Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition.

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

Regulation of blood flow by aspirin following muscle ischemia.

BACKGROUND: The vascular endothelium secretes a balance of dilator and constrictor substances which regulate vascular tone. During ischemic stress, this balance changes. After a short period of ischemia, a protective mechanism known as reactive hyperemia (RH) contributes to a post-ischemic increase in blood flow. The agents regulating this phenomenon remain controversial. AIM: The purpose of this study was to examine whether aspirin regulates vascular endothelial function following ischemia. METHODS: Sixteen healthy volunteers presented for two visits, each serving as their own control, and randomized to receive 500 mg aspirin or placebo. Forearm blood flow (FBF) was measured at baseline and during reactive hyperemia (RH) which was induced by five minutes of arterial occlusion. Blood samples were analyzed for vWF and lipids. RESULTS: After ischemia, RH was attenuated when subjects were pre-medicated with 500 mg aspirin compared to placebo: AUC[aspirin] = 1450 +/- 201 mL/100 mL tissue/min vs. AUC[pIacebo] = 2207 +/- 294 mL/100 mL tissue/min; (p < 0.05). Separation of the subjects with high HDL or low HDL levels resulted in a similar peak FBF response with placebo, but in the high-HDL group only, aspirin ingestion attenuated peak FBF after ischemia compared to the placebo condition (22.6 +/- 1.7 m/100 mL tissue/min vs. 33.5 +/- 3.2 mL/100 mL tissue/min, respectively) (p < 0.05). CONCLUSIONS: Aspirin partially regulates the RH response following ischemia compared to placebo, and this effect appears to be more profound when adjusting for plasma HDL concentration in healthy individuals. This suggests that the post-ischemic RH response may be partially mediated by arachidonic acid-derived mediators such as the prostaglandins.

MIPS: a database for genomes and protein sequences.

The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz-Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91-93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155-158; Barker et al. (2001) Nucleic Acids Res., 29, 29-32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de).

Impaired endogenous fibrinolytic capacity in prehypertensive men.

Prehypertension (blood pressure (BP) 120-139/80-89 mm Hg) is associated with an increased risk for future atherothrombotic events. Although the mechanisms underlying this elevated risk are not completely understood, one possibility is that prehypertension is associated with impaired endothelial fibrinolytic capacity. We tested the hypothesis that vascular endothelial release of tissue-type plasminogen activator (t-PA) is impaired in prehypertensive men. Net endothelial release of t-PA was determined, in vivo, in response to intrabrachial infusions of bradykinin (12.5, 25, 50 ng per 100 ml tissue per min) and sodium nitroprusside at (1.0, 2.0, 4.0 µg per 100 ml tissue per min) in 42 middle-age and older men: 16 normotensive (BP range: 100-119/57-79 mm Hg); 16 prehypertensive (BP range: 120-139/76-89 mm Hg); and 10 hypertensive (BP range: 140-150/74-100 mm Hg). Net release of t-PA antigen was ~25% lower (P<0.05) in the prehypertensive (-0.9 ± 0.8 to 42.4 ± 5.3 ng per 100 ml tissue per min) compared with the normotensive (0.5 ± 1.0 to 53.9 ± 6.5 ng per 100 ml tissue per min) men. There was no significant difference in t-PA release between the hypertensive (-1.8 ± 1.6 to 40.8 ± 6.6 ng per 100 ml tissue per min) and prehypertensive groups. Sodium nitroprusside did not significantly alter the t-PA release in any group. These data indicate that endothelial t-PA release is diminished in prehypertensive men. Further, the level of impairment in t-PA release seen with clinical hypertension is already apparent in the prehypertensive state. Impaired endothelial fibrinolytic function may underlie the increased atherothrombotic risk associated with BP in the prehypertensive range.

The new gene DmX from Drosophila melanogaster encodes a novel WD-repeat protein.

DmX is a novel gene from Drosophila melanogaster located on the X chromosome in region 5D5/6-E1. The molecular analysis of the genomic and cDNA sequences of DmX shows that the gene spans appr. 16kb and displays a mosaic structure with 15 exons. The 12kb long DmX transcript is present in Drosophila embryos, larvae and adults of both sexes. The open reading frame of DmX encodes a novel WD-repeat protein, containing at least 30 WD-repeat units. WD-repeat proteins contain a conserved motif of approximately 40 amino acids (aa), usually ending with the dipeptide Trp-Asp (WD). Homologues of the DmX gene exist in other dipteran species, in Caenorhabditis elegans and human, revealing that DmX is an evolutionarily well conserved gene. The inferred DMX amino acid sequence shows also limited, but significant similarity to a yeast ORF with unknown function. 1998 Elsevier Science B.V.

