Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn's disease.

BACKGROUND: Patients with Crohn's disease have defects in intestinal epithelial permeability that are inadequately explained by known inflammatory bowel disease (IBD) susceptibility genes. E-cadherin (CDH1) plays a vital role in maintaining the integrity of the intestinal barrier and its cellular localisation is disrupted in patients with Crohn's disease. AIM: To determine if polymorphisms in the CDH1 gene are associated with Crohn's disease and to determine the function associated with these polymorphisms. METHODS: The hypothesis was tested using a candidate gene approach using 20 Tag SNPs derived from the HapMap and Crohn's disease trios. Functional studies were carried out using HapMap cell lines and polarised epithelial cell lines (MDCK-1 and Caco2). RESULTS: Here we show that CDH1 is associated with Crohn's disease in 327 trios (rs10431923 excess transmission of "TT" genotype; p = 0.0020) and is replicated in the Wellcome Trust Case Control Consortium CD data set (TT risk allele; OR 1.2, p = 0.005). Patients with the Crohn's disease risk haplotype (rs12597188, rs10431923 and rs9935563; GTC allelic frequency 21%; p = 0.000016) exhibited increased E-cadherin cytoplasmic accumulation in their intestinal epithelium which may be explained by the presence of a novel truncated form of E-cadherin. Accordingly, expression of this truncated E-cadherin in cultured polarised epithelial cells resulted in abnormal intracellular accumulation and impaired plasma membrane localisation of both E-cadherin and beta-catenin. CONCLUSION: The mis-localisation of E-cadherin and beta-catenin may explain the increased permeability seen in some patients with Crohn's disease. Thus, the polymorphisms identified in CDH1 are important for understanding the pathogenesis of Crohn's disease and point to a defect in barrier defence.

Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress.

BACKGROUND AND AIMS: Chronic psychological stress, including water avoidance stress (WAS), induces intestinal mucosal barrier dysfunction and impairs mucosal defences against luminal bacteria. The aim of this study was to determine the ability of a defined probiotic regimen to prevent WAS induced intestinal pathophysiology. METHODS: Male rats were subjected to either WAS or sham stress for one hour per day for 10 consecutive days. Additional animals received seven days of Lactobacillus helveticus and L rhamnosus in the drinking water prior to stress and remained on these probiotics for the duration of the study. Rats were then sacrificed, intestinal segments assessed in Ussing chambers, and mesenteric lymph nodes cultured to determine bacterial translocation. RESULTS: All animals remained healthy for the duration of the study. Chronic WAS induced excess ion secretion (elevated baseline short circuit current) and barrier dysfunction (increased conductance) in both the ileum and colon, associated with increased bacterial adhesion and penetration into surface epithelial cells. Approximately 70% of rats subjected to WAS had bacterial translocation to mesenteric lymph nodes while there was no bacterial translocation in controls. Probiotic pretreatment alone had no effect on intestinal barrier function. However, WAS induced increased ileal short circuit current was reduced with probiotics whereas there was no impact on altered conductance. Pretreatment of animals with probiotics also completely abrogated WAS induced bacterial adhesion and prevented translocation of bacteria to mesenteric lymph nodes. CONCLUSION: These findings indicate that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract.

Anastomotic ulceration following small bowel transplantation.

We describe, for the first time, anastomotic ulcers (AU) following bowel transplantation at the Hospital for Sick Children. Two children presented with rectal bleeding, 6 and 9 months, following the transplantation. Isolated ulcers were identified at the ileo-colonic and the colo-colonic anastomosis site. The ulcers resolved, but recurred 6 and 7 months after the initial presentation. Both patients were positive for adenovirus in the stool and were treated with rapamycin. The histology revealed granulation tissue formation with mild inflammation in the adjacent mucosa, without evidence of rejection or infection. A literature search revealed 10 studies reporting 29 patients who developed AU following various surgical etiologies, none of which was bowel transplant. Numerous factors that are unique to the post-transplant period may predispose to such ulcer and are discussed in detail. Physicians and surgeons should be aware of this multifactorial complication, among other etiologies, as a cause of anemia or rectal bleeding following intestinal transplantation.

A case of alpha-fetoprotein-producing gastric cancer.

