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Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1-positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons.

Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide–cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.

HISTORY AND CLINICAL FINDINGS About 10 weeks before admission to hospital a 73-year-old woman developed a fever of up to 40 degrees C for three days. She then had subfebrile temperature for several weeks with some rises to 39 degrees C. She was known to have type II a diabetes mellitus and pulmonary silicosis, having worked in a porcelain and ceramic factory for many years. Before admission her cerebral functions were rapidly deteriorating, especially short-term memory. This was followed by increasing paraplegia of the legs with inability to walk. She finally had urinary and faecal incontinence and swallowing difficulties with tendency to aspiration, which necessitated hospitalisation. INVESTIGATIONS Both lactate dehydrogenase (339 U/l) and C-reactive protein (112 mg/l) were elevated; the platelet count was low (73000/microliters). Cerebrospinal fluid was unremarkable, as was computed tomography of the skull. But magnetic resonance imaging revealed multiple spotty lesions with low contrast-medium uptake throughout the brain, pointing to a disseminated bacterial or mycotic infection. 3 days later the chest-ray showed small nodular soft shadows in the lungs, and lung functions had decreased. Mycobacteria were found in the urine and liver biopsy showed granulomatous hepatitis, establishing the diagnosis of miliary tuberculosis in the presence of silicosis. TREATMENT AND COURSE Tuberculostatic treatment was instituted with four drugs (pyrazinamide, ethambutol, isoniazid and streptomycin. After 6 weeks the patient was again able to walk and continent of urine during the day. All cerebral functions gradually improved. CONCLUSION Miliary tuberculosis should be included in the differential diagnosis of ill-defined feverish disease, especially in the elderly.

Anamnese und klinischer Befund: Bei einem 57jahrigen Patienten mit chronischer Diarrhoe war seit Jahren keine Diagnose gefunden worden. Die bis zu 15 wasrigen Stuhle pro Tag waren unabhangig von der Nahrungsaufnahme und ohne Blutbeimengung. Begleitend waren Muskelkrampfe vor allem in den Beinen aufgetreten. Seit 15 Jahren waren rezidivierende Ulcera ventriculi et duodeni bekannt, deswegen war vor 13 Jahren eine selektive proximale Vagotomie durchgefuhrt worden. Der korperliche Untersuchungsbefund war unauffallig. Untersuchungen: Leicht erhoht war die Aktivitat der alkalischen Phosphatase (182 U/l) und der Spiegel des C-reaktiven Proteins (9,3 mg/l); der Serumeisenspiegel betrug 42 µg/dl. Alle anderen Parameter des Routinelabors einschlieslich Eiweiselektrophorese, Blutbild und Differentialblutbild, lagen im Normbereich. Eine Gastroskopie erbrachte eine ulzerierende Duodenitis, eine erosive Gastritis mit zahlreichen Ulcera und eine Refluxosophagitis III°-IV°. Endosonographisch fiel ein vergrobertes Schleimhautrelief des Magens auf als Zeichen der foveolaren Hyperplasie und ein etwa 4 × 3 cm groser Tumor neben dem Bulbus duodeni. Der daraufhin bestimmte Gastrinspiegel war auf 7537 pg/ml erhoht (Normwert

The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG. Surprisingly, MSI was not detected in 86MI and it appears to express all the major MMR components hMSH2, hMSH6, hMLH1, hPMS2, hMSH3, hMLH3, MBD4 (MED1) and hExo1. These data are consistent with at least two possibilities: (1) A missense mutation in one of the MMR genes, which dissociates MSI from drug resistance, or (2) inactivation of a second pathway that leads to MMR-deficiency and MNNG resistance, but induces negligible levels of MSI. We conclude that MMR deficiency is largely not associated with the pathogenesis of SCLC.