Nonprocurement of transplantable organs in a tertiary care hospital: a focus on sociological causes.

BACKGROUND: The purpose of this study was to investigate prospectively the medical and organizational causes of nonprocurement of transplantable organs and to provide explicit information on the determining factors of family response to donation request. METHODS: Medical causes investigated were age, human immunodeficiency virus, human T-cell lymphoma virus and hepatitis C virus status, documented malignancy, and chronic cardiac or renal failure. Organizational aspects investigated were cause and place of death and number of referrals. Sociological aspects were investigated by semidirective interviews with the families of the deceased. RESULTS: A total of 105 brain-dead patients and 42 families were included. Of the 105 patients, 9 were not eligible for donation because of medical reasons; cardiac arrest occurred before organ procurement in 6 cases. Denial of consent from the coroner occurred in 7 cases. Consent was requested from the families in 82 cases, obtained in 53 cases, and denied in 29 cases. Consent to donation was associated with openness of the process, information about brain death and transplantation, previously stated will of the deceased, favorable attitude of the deceased toward the medical profession, and a generally altruistic attitude of the deceased. Denial of donation was associated with poor understanding of brain death and fear of being deprived of the deceased body. CONCLUSIONS: Practical implications of this study include encouraging people to state their attitudes toward organ donation and improving the level of information available to the general public on the meaning of brain death and on the medical aspects of organ transplantation.

Renal transplantation in a patient with hypocomplementemic urticarial vasculitis syndrome.

We describe a 36-year-old man who presented with hypocomplementemic urticarial vasculitis syndrome (HUVS) with severe renal involvement. Despite steroid therapy, the patient developed end-stage renal disease (ESRD) leading to chronic hemodialysis therapy. Renal transplantation was performed after hemodialysis therapy (secondary), and the patient developed a typical HUVS relapse 9 months after transplantation despite conventional immunosuppressive therapy that was successfully treated with plasma exchange. This case shows for the first time that HUVS can induce severe renal involvement responsible for ESRD and that HUVS can relapse after renal transplantation. It also suggests that plasma exchange therapy may be of value for rapidly controlling the clinical symptoms.

Adherence and migration of guinea pig granulocytes after enzyme treatment of the cell surface.

Glycogen-induced polymorphonuclear granulocytes (PMN) from the peritoneal cavity of guinea pigs were examined (1) for their adherence to nylon fibers in the absence and presence of the adherence-enhancing chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-MLP), (2) for their random migration through the filter of a Boyden chamber and (3) for their chemotactic migration towards f-MLP. The cells were analyzed before and after treatment with the enzymes neuraminidase, papain and trypsin. PMN adhesiveness was increased by neuraminidase digestion but reduced by treatment with the proteolytic enzymes. Neuraminidase and trypsin had no effect on cell migration, while papain reduced random migration without affecting f-MLP-induced chemotaxis. The data suggest that the type of adherence measured by the nylon fiber method differs from the temporary attachment of cells migrating through a chemotaxis filter towards an attracting substance.

The effects of bovine serum albumin and the chemotactic peptide formyl-methionyl-leucyl-phenylalanine on the adherence of guinea pig polymorphonuclear leukocytes to nylon fiber columns.

Glycogen-induced guinea pig polymorphonuclear leukocytes were passed over nylon fiber columns and adherent and nonadherent fractions were quantitated by determination of hexosaminidase activity after lysis with Triton. Bovine serum albumin reduced the number of adherent cells at 37 degrees C as well as 4 degrees C, after fixation of the cells with glutaraldehyde, and in the absence and presence of EDTA. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine increased the adherent cell fraction at 37 degrees C, but failed to do so at 4 degrees C, after glutaraldehyde fixation and in the presence of 10 mmol/EDTA. Since metabolic inhibitors had no effect on the rate of adherence it is suggested that increased cell deformability is responsible for chemotactic factor-induced increased cell attachment to nylon fibers.

Bcl-2 protein in normal, hyperplastic and neoplastic breast tissues. A metabolite of the putative stem-cell subpopulation of the mammary gland.