INTRODUCTION: A case of alpha-fetoprotein (AFP)-producing gastric cancer is described in a 57-year-old Chinese woman. CLINICAL PICTURE: She presented with bleeding tendency and bone pain, and was found to have haematological evidence of disseminated intravascular coagulation and spinal metastasis. Her tumour markers, including AFP, Ca 19-9 and carcinoembryonic antigen (CEA) were elevated. In view of the elevated tumour markers, there was an exhaustive search for a primary lesion in the gastrointestinal tract, liver and ovaries. There was no radiological evidence to suggest any lesion in the chest, liver or pelvis. Lectin affinity electrophoresis of the AFP showed AFP-L2 and AFP-L3 bands, which are suggestive of a non-hepatoma malignancy. MANAGEMENT: Gastroscopy showed a gastric ulcer and she developed bleeding after the gastric biopsy which required urgent surgery. Intraoperatively she was found to have carcinomatous peritone and a malignant ulcer in the greater curve of the stomach. Histology confirmed a linitis plastica like adenocarcinoma which stains for AFP. OUTCOME: She died from multi-organ failure 3 days after surgery. CONCLUSION: AFP-producing adenocarcinoma of the stomach is not uncommon. Lectin affinity electrophoresis of AFP is helpful in the differentiation between hepatoma and non-hepatoma malignancies.

B and T cells are not required for the viable motheaten phenotype.

Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease.

Hematopoietic transforming potential of activated ras in chimeric mice.

Although activating mutations in ras genes are the most common genetic abnormality in human hematologic malignancies, the role of ras mutations as an initiating event in leukemogenesis remains unclear. To assess the consequences of ectopic expression of an activated ras gene in normal hematopoietic cells in vivo, lethally irradiated mice were reconstituted with bone marrow cells infected with a mutant ras-containing retrovirus [murine stem cell virus (MSCV)-v-H-ras] based on the MSCV retroviral vector which efficiently transduces functional genes into hematopoietic stem/progenitor cells. Despite a marked myeloid leukocytosis detectable in the peripheral blood within 4 weeks of engraftment, none of 22 primary or secondary transplant recipients studied for longer periods of time presented with myeloid neoplasms. Instead, 18 of the MSCV-v-H-ras mice developed pre-T-cell thymic lymphomas and/or pre-B-cell lymphoblastic leukemia/lymphomas between 7 and 12 weeks post-transplantation. The pre-B and pre-T lymphoid tumors that arose in one animal were shown to harbor a common MSCV-v-H-ras provirus, indicating that the target cell for transformation was a bipotential lymphoid precursor. To more precisely examine the effects of activated ras expression on the behavior of hematopoietic progenitors, infected bone marrow cells were assayed in methylcellulose cultures under conditions favorable for growth of multilineage myeloid colonies or were passaged as bulk suspension cultures in the presence of various hematopoietic growth factors, including interleukin (IL)-3, IL-4, IL-6 and IL-7. MSCV-directed expression of v-H-ras selectively promoted the formation of large dense colonies comprised of monocyte-macrophages in methylcellulose cultures. When transferred to liquid cultures, the vast majority of the cells underwent terminal macrophage differentiation. By comparison, tumorigenic B-lymphoid and mixed lymphoid/myeloid cell lines were routinely established from the bulk suspension cultures, with cell lines of predominantly myeloid phenotype emerging only in IL-6-supplemented cultures. These results, considered together with previous findings, suggest that activating ras mutations could be an initiating genetic alteration in human acute lymphoblastic leukemia but are more likely to be a post-initiation change in human acute myeloid leukemia.

Isolated chloroma: the effect of early antileukemic therapy.

