTS: Eighty-eight patients (33 female, 55 male; median age 5.20 years) fulfilled our predetermined diagnostic criteria for Shwachman syndrome; 63 patients were isolated cases, and 25 affected siblings were from 12 multiplex families. Steatorrhea was present in 86% (57 of 66), and 91% (78 of 86) displayed a low serum trypsinogen concentration. Patients older than 4 years more often had pancreatic sufficiency. Neutropenia occurred in 98%, anemia in 42%, and thrombocytopenia in 34%. Myelodysplasia or cytogenetic abnormalities were reported in 7 patients. Short stature with normal nutritional status was a prominent feature. CONCLUSIONS: Clinical features among patients with Shwachman syndrome varied between patients and with age. Similarities in phenotype between isolated cases and affected sibling sets support the hypothesis that Shwachman syndrome is a single disease entity.

Exclusion of linkage of Shwachman-Diamond syndrome to chromosome regions 6q and 12q implicated by a de novo translocation.

Shwachman-Diamond syndrome is a rare genetic disorder of unknown pathogenesis involving exocrine pancreatic insufficiency and hematological and skeletal abnormalities. There is broad clinical variability; the extent of heterogeneity is unknown but comparisons within a large cohort of patients show no striking differences between patients of families with single or multiple affected offspring. Segregation analysis of a cohort of 69 families has suggested an autosomal recessive mode of inheritance. A single constitutional de novo chromosome rearrangement was reported in a Japanese patient involving a balanced translocation, t(6;12)(q16.2;q21.2), thereby suggesting possible loci for a genetic defect. Evenly spaced microsatellite markers spanning 26-32 cM intervals from D6S1056 to D6S304 and D12S375 to D12S346 were analyzed for linkage in members of 13 Shwachman-Diamond syndrome families with two or three affected children. Two-point lod scores were calculated for each marker under assumptions of recessive inheritance and complete penetrance. Negative lod scores indicated exclusion of both chromosome regions. Further, affected sibs were discordant for inheritance of chromosomes in most families based on constructed haplotypes. The cytogenetic abnormality is not associated with most cases of Shwachman-Diamond syndrome.

Unconventional treatment of neuroenteric cyst in a newborn.

Neuroenteric cysts are uncommon congenital malformations that can require early surgical treatment. The authors report on an unusual treatment of a very large neuroenteric cyst that involved most of the small bowel and extended into the chest. A 1-day-old boy was admitted because of abdominal distension. The prenatal ultrasound results at 8 and 36 weeks had been normal. Examination showed right upper quadrant fullness and mild respiratory distress. A malformed sternum and asymmetric upper thoracic vertebra were seen on the initial x-rays. The possibility of a midthoracic right paravertebral mass was raised. Abdominal ultrasound findings were consistent with a large bowel duplication cyst. Laboratory results were all normal except the bilirubin level, which was (59 mmol/L). During laparotomy, the second part of the duodenum was found to enter a dilated cyst, and the terminal ileum arose from the cyst. The total length of the intact small bowel was 20 cm including a competent ileocecal valve. The site of the biliary duct entering the cyst was not clear. The surgical procedure involved partial resection of the anterior wall of the cyst, creation of an enteric tube from the posterior cyst wall to communicate between the duodenum and the ileum, and addition of another 25 cm of length to the small bowel. An enterocutaneous fistula was created with the proximal portion of the cyst because the site of the papilla of Vater was suspected to enter this part of the cyst. A postoperative HIDA scan showed good uptake with no excretion into the gut or the proximal pouch.(ABSTRACT TRUNCATED AT 250 WORDS)