This investigation, though initially designed to examine the possible influence of the Bcl-2 protein on the node-metastasizing capacity of breast carcinomas, was amplified to study the expression of this anti-apoptotic protein in normal breast lobules and hyperplastic lesions. We examined paraffin sections of 508 breast carcinomas, stained for Bcl-2, estrogen (ER) and progesterone receptors (PgR) and epithelial membrane antigen, and occasionally for other antigens as well. Only a few cells showing a strong Bcl-2 positivity spotted the tubulo-lobular units of normal resting glands, whereas such cells were relatively numerous in atrophic lobules, and very scarce in the terminally differentiated lactating breast. Columnar and usual types of hyperplasia were exclusively, or almost exclusively, composed of Bcl-2(+), ER(+) and PgR(+) cells. The foci of carcinoma in situ and those of invasive carcinomas were respectively 83% and 66% positive for Bcl-2 in at least 25% of their cells. Even among the invasive carcinomas, Bcl-2(+) cases included 83% and 87% of the ER(+) and PgR(+) cases, respectively (p=0.0001). Though there was a statistically significant inverse relation between Bcl-2 and tumor grade (p=0.0001), no significant association was found between Bcl-2 and lymph node stage. In conclusion, we suggest that normal, hyperplastic and neoplastic breast epithelial cells expressing the anti-apoptotic protein Bcl-2 are immature cells that ought to form part of the stem-cell subpopulation, which is committed to the development and to the maintenance of the normal gland and which gives rise to hyperplastic and neoplastic disorders when its proliferation is deregulated. In ductal proliferative changes Bcl-2 assays may be useful for diagnostic but not for prognostic purposes.

Na(+)-dependent succinate uptake in Corynebacterium glutamicum.

Succinate is effectively taken up by washed cells of Corynebacterium glutamicum. The apparent Km value of uptake is about 150 microM and the Vmax 4-7 nmol (mg dry weight)-1 min-1 and uptake can be competetively inhibited by fumarate and oxaloacetate. The activation energy was determined to be 50 kJ/mol. The transport activity is clearly dependent on the presence of Na+ ions in the incubation medium and on the membrane potential and has a pH optimum around 8.5. It is concluded that succinate is taken up in C. glutamicum via a specific carrier by a secondary active, Na+ coupled mechanism.

Lymphopenia and abnormal balance of T-lymphocyte subpopulations in toxic epidermal necrolysis.

A lymphopenia (peripheral-blood-lymphocyte count less than 1,000/mm3) was observed in seven out of ten patients with toxic epidermal necrolysis (TEN). The enumeration of T-lymphocyte subsets with monoclonal antibodies showed a decreased number of pan T-lymphocytes (OKT3-positive), which was related to a profound depletion of OKT4-positive cells. In contrast, OKT8-positive cell counts were not significantly changed. This abnormal balance of T-lymphocytes was linked to the acute phase of the disease and was not found after recovery. The pathogenetic mechanisms of such T-lymphocyte abnormalities in TEN remain unclear.

A cost analysis of the prevention of end-stage renal disease: immunoglobulin therapy for IgA nephropathy.

A cost analysis was used to evaluate three possible immunoglobulin (IgG) treatment protocols for end-stage renal disease due to IgA nephropathy. The perspective chosen for the cost analysis was that of the health-care delivery system. The baseline strategy was the absence of IgG treatment, and alternative strategies corresponded to three protocols presently on trial: all three included a high initial dose of intravenous IgG. Protocol 1 followed with intramuscular IgG injections only, protocol 2 with intramuscular plus intravenous injections, and protocol 3 with intravenous injections only. The costs of treatment included the costs of immunoglobulins, outpatient hospital costs, and the costs of tests; the saving (costs averted) resulted from kidney dialysis averted. The bottom line for the health-care system is a net savings of $233,000, $213,000, or $83,000, depending on the protocol chosen. The computation of costs did not value physical and psychological health benefits. Thus, any subjective benefit, such as improved comfort, or objective benefit, such as longer life expectancy, would be an improvement over the results presented here.

Iguana-associated salmonellosis in a young adult.

PURPOSE: Review a case of Salmonella infection in a young adult related to handling of an infected iguana. METHODS: Case report. RESULTS: Most cases of Salmonella infection related to handling of reptiles have occurred in children. We report a case of Salmonella diarrhea in a 19-year-old male who kept a pet iguana. The iguana was asymptomatic, but Salmonella grew from stool specimens. CONCLUSIONS: Those who keep iguanas as pets, which are particularly attractive to adolescents and young adults, should be aware that iguanas frequently carry Salmonella. Those caring for adolescents and young adults should always inquire into the pet-keeping habits of their patients when illnesses develop.

EMA: a differentiation antigen related to node metastatic capacity of breast carcinomas.