OBJECTIVE: To evaluate the effect of antileukemic chemotherapy administered at diagnosis on the survival of patients with isolated chloroma. DESIGN: Retrospective review of locally identified patients and analysis of cases from the medical literature. PATIENTS: The records of all patients with isolated chloroma identified at three teaching hospitals in Toronto between 1980 and 1994 were reviewed. A MEDLINE search was done to identify all cases of isolated chloroma reported in the English-language medical literature. Patients with a previous known hematologic disorder were excluded. MEASUREMENTS: The effect of therapy on 1) the interval between diagnosis of chloroma and diagnosis of acute myeloid leukemia and 2) survival was determined. RESULTS: 7 local patients and 83 published cases were identified, for a total of 90 evaluable patients. For the entire group, the median time to the diagnosis of acute myeloid leukemia was 9 months, and median survival was 22 months. Chemotherapy was administered to 49 patients (54%) at diagnosis of chloroma. Significantly fewer patients treated with chemotherapy subsequently developed acute myeloid leukemia (41% compared with 71%; P = 0.001). Survival was longer in patients treated with chemotherapy (> 50% alive with a median follow-up of 25 months compared with a median survival of 13 months for those initially untreated; P = 0.001). Multivariate analysis showed that neither local radiotherapy nor surgery had an effect on survival. CONCLUSIONS: Administration of antileukemic chemotherapy at diagnosis of chloroma is associated with a significantly lower probability of developing acute myeloid leukemia and with longer survival.

Sequence preservation of the third exon of the bcl-2 gene in non-Hodgkin's lymphoma: absence of somatic hypermutation.

The t(14;18) translocation juxtaposes the bcl-2 gene on chromosome 18 to a joining (J) gene segment of the immunoglobulin heavy chain gene (IgH) on chromosome 14. Up to 85% of non-Hodgkin's lymphomas (NHL) are t(14;18) positive. Recent reports have documented point mutations in the second exon of translocated bcl-2 alleles and postulated that immunoglobulin variable (V) region somatic hypermutation, related to Ig sequences approximately 250 Kb downstream, may be mediating these mutations. We have examined the third exon of bcl-2, directly adjacent to Ig sequences in the t(14;18), for point mutations. In particular, we studied the translated region of exon 3 in 45 NHLs by SSCP analysis and failed to detect a single point mutation. Further, we sequenced eleven t(14;18) breakpoints, including both bcl-2 and JH sequences, and detected only one point mutation, in a JH-derived sequence. We conclude that immunoglobulin V region somatic hypermutation does not induce point mutations into the t(14;18) breakpoint region or into the translated region of the third exon of bcl-2 alleles involved in the t(14;18) translocation, conserving the membrane insertion properties of the carboxyl tail of this protein.

H-ras oncogene mutation and human papillomavirus infection in oral carcinomas.

OBJECTIVE: Previous in vitro studies have demonstrated that transfection of an activated ras gene induces malignant transformation in epithelial cell lines infected with the human papillomavirus (HPV). The results of these studies support the hypothesis that HPV may cooperate with an activated ras gene in epithelial tumor carcinogenesis. To test this hypothesis in head and neck cancers, we screened 35 oral carcinomas for the presence of HPV DNA and for a mutated H-ras gene. DESIGN: The design of the study was screening survey type. Twenty-seven oral squamous cell carcinomas and eight verrucous carcinomas were analyzed for the presence of HPV DNA using the polymerase chain reaction, followed by Southern blot and probe hybridization. The tumors were also screened for point mutations of the H-ras gene using the polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Six (22%) of the 27 oral squamous cell carcinomas demonstrated point mutation in the H-ras gene. In addition, six tumors (22%) were positive for HPV DNA, with three tumors (11%) demonstrating both HPV DNA and H-ras gene point mutation. While the rate of simultaneous HPV infection and ras gene activation by point mutation was 11% in oral squamous cell carcinomas, 25% of oral verrucous carcinomas contained both HPV DNA and mutation in the H-ras gene. CONCLUSIONS: These results suggest a stronger association between HPV infection and activation of the H-ras gene in oral verrucous carcinomas. These results continue to confirm the multihit hypothesis of tumorigenesis and suggest that in some cases of oral cancer at least two of these events are H-ras gene mutation and HPV infection.

Alterations of the p53 tumor suppressor gene in diffuse large cell lymphomas with translocations of the c-MYC and BCL-2 proto-oncogenes.

Diffuse large cell lymphomas are aggressive tumors of B-cell origin. In some cases they arise from low-grade follicular lymphomas carrying the t(14;18) translocation, an event that leads to the overexpression of the BCL-2 gene product. More frequently, however, they lack the t(14;18) translocation. Rearrangements of the c-MYC proto-oncogene and mutations of the p53 tumor suppressor gene have also been documented in these lymphomas. This study examines the extent to which alterations in the BCL-2, c-MYC, and p53 genes co-exist within individual lymphomas. Eight diffuse large cell lymphoma cell lines and 11 diffuse large cell lymphoma tumors were assessed for genetic alterations in these three genes. Our results indicate that there is a heterogeneity in the oncogene/suppressor gene profile among diffuse large cell lymphomas. Two cell lines and one tumor carried alterations in all three genes, one cell line carried alterations of c-MYC and p53, and one primary tumor and one cell line carried p53 mutations and the t(14;18). Single alterations of BCL-2 and p53 were also observed. Another cell line had no alterations in any of these genes. The heterogeneity indicates that varied mechanisms may be involved in the generation of diffuse large cell lymphomas.