Objective. MLH1 methylation is associated with the microsatellite instability (MSI) phenotype in endometrial cancer and atypical endometrial hyperplasia, a premalignant precursor to carcinoma. The observation that methylation is also seen in atypical endometrial hyperplasia without MSI suggests that methylation is an early event in endometrial tumorigenesis. Our objective was to determine if methylation is always present in MSI-positive atypical hyperplasia concomitant with MSI-positive, methylation-positive carcinoma. Methods. We used laser capture microdissection to study MLH1 methylation and MSI in a large series of endometrial cancer cases that had previously been shown to have methylation and the MSI-high (MSI-H) phenotype. We resampled areas of carcinoma from 27 patients along with 51 foci of concomitant atypical endometrial hyperplasia. Results. Consistent with previous reports, we saw MLH1 methylation in areas of atypical endometrial hyperplasia that did not show MSI. In addition, we noted that 18% of the MSI-H atypical endometrial hyperplasia DNAs lacked methylation of critical cytosines in the MLH1 promoter. Immunohistochemistry studies showed that these MSI-H unmethylated foci of atypical endometrial hyperplasia failed to express MLH1, as did regions of simple hyperplasia. Conclusion. Methylation of the MLH1 promoter is an early event in endometrial tumorigenesis. Given that not all MSI-positive tissues had methylation at cytosines −229 and −231, it appears that methylation may not be required for MLH1 silencing and loss of mismatch repair.

In order to demonstrate the relationship between microsatellite instability and other types of genomic instability, a series of 56 sporadic colorectal carcinomas was investigated by flow cytometrical ploidy analysis, oligonucleotide fingerprinting, and microsatellite polymerase chain reaction (PCR). Stabilization of the p53 gene product was analysed by immunohistochemistry and proliferative activity was determined flow cytometrically and by silver staining of nucleolar organizer regions (AgNORs). Of the 56 carcinomas, 11 (19 per cent) exhibited microsatellite instability ; 33 of the cases were aneuploid (59 per cent) and 29 (52 per cent) showed alterations of the oligonucleotide fingerprints. There was a significant correlation of microsatellite instability with localization of these tumours proximal to the splenic flexure, diploid DNA content, and less frequent p53 stabilization. A solid growth pattern, mucinous differentiation, and a Crohn's-like lymphoid infiltrate were also characteristic for those tumours. The results demonstrate for the first time a significantly lower proliferative activity in tumours with microsatellite instability. Data obtained from DNA flow cytometry or from oligonucleotide fingerprinting did not correlate with such tumour characteristics. It is proposed that the use of microsatellite PCR facilitates specifically the detection of a group of colorectal cancers which may differ in pathogenesis and perhaps prognosis.

Objective : Previous studies have revealed large variations in the interobserver agreement of dysplasia grading in inflammatory bowel disease. Therefore, we investigated the diagnostic value of two novel monoclonal antibodies (MIB 1 against Ki-67 and PC 10 against PCNA) in the detection of dysplasia. Methods : A total of 62 biopsies were investigated and histologically classified as follows : 13 probably positive for dysplasia ; 15 low-grade dysplasia ; five high-grade dysplasia ; and 15 inflammation without dysplasia and 14 normal controls. The percentage of positive Ki-67- or PCNA-stained nuclei (= labelling index) was determined in relation to the distribution throughout the mucosa. Results : In all biopsies PCNA-labelling index exceeded that of Ki-67-labelling index. In the superficial half of the crypt PCNA- and Ki-67-labelling indices in the biopsies with 'indefinite for dysplasia, probably positive' or low-grade dysplasia exceeded that of normal tissue (P

Three cases of solitary circumscribed neuroma (so-called palisaded, encapsulated neuroma) arising in the dermis have been studied. Two patients were male and one was female, with ages ranging from 27 to 59 years. All lesions arose on the forehead. Histologically, the neoplasms were partly encapsulated and were composed of broad fascicles, nests, and whorl-like structures of bland appearing spindle cells often separated by artefactual clefts. Immunohistochemically, most of the tumour cells stained positive for S-100 protein, and a considerable number also for CD 34, but failed to stain for epithelial membrane antigen and melanoma associated antigens. Solitary circumscribed neuroma has to be distinguished from long-standing dermal melanocytic nevus, mucosal neuromas seen in type IIb multiple endocrine neoplasia (MEN) syndrome, classical and cellular schwannoma, neurofibroma, perineurioma, traumatic neuroma, and angioleiomyoma.

The analysis of multifactorial diseases requires the efficient investigation of large numbers of (gene) loci and patient (family) samples. Since simple repetitive DNA markers are dispersed all over the chromosomes, molecular techniques employing these tools render most conventional screening procedures obsolete. Examples of tumors, autoimmune diseases and infections are presented to validate concepts of indirect gene diagnoses via simple, tandemly arranged, repetitive DNA sequences. The salient advantages of microsatellite technologies vs. those of multilocus DNA fingerprinting are weighed.