Epithelial membrane antigen (EMA), also known as MUC1, is a mucinous glycoprotein fixed to the luminal domain of the epithelial cell membrane of normal breast ducts. However, in breast cancer cells, it is usually dispersed in the cytoplasm. EMA staining patterns of 330 breast carcinomas were examined, and three groups formed: lineal (16%), cytoplasmic (75%), and negative (9%). Although these patterns were somewhat related to histological cancer types, this was not statistically significant. However, EMA showed statistically significant univariate relationships to tumor grade, tumor size, estrogen and progesterone receptors, and nodal stage. Logistic regression analysis showed that among these variables, all of which were univariately related to node metastasis, only tumor size and EMA were independent nodal stage predictors. A combined analysis of these two factors revealed that the statistical probability of a tumor metastasizing to four or more nodes increased in each tumor size group from 0.9% to 12% for pT1, from 2% to 29% for pT2 and from 10% to 63% for pT3, depending on the EMA staining. The tumors showing a lineal pattern were the least metastasizing, while the EMA-negative tumors were the most. After recognizing these relationships between EMA staining patterns and other well-known differentiation markers and the lymph node metastatic capacity of carcinomas, and considering the results obtained by others on survival, one might conclude that EMA is both a differentiation marker and a histological prognostic factor.

[Immune deficiency associated with experimental graft-versus-host reaction. Lack of correlation with T-cell cytotoxic activity]. / Déficit immunitaire associé à la réaction expérimentale du greffon contre l'hôte. Absence de corrélation avec l'activité cytotoxique des lymphocytes T.

Graft-versus-host reactions (GvHR) are initiated by T lymphocytes. In mice, the T cell subset involved depends on the incompatibility for minor or major histocompatibility antigens between donor and host. However, the correlation between phenotype and function is not absolute, and anti-host cytotoxic T cells can be detected in recipients without GvHR. We have presently investigated in a P----F1 model differing at the MHC, whether GvH associated immunosuppression was correlated with donor cytotoxic T cell activity. The immunodeficiency was tested by the ability of the F1 mice to generate a cytotoxic T cell response against TNP self or an alloantigen. F1 specific parental cytotoxic T cells generated in vitro induced less immunosuppression than naive parental spleen cells. Specific in vivo priming increased the cytotoxicity of parental spleen cells, but decreased their capacity to induce GvH associated immunosuppression. In contrast, non specific priming resulted the usual immunodeficiency. Spleens of the F1 mice injected with specific cytotoxic T cells were very enlarged, suggesting that these cells remained capable of inducing a GvHR without generating immunosuppression.

[Immunodeficiency caused by graft versus host reaction. Extra-thymic effect of thymus in restoring immunity]. / Déficit immunitaire lié à la réaction du greffon contre l'hôte. Influence extra-thymique du thymus dans la restauration de l'immunité.

Cell-mediated immunity in lethally irradiated graft-versus-host mice can be restored after repopulation with syngeneic bone marrow cells and thymus grafting. The thymus is not only required for the maturation of T lymphocytes, but also for the inhibition of a radioresistant mechanism resulting in lymphoid cell rejection.

[Mono- and oligoclonal immunoglobulin anomalies in kidney transplant patients]. / Les anomalies immunoglobulinémiques mono- et oligoclonales chez les transplantés rénaux.

Serum from 115 HIV negative renal transplant recipients having more than 6 months follow-up was tested for the presence of mono- or oligoclonal immunoglobulins (moIg) by immunoelectrophoresis or immunofixation. Mono/oligoclonal gammapathy was detected in 16 patients (13.9%). Eight of these patients had only one monoclonal band, whereas the other eight had two or more bands. Thirteen of the 16 patients (81.3%) were IgG kappa positive, nine (56.3%) were IgG lambda positive, four (25.0%) were IgM lambda positive and only one (6.3%) was IgM kappa positive. Six monoclonal patients (37.5%) were IgG kappa positive and two monoclonal patients (12.5%) were IgG lambda positive. The oligoclonal combination IgG kappa lambda was present in three patients (18.8%), the combination IgG lambda + IgM lambda was present in two patients (12.5%) and IgG lambda + IgM lambda was present in one patient. The triple combination IgM kappa lambda + IgG kappa lambda and IgM lambda + IgG kappa lambda was found in two patients (12.5%). Ninety percent of these moIg did not exceed 2 g/L. MoIg appeared between 1 and 28 months after the kidney transplantation (mean value: 8.5 5.9 months) but were often transient, disappearing within 1 to 19 months in 13 patients (81.3%). Nine of the 16 cases (56.3%) disappeared before the end of the first year after detection. Risk factors for the appearance of these immunoglobulins have been identified as: the patient's age, the duration of haemodialysis, the occurrence of prior (anti-cytomegalovirus [CMV]) infection, and therapy with cyclosporin A (CsA). The persistence of monoclonal gammapathy was associated with acute or reactivated Epstein-Barr virus (EBV) infection and inability to convert IgM to IgG CMV antibodies. Furthermore, no association was established with previous hepatitis B or C infection or the number of rejection episodes. Kaposi's sarcoma was found in one patient (6.3%) but had no correlation with the presence of moIg. We recommend careful follow up of renal transplant patients in whom moIg have been discovered.