The bcl-2 oncogene in Hodgkin's disease arising in the setting of follicular non-Hodgkin's lymphoma.

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.

Progenitor cell hyperplasia with rare development of myeloid leukemia in interleukin 11 bone marrow chimeras.

Post 5-fluorouracil-treated murine bone marrow cells infected with a recombinant retrovirus (murine stem cell virus-interleukin 11 [MSCV-IL-11]) bearing a human IL-11 gene were transplanted into lethally irradiated syngeneic mice. Analysis of proviral integration sites in DNA prepared from hematopoietic tissues and purified cell populations of long-term reconstituted primary and secondary recipients demonstrated polyclonal engraftment by multipotential stem cells. High levels (100-1,500 U/ml) of IL-11 were detected in the plasma of the MSCV-IL-11 mice. Systemic effects of chronic IL-11 exposure included loss of body fat, thymus atrophy, some alterations in plasma protein levels, frequent inflammation of the eyelids, and often a hyperactive state. A sustained rise in peripheral platelet levels (approximately 1.5-fold) was seen throughout the observation period (4-17 wk). No changes were observed in the total number of circulating leukocytes in the majority of the transplanted animals (including 10 primary and 18 secondary recipients) despite a > 20-fold elevation in myeloid progenitor cell content in the spleen. The exceptions were members of one transplant pedigree which presented with myeloid leukemia during the secondary transplant phase. A clonal origin of the disease was determined, with significant expansion of the MSCV-IL-11-marked clone having occurred in the spleen of the primary host. Culturing of leukemic spleen cells from a quaternary recipient led to the establishment of a permanent cell line (denoted PGMD1). IL-11-producing PGMD1 myeloid leukemic cells are dependent on IL-3 for continuous growth in vitro and they differentiate into granulocytes and macrophages in response to granulocyte/macrophage colony-stimulating factor. The inability of autogenously produced IL-11 to support autonomous growth of PGMD1 cells argues against a mechanism of transformation involving a classical autocrine loop.

Detection of occult lymphoma in the peripheral blood and bone marrow of patients with untreated early-stage and advanced-stage follicular lymphoma.

PURPOSE: The object of this study was to compare the relative sensitivities of morphologic, immunophenotypic, gene rearrangement, cytogenetic, and polymerase chain reaction (PCR) analyses in the detection of lymphoma cells in the bone marrow and peripheral blood of patients with follicular lymphoma. PATIENTS AND METHODS: Bone marrow and peripheral-blood samples from 28 newly diagnosed patients with follicular lymphoma referred from several different medical centers were assessed. Routine morphologic assessment was performed initially and the remainder of the sample was aliquoted for DNA extraction to be used for gene rearrangement and PCR analyses and for immunophenotypic and cytogenetic analyses where a sufficient amount of sample remained. RESULTS: Morphologic assessment of the bone marrow was positive for lymphoma cells in 11 of 28 patients. PCR amplification of t(14;18) breakpoint DNA detected lymphoma cells in 17 of 24 patients assessed. Morphologic assessment detected lymphoma cells in three bone marrow samples that were negative by PCR. PCR analysis was the only method able to detect circulating lymphoma cells in peripheral blood at diagnosis and was positive in 15 of 24 samples. The other methods of assessment did not show lymphoma in any samples in which lymphoma was not detected by morphologic or PCR analysis. Lymphoma cells were found in the bone marrow and/or peripheral blood as frequently in early-stage patients as in advanced-stage patients. CONCLUSION: PCR amplification of t(14;18) breakpoint DNA together with morphologic assessment had the highest yield of detecting lymphoma cells in the bone marrow and/or peripheral blood of our population of newly diagnosed patients with follicular lymphoma. The clinical significance and prognostic importance of lymphoma cells detected by PCR in the bone marrow and/or peripheral blood of newly diagnosed follicular lymphoma patients awaits long-term follow-up data of these and additional patients.