The effect of the microbial hyaluronic acid splitting enzyme hyaluronate lyase produced by Streptococcus agalactiae was investigated in vitro in human atherosclerotic plaque specimens and in vivo on Watanabe heritable hyperlipidaemic rabbits (WHHL) as an animal model for familiar hypercholesteraemia. The in vitro presence of the enzyme caused a partial destruction of the atherosclerotic plaque surfaces as well as releasing of glucuronic acid and solid calcium-containing materials from pieces of atherosclerotic plaques in human arteries. Accordingly hyaluronic acid seems to be the main component for anchoring of calcium deposits on the plaque surfaces. Repeated intravenous injections of hyaluronate lyase in WHHL rabbits resulted in a tendency of decreased formation of atherosclerotic plaques. The observed effects are discussed to be primary the result of the splitting of hyaluronic acid in the vessels.

Adenomas are well established as precursors of colorectal cancer. In most cases the progression to malignancy is not a rapid process. However, in Hereditary Non-Polyposis Colorectal Carcinoma (HNPCC) an earlier onset of adenomas and a faster progression to cancer is well known. 122 known HNPCC patients were followed for five years to detect colorectal adenomas. The goal of the study was to determine whether microsatellite instability (MSI) can be identified at the premalignant stage. 36 patients were identified who had developed a total of 71 adenomas. The adenomas were analyzed for MSI and loss of mismatch repair (MMR) protein expression by immunohistochemistry.

Since gastric cancer is an exceptional heterogeneous tumor conflicting results have been obtained about the relationship between genotype and phenotype. From the molecular point of view gastric carcinoma diffuse type forms a distinct entity which is microsatellite stable, has almost no p53 mutations and exhibits in at least half of the cases mutations in the E-cadherin gene. In contrast, all other gastric carcinomas comprise a heterogeneous group of which about one third exhibits microsatellite instability (MSI) but no p53 protein stabilization or gene mutations. These tumors are either of pure intestinal (glandular) type or show large solid (medullary) tumor cell clusters. Thereby, in sporadic gastric cancer MSI is caused by loss of hMLH1 expression due to hypermethylation of the promotor region rather than by mutation of the gene itself. Tumors that are microsatellite stable (MSS) and show p53 alterations are either intestinal (about 70%) or a mixed-type encompassing at least 5% glandular and poorly differentiated diffuse components (about 30%). Whereas pure diffuse type gastric cancer is unlikely to develop from intestinal type carcinoma, this may, however, be the case in some advanced mixed-type gastric cancers.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit cancer preventive effects and have been shown to induce regression of adenomas in FAP patients. In order to elucidate the probable underlying mechanism, the effect of NSAIDs on mismatch repair related microsatellite instability was investigated. Six colorectal cancer cell lines all but one deficient for human mismatch repair (MMR) genes were examined for microsatellite instability (MSI) prior and after treatment with Aspirin or Sulindac. For rapid in vitro analysis of MSI a microcloning assay was developed by combining Laser microdissection and random (PEP-) PCR prior to specific MSI-PCR. Effects of NSAIDs on cell cycle and apoptosis were systematically investigated by using flow cytometry and cell-sorting. MSI frequency in cells deficient of MMR genes (hMSH2, hMLH1, hMSH6) was markedly reduced after long-term (10 weeks) NSAID treatment. This effect was reversible, time- and concentration dependent. However, in the hPMS2 deficient endometrial cancer cell line (HEC-1-A) the MSI phenotype kept unchanged. According to cell sorting, non-apoptotic cells were stable and apoptotic cells were unstable. These results suggest that aspirin/sulindac induces a genetic selection for microsatellite stability in a subset of MMR-deficient cells and may thus provide an effective prophylactic therapy for HNPCC related colorectal carcinomas.

