Parvovirus B19 detected in Rosai-Dorfman disease in nodal and extranodal manifestations.

Sinus histiocytosis with massive lymphadenopathy (SHML), also designated as Rosai-Dorfman disease (RDD), is a rare benign reactive lymphoproliferative disorder. It is defined by a characteristic histopathology with sinus histiocytosis and haemophagocytosis known as emperipolesis. In histiocytes S100 is strongly expressed, whereas CD1a staining typically is negative. The disease mainly manifests at a single lymph node; however, multilocular and extranodal affection can occur. Causative infectious agents, and virus infections in particular, have repeatedly been suspected, although until now the origin of the disease has been unclear. Four cases of RDD (two nodal sites and two extranodal upper respiratory tract sites) were analysed for parvovirus B19 (B19) infection by immunohistochemistry to detect B19 capsid proteins VP1/VP2. In all the four cases, huge numbers of B19-positive cells were partly detected. The positive cells were identified either as lymphocytes or, in one extranodal case, also as respiratory epithelial cells. This is the first report of B19 infection in RDD tissue, indicating that B19 may be associated with the pathogenesis of SHML.

Expression of the nm23 homologues nm23-H4, nm23-H6, and nm23-H7 in human gastric and colon cancer.

Eight members of the nm23-gene family have been described. The involvement of nm23-H1 and nm23-H2 in tumour progression and metastasis, as well as in gene regulation and apoptosis, has been shown in numerous studies. Whether nm23-H4, -H6, and -H7 play a role in tumours is, however, largely unknown. This study describes data on the expression of these three nm23 homologues in human colon and gastric cancer by real-time RT-PCR and immunohistochemistry. Increased expression of these genes, most strikingly nm23-H4 and -H7, was observed in the majority of tumours analysed. No correlation with tumour stage according to the TNM classification was found. In contrast, by immunohistochemical analysis, nm23-H4 and -H6 overexpression correlated with the intestinal tumour type in gastric cancer tissues, whereas no increased immunoreactivity for the three nm23 proteins was noted in the diffuse type tumour specimens. These findings indicate that nm23-H6, and particularly nm23-H4 and -H7, may be involved in the development of colon and gastric carcinoma, the latter possibly in a type-specific manner. A contribution to tumour progression or metastasis could not, however, be proven. Elucidation of the specific mechanisms by which the nm23 homologues nm23-H4, -H6, and -H7 are involved in tumour development requires further studies.

Search for deletion 22q11.2 in interphase nuclei of buccal mucosa of patients ascertained by isolated cleft palate: a new diagnostic approach.

A new approach for the detection of chromosome deletion 22q11.2 in interphase nuclei from buccal mucosa cells obtained by a non-invasive procedure is described. FISH analysis has been performed on samples from a group of 101 patients that presented consecutively for speech therapy and/or surgical correction of cleft palate. A normal result has been obtained in 98 patients; a deletion 22q11.2 was present in three patients (2.8%) with cleft palate.

HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues.

We have developed a novel method for tissue fixation, including subsequent paraffin-embedding and sectioning, that allows the complete pathological analysis of all types of human soft tissues. Furthermore, it maintains additional positive features relevant to immunohistochemistry and molecular pathology. The so-called HOPE-technique (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) comprises a protection-solution with an organic buffer, acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature. Although the exact mechanism of protection has still to be elucidated, it seems rather unlikely that chemical bindings occur during the whole process of fixation, which is described and compared with the standard formalin-paraffin technique. Essentially, HOPE-fixed sections show formalin-like morphology. However, the sections are somewhat difficult to handle because of their fragility. This is due to the absence of any type of protein cross-linking and the dynamic processes of immersion and outflow of the HOPE protection solution. HOPE-fixed sections provide an excellent preservation of proteins and antigenic structures for differential analysis by immunohistochemical and/or enzyme histochemical techniques. However, their most remarkable feature is the extremely low degradation of nucleic acids (DNA and RNA) combined with good results obtained by in situ hybridization techniques. In conclusion, HOPE fixation may become a valuable additional tool in modern pathology.

[Identification of new substrates of cyclin-activated kinases in breast cancers]. / Identifizierung neuer Substrate Cyclin-abhängiger Kinasen in Mammakarzinomen.

Mitogenic and growth-inhibitory signals influence cell-cycle progression through their action on a family of cyclin-dependent kinases (cdks). The activity of cdk complexes is regulated in part by the association of a cyclin partner that acts as a positive effector. These cyclin/CDK complexes promote cell cycle progression by the phosphorylation of key substrates. Cyclin D1 and E are frequently overexpressed in breast cancers and cyclin E overexpression has been correlated with a poor prognostic outcome. The in vivo substrates of cyclin