Prevalence of RAS oncogene mutation in head and neck carcinomas.

RAS genes encode for a protein (p21) known to play an important role in the regulation of normal signal transduction and cell growth. Activation of RAS genes have been strongly implicated in the pathogenesis of cancer in cell line studies, animal models and in human tumors. RAS genes have been shown to be mutated in 10 to 15% of human solid tumors but the frequency of mutation varies widely depending on the tumor type. The prevalence of RAS mutation has not been well-established in head and neck squamous cell carcinomas (SCC). The purpose of our study was to screen a relatively large number (50) SCC tumors using a gene amplification technique, the polymerase chain reaction (PCR). H-RAS gene mutation is identified by diagnostic restriction length polymorphism, created by introducing specific mismatched primers in the PCR. The first 20 tumors were also amplified and directly sequenced for K-RAS codon 12 and 13. Four of the 50 screened tumors were positive for H-RAS codon 12 mutation. All tumor DNA screened normal at codon 61 and the first 20 tumors were also normal at K-RAS codon 12 and 13. The prevalence of RAS mutations appears to be low in head and neck squamous cell carcinomas. Tumors positive for point mutation in the H-RAS gene revealed some unusual clinical characteristics.

Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization.

Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.

Castleman's disease associated with pemphigus vulgaris.

Castleman's disease is a rare, benign, lymphoproliferative disorder of unknown cause. The hyaline-vascular type is frequently associated with a localized mediastinal mass. The plasma-cell type is associated with constitutional symptoms, multicentric lymph node involvement, lymphoma development, and autoimmune disease-like laboratory abnormalities such as elevated erythrocyte sedimentation rate, anemia, and thrombocytopenia. We report a case of hyaline-vascular Castleman's disease associated with a cutaneous autoimmune disease, pemphigus vulgaris. We also reviewed the clinicopathologic features of four similar cases. Among these five reports of Castleman's disease, five patients had severe erosive stomatitis diagnosed as oral pemphigus, three had keratoconjunctivitis, and three had circulating pemphigus antibodies. All were young, ranging in age from 15 to 21 years, and four of the five were women. Two had hyaline-vascular Castleman's disease, whereas three had plasma-cell Castleman's disease. All five had surgical resection of the Castleman's disease mass. After surgery, remission of pemphigus vulgaris could be achieved with reduced dosages of steroids in all cases. In at least two cases steroid treatment could be completely discontinued. We postulate that an underlying immune dysfunction in Castleman's disease facilitates the expression of pemphigus.

Monocytoid B-cell lymphoma: a study of 36 cases.

We have studied 36 cases of monocytoid B-cell lymphoma (MBCL). We confirm the predilection for females (30 of 36; ratio, five women to one man). The median age was 65 years (range, 29 to 85 years). Monocytoid B-cell lymphoma characteristically involves peripheral lymph nodes (30 of 36) with a propensity for paraparotid or intraparotid nodes. Salivary glands were affected in five patients. Other extranodal sites of involvement included breast, thyroid, stomach, and soft tissue of chest wall. Eight patients manifested with Sjögren's syndrome, one had systemic lupus erythematosus, one presented initially with Raynaud's phenomenon, and two had a monoclonal gammopathy. "Composite lymphomas" were encountered in seven patients. In addition, association with or progression to a higher-grade lymphoma, ie, mixed small and large cell (one) and large cell (six), was observed in seven patients and was associated with a more aggressive behavior of the lymphoma. Immunohistochemical studies performed on biopsy sections from 20 patients confirmed the B-cell nature of MBCL. An average reactivity of less than 10% of the monocytoid B cells with the proliferation marker Ki-67 was demonstrated, in keeping with the indolent behavior of MBCL. Despite our observation of follicular lymphomas frequently accompanying MBCL, the t(14;18) chromosomal translocation does not appear to play a pathogenetic role for MBCL, as determined by molecular studies for the t(14;18) chromosomal translocation and immunologic studies for the BCL2 protein. Our observations also provide support for the proposal that there is an overlap between MBCL and "MALT lymphomas" (those arising from mucosa-associated lymphoid tissue).