During the last few years, the molecular basis of several cancer predisposition syndromes has been discovered which offers new tools for cancer prevention and early detection. This will be demonstrated in one of the most frequent hereditary cancer syndromes, namely the hereditary nonpolyposis colorectal cancer (HNPCC) which accounts for about 5% to 8% of CRC. Thereby, families with exclusively CRC (Lynch type I syndrome) and those with extracolonic cancers especially of endometrium, stomach, small bowel and upper urinary tract (Lynch type II syndrome) can be discriminated. At the molecular level, HNPCC is caused by germline mutations in one of the mismatch repair genes (hMSH2, hMLH1, hMSH6, hPMS2). Thus, nucleotide mispairings occurring particularly within simple repetitive genomic sequences (microsatellites) during replication are no longer be repaired properly and can be demonstrated by PCR as so-called microsatellite instability (MSI). Since more than 90% of HNPCC associated and only about 15% of sporadic CRC show MSI, this test is a useful tool for HNPCC screening. In case of a negative result HNPCC is highly unlikely. In positive cases (withor = 2 out of 5 unstable defined microsatellite markers) the definite molecular diagnosis can only be obtained by sequencing the mismatch repair genes from the patient's blood or normal DNA. As immunohistochemistry reveals loss of hMSH2 or hMLH1 expression in most MSI positive CRC, these data provide useful information for the sequencing strategy. Molecular tumor screening by MSI test and immunohistochemistry is recommended in patients i.) with a positive family history (acc. to the Amsterdam criteria), ii.) suffering from multiple HNPCC related carcinomas, iii.) with HNPCC related cancer before 45 ys of age, and iv.) with right-sided CRC exhibiting medullary, signet-ring or mucinous differentiation. Finally, these tests as well as genetic counseling and treatment of the patient need to be done by an interdisciplinary approach. Thereby, the pathologist can substantially contribute to identify HNPCC related carcinomas either by clinical or morphological criteria and to initiate the molecular screening test.

Alterations of the length of simple repetitive genomic sequences (microsatellite instability, MSI) characterize a distinct mechanism of colorectal carcinogenesis. Such MSI has been found to be associated with hereditary nonpolyposis colorectal cancer (HNPCC) that involves mutation of the human mismatch repair genes hMSH2 and hMLH1 as well as many sporadic cancers of most tissue types. Although the study of MSI status is a useful tool for HNPCC screening and for the determination of tumor prognosis in sporadic cases of colorectal cancer, the reliability of MSI diagnosis is still a subject of debate. Here we have examined 58 primary colorectal tumors (selected from a cohort of 200) using 31 microsatellite markers that comprised the most frequent simple repeat types. The expression of the hMSH2 and hMLH1 mismatch repair proteins was studied by immunohistochemistry, and most patients were surveyed for at least 2 years. Reproducibility of gel interpretation, as well as diagnostic sensitivity and specificity of the MSI status, were determined. We found that unambiguous determination of band shifts as well as MSI diagnosis were closely related to the type of the marker repeat and that MSI could be subdivided into "high" MSI (20% unstable loci), "low" MSI (10% unstable loci), and microsatellite stable (0% unstable loci). One-half of the patients with high MSI tumors (n = 8) fulfilled either the Amsterdam criteria (n = 4), had at least one relative with HNPCC-related carcinoma (n = 2), or were diagnosed with colorectal cancer at an age below 45 years (n = 2). Fourteen of the 15 high MSI tumors had lost either hMSH2 (n = 8) or hMLH1 (n = 6) protein expression. In contrast, all of the low MSI tumors and the MSI-negative tumors displayed normal expression of hMSH2 and hMLH1. These studies provide a clear recommendation for the uniform use of a panel of 10 microsatellites and a definition of at least 40% instability (using these defined marker loci) in the diagnostic analysis of MSI.

The molecular biology section of the Hereditary Non-Polyposis Colorectal Cancer study group-Germany, instituted a multicenter study to test the reliability and quality of microsatellite instability (MSI) analysis. Eight laboratories compared MSI analyses performed on 10 matched pairs of normal and tumor DNA from patients with colorectal carcinomas. A variety of techniques were applied to the detection of microsatellite changes: (a) silver and ethidium bromide staining of polyacrylamide gels; (b) radioactive labeling; and (c) automated fluorescence detection. The identification of highly unstable tumors and tumors without MSI was achieved in high concordance. However, the interpretation of the band patterns resulted in divergent classifications at several microsatellite marker loci for a large fraction of this tumor/normal panel. The data on more than 30 primers per case suggest that the enlargement of the microsatellite panel to more than 10 loci does not influence the results. In this study, cases with MSI in less than 10% of loci were classified as microsatellite stable, whereas MSI was diagnosed in cases with more than 40% of all markers unstable. We propose that a panel of five microsatellite loci consisting of repeats with different lengths should be analyzed in an initial analysis. When less than two marker loci display shifts in the microsatellite bands from tumor DNA, the panel should be enlarged to include an additional set of five marker loci. The number of marker loci analyzed as well as the number of unstable marker loci found should always be identified. These criteria should result in reports of MSI that are more comparable between studies.

No cause had been found for chronic diarrhoea in a 57-year-old man. Up to 15 watery stools daily had been without relation to food intake and without blood admixture. But muscular cramps had developed, especially in the legs. The patient had a history of recurrent peptic ulcers for which a selective proximal vagotomy had been performed 13 years ago. Physical examination was unremarkable.Alkaline phosphatase activity (182 U/l) and C-reactive protein (9.3 mg/l) were slightly raised; serum iron was 42 micrograms/dl, while all other routine laboratory tests, including protein electrophoresis, blood picture and differential count were within normal limits. Gastroscopy revealed ulcerative duodenitis, gastritis with erosions and numerous ulcers and reflux oesophagitis, grade III-IV. Endosonography showed enlarged gastric mucosal relief as sign of foveolar hyperplasia and a ca. 4 x 3 cm tumour next to the duodenal bulb. Gastrin level was 7537 pg/ml (normal150 pg/ml). Computed tomography and somatostatin receptor scintigraphy confirmed the site and size of the gastrinoma.Treatment with omeprazole (40 mg three times daily) slightly improved the symptoms. The tumour was excised a week after diagnosis. The patient has been symptom-free since then.Chronic diarrhoea of unknown aetiology can be caused by an endocrine tumour; endosonography can often provide information on the diagnosis and location of such a tumour.

Microsatellite instability (or replication error phenotyp) is a new molecular phenotyp of a substantial fraction of human cancers. The microsatellite instability in these cancers arises from alterations in normal regions of the genome consisting short sequences of repeated DNA. Ubiquitous changes in length of microsatellite sequences between constitutional and tumor DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal cancer. Microsatellite instability is also found in a substantial percentage of sporadic endometrial, and gastric cancer, as well as in additional sporadic cancers, such as lung cancer which is usually not associated with HNPCC. Thus far, four different mismatch repair genes (hPMS1, hPMS2, hMLH1 hMSH2), all homologous to bacterial DNA repair genes have been identified as involved in HNPCC kindreds, and consequently they are associated with microsatellite instability. In conclusion, these basic genetic informations provide new insights into a new molecular pathway in oncogenesis, i.e. the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate (replication error phenotyp) and thus to cancer.

The in vivo effect of dehydroepiandrosterone (DHEA) on hepatic glycogen content and on glucose metabolizing enzymes was investigated in male and female Sprague-Dawley rats treated with 0.6% (w/w) DHEA in the diet for 3, 7, 14, 28 and 140 days. The glycolytic enzymes studied (glucokinase, hexokinase, pyruvate kinase) showed a significant persistent decrease in activity in both sexes after 3-7 days of treatment. Gluconeogenic enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase) were increased after 3 days, but decreased after 7-14 days. Glycolytic enzymes showed a stronger reduction than gluconeogenic enzymes. Females were slightly more affected than males. Glucose-6-phosphate dehydrogenase was unchanged in females, but increased in males. Glycogen content and the activity of glycogen phosphorylase were reduced after 3 days of treatment. mRNA analysis of glucokinase and phosphorylase indicated that these enzyme alterations were accompanied by reduced transcriptional expression, while glucose-6-phosphate dehydrogenase mRNA levels were unchanged. Withdrawal of DHEA from 4 week-treated rats was associated with an almost complete reversibility of the enzyme alterations after 2 weeks. After long-term treatment (140 days) glucokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase activities were no longer altered. Since DHEA treatment affects the key enzymes of glucose metabolic pathways in the same sense, it is suggested that DHEA does not regulate individual enzymes but rather common regulatory factors or signalling pathways.

About 10 weeks before admission to hospital a 73-year-old woman developed a fever of up to 40 degrees C for three days. She then had subfebrile temperature for several weeks with some rises to 39 degrees C. She was known to have type II a diabetes mellitus and pulmonary silicosis, having worked in a porcelain and ceramic factory for many years. Before admission her cerebral functions were rapidly deteriorating, especially short-term memory. This was followed by increasing paraplegia of the legs with inability to walk. She finally had urinary and faecal incontinence and swallowing difficulties with tendency to aspiration, which necessitated hospitalisation.Both lactate dehydrogenase (339 U/l) and C-reactive protein (112 mg/l) were elevated; the platelet count was low (73000/microliters). Cerebrospinal fluid was unremarkable, as was computed tomography of the skull. But magnetic resonance imaging revealed multiple spotty lesions with low contrast-medium uptake throughout the brain, pointing to a disseminated bacterial or mycotic infection. 3 days later the chest-ray showed small nodular soft shadows in the lungs, and lung functions had decreased. Mycobacteria were found in the urine and liver biopsy showed granulomatous hepatitis, establishing the diagnosis of miliary tuberculosis in the presence of silicosis.Tuberculostatic treatment was instituted with four drugs (pyrazinamide, ethambutol, isoniazid and streptomycin. After 6 weeks the patient was again able to walk and continent of urine during the day. All cerebral functions gradually improved.Miliary tuberculosis should be included in the differential diagnosis of ill-defined feverish disease, especially in the elderly.

In order to demonstrate the relationship between microsatellite instability and other types of genomic instability, a series of 56 sporadic colorectal carcinomas was investigated by flow cytometrical ploidy analysis, oligonucleotide fingerprinting, and microsatellite polymerase chain reaction (PCR). Stabilization of the p53 gene product was analysed by immunohistochemistry and proliferative activity was determined flow cytometrically and by silver staining of nucleolar organizer regions (AgNORs). Of the 56 carcinomas, 11 (19 per cent) exhibited microsatellite instability; 33 of the cases were aneuploid (59 per cent) and 29 (52 per cent) showed alterations of the oligonucleotide fingerprints. There was a significant correlation of microsatellite instability with localization of these tumours proximal to the splenic flexure, diploid DNA content, and less frequent p53 stabilization. A solid growth pattern, mucinous differentiation, and a Crohn's-like lymphoid infiltrate were also characteristic for those tumours. The results demonstrate for the first time a significantly lower proliferative activity in tumours with microsatellite instability. Data obtained from DNA flow cytometry or from oligonucleotide fingerprinting did not correlate with such tumour characteristics. It is proposed that the use of microsatellite PCR facilitates specifically the detection of a group of colorectal cancers which may differ in pathogenesis and perhaps prognosis.

Histological type, malignancy grade, and tumor stage are among the most important parameters predicting outcome in cancer patients. Making use of immunocytochemistry as well as polymerase chain reaction-based techniques the demonstration of micrometastatic tumor spread, for example, into bone marrow, lymph nodes, and peritoneal cavity, is a new staging parameter of prognostic significance. In contrast, the prognostic value of different proliferation markers such as Ki67 (Mib 1), PCNA, and AgNOR has not yet been unequivocally established. A series of genetic change has been described in the development of cancer. In general, these changes seem to be of predictive value within defined tumor stages and it might be helpful to determine several genetic lesions within one tumor. Very recently a new mechanism of carcinogenesis closely related to the hereditary nonpolyposis cancer syndrome (HNPCC) was detected. Due to mutations in mismatch repair genes (hMSH 2, hMLH1, hPMS1,2) instabilities in simple repetitive genomic sequences occur, which are the genetic hallmark of most HNPCC tumors. This opens a new field to cancer prevention.

Histological type, malignancy grade, and tumor stage are among the most important parameters predicting outcome in cancer patients Making use of immunocytochemistry as well as polymerase chain reaction-based techniques the demonstration of micrometastatic tumor spread, for example, into bone marrow, lymph nodes, and peritoneal cavity, is a new staging parameter of prognostic significance. In contrast, the prognostic value of different proliferation markers such as Ki67 (Mib 1), PCNA, and AgNOR has not yet been unequivocally established.

The expression of two specific nucleolar antigens, p120 and B23, has been investigated in the prostatic carcinoma cell line LNCaP as well as in 40 frozen and 40 formalin-fixed tissue samples of benign and malignant prostatic lesions (15 benign hyperplasias, 5 grade 1, 15 grade 2, and 5 grade 3 carcinomas). In vitro, immunoreactivity of p120 was confined to nucleoli of proliferating cells, with virtually no negative staining during S and G2/M phases. Unlike p120, B23 was expressed in the nucleoli of all LNCaP cells independently of growth and cell cycle phases. Hence, B23 was detectable in all stromal as well as in normal and malignant epithelial prostatic cells, both in fresh and in formalin-fixed tissue sections after microwave treatment. In contrast, the immunoreactivity of p120 was almost completely restricted to the nucleoli of prostate carcinoma cells: frozen sections of benign prostatic hyperplasia (n = 15) were either totally negative for p120 (n = 13) or had a low percentage of positively stained cells (labeling index = 3.3% in 3 cases). In the carcinoma group 76% (19/25) of the specimens were p120 positive, and there was a significant rise of labeling index from 18.1% in grade 1 to 82.2% in grade 3 carcinomas (P0.001). In contrast to B23, p120 could not be reliably demonstrated in formalin-fixed and paraffin-embedded tissue. We therefore conclude that anti-B23 is a general marker of nucleoli, whereas expression of p120 appears to correlate with "hyperactivity" of the nucleolus and provides a new tool for flow cytometrical and immunohistochemical assessment of nucleolar activity in tumor pathology.

The aim of the present study was to establish a rapid, non-radioactive screening method for the detection of microsatellite instability (MIN). MIN is the primary characteristic of the mutator phenotype in tumours constituting hereditary non-polyposis colon cancers (HNPCC). We investigated 30 patients suffering from colorectal cancer using a non-radioactive PCR-based technique. MIN was present in 7 of 30 (23%) of the cases. There was a statistically significant correlation between MIN and localization of the tumour. Five of 7 (72%) tumours with MIN but only 4 of 23 (17%) tumours without MIN were localized in the proximal colon (P0.01). There was a tendency to higher MIN frequency in tumours of patients with familial clustering of cancers. However, this was statistically not significant (P0.05). In addition, no correlation between MIN and tumour grade and stage was found. For the investigations in the present study we used a non-radioactive PCR-based method followed by denaturating polyacrylamide gel electrophoresis and silver staining. This method is highly sensitive and reproducible. Thus, PCR-based analysis using a non-radioactive staining technique represents a comprehensive tool for MIN screening in diagnostic pathology.

Very recently a new molecular mechanism in the tumorigenesis of colorectal carcinoma has been described which is closely linked to hereditary non-polyposis colonic cancer (HNPCC). Ubiquitous changes in the length of simple repetitive DNA sequences between constitutional and tumour DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal carcinoma. Such microsatellite instabilities have been shown to be the phenotypical marker of mutations in the human homologues of prokaryotic mismatch repair genes (MutS, MutL, MutH). These data provide crucial new tools in the detection of patients at high risk of developing colon cancer and other HNPCC-related carcinomas. In addition, these developments provide new insights into a new, presumably primary event in oncogenesis, i.e. the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate (“mutator phenotype”) and thus to cancer.

Etwa 8–10% der kolorektalen Karzinome (CRC) werden zu den „hereditary non-polyposis colorectal carcinomas“ (HNPCC) gerechnet. Dabei handelt es sich um ein autosomal dominant vererbtes Krebsleiden, welches ursprunglich von H. Lynch beschrieben wurde und klinisch durch die sogenannten „Amsterdam Kriterien“ wie folgt definiert ist: 1. Drei Verwandte ersten Grades aus mindestens zwei Generationen sind an einem CRC erkrankt und 2. mindestens einer der Patienten ist bei Erkrankungsbeginn junger als 50 Jahre [1].

To investigate the effects of social isolation on host resistance male mice were housed either individually (IH) or in groups of four or five (GH). All animals were infected with MengoM,L virus. Incubation time (INCUB), duration of illness (ILL), death rate (DR), histopathological changes, and serum corticosterone levels (CORT) were recorded. First, the effect of IH starting 4 days prior to infection was studied in 5 different inbred strains. Next, the effect of different IH length was examined, and the role of T-cells was investigated by comparing euthymic (+/+) and athymic (nu/nu) NMRI mice. Finally, the effects of the infection on CORT in IH and GH mice were compared in C57BL/6 mice. The major findings were: 1. IH significantly increased ILL in all but the DBA/2 strain, whereas DR was not affected except in C57BL/6. 2. Longer IH (starting 35 [DBA/2] or 10 [NMRI] days prior to virus inoculation) significantly shortened INCUB and prolonged ILL, but IH starting on the day of virus inoculation [DBA/2] significantly prolonged INCUB and shortened ILL. 3. NMRI nude mice exhibited an unaltered DR accompanied by a tremendously prolonged INCUB. 4. Investigations in C57BL/6 mice revealed a significant rise of CORT after infection. This increase was higher in IH compared to GH mice. It is suggested that IH attenuates T-cell mediated inflammatory processes and/or increases macrophage activation, which in turn results in a prolonged course of the disease.

Expression of two nucleolar antigens, p120 and B23, was studied in a prostatic carcinoma cell line (LNCaP) and in frozen and paraffin embedded tissue sections of 40 benign and malignant prostatic lesions. The percentage of p120 negative G0/G1 phase cells rose significantly during transition from exponential to plateau growth phase in vitro (from 9% to 32%). In contrast, B23 was equally expressed throughout different cell cycle and growth phases. Thus, nucleoli of almost all stromal and epithelial cells were stained by B23 in tissue sections. P120, however, selectively stained nucleoli of proliferating prostatic epithelium. Whereas 88% (13/15) of benign hyperplasia were p120 negative this was the case in only 24% (6/25) of carcinomas. Using microwave procedure both MoAbs reacted in paraffin sections, but the percentage of p120 negative cases doubled. A routine application to formalin fixed and paraffin embedded tissue cannot be recommended thus far.

In einer prospektiven Studie wurde die DNA bei 155 Patienten mit kolorektalem Karzinom aus Tumorgewebe und Normalgewebe auf Mikrosatelliteninsta-bilitat untersucht und die Daten mit dem klinischen Verlauf korreliert. 30 kolorektale Karzinome zeigten Mikrosatelliteninstabilitat an mindestens einem Locus, 10 der Tumore wiesen an mindestens 3 Loci Instabilitaten auf („hochinstabil”), 4 der 10 Patienten mit hochinstabilen Tumoren wiesen eine positive HNPCC Anamnese auf. Hochinstabile Tumore sind haufiger im rechtsseitigen Kolon gelegen (p

A patient with Hodgkin's disease (clinical stage IIIB) received chemotherapy and total nodal irradiation. After the transfusion of filtered packed red cells, this patient developed transfusion-associated graft-versus-host disease (TA-GVHD). The genetic fingerprint of the patient's peripheral blood lymphocytes (PBLs) differed completely from that of her other body tissues. Normally, after transfusion, only the patient's own genetic fingerprints are observed in the PBLs, as exemplified in more than 10 control cases in which the transfused blood had not been filtered before transfusion. No signal bands corresponding to those of the blood donor could be demonstrated in samples of the patient's tissue DNA. Moreover, chimerism was detected in the hybridization pattern of the patient's PBLs on the ninth day after the onset of symptoms. Polymorphic simple repeats in the HLA-DRB gene after amplification by polymerase chain reaction were also investigated, which confirmed the fingerprinting results. The advantages of these methods for the diagnosis of TA-GVHD include the rapid and unequivocal diagnosis as well as the fact that there is no need for the relatives to be HLA typed.

It is reported on 191 patients of an intensive care unit, in whom for the purpose of the prophylaxis of thromboses and thromboembolic complications a low-dose heparinisation was carried out. For comparison served the documents of 137 patients who were admitted to the same department for homogeneous reasons but had not been treated prophylactically with anticoagulants. In no case more massive haemorrhages developed which might have compelled us to interrupt the therapy. In spite of the low-dose heparinisation 4 pulmonary embolisms as well as thromboses of the abdominal aorta, the radial artery and the coronary arteries were the result; they were confirmed autoptically . Thus the low dose heparinisation does not represent a sufficient protection against the thromboembolic complications for patients with internal diseases who are confined to bed. The demand is made to establish the individual need of heparin and to adjust the heparin dose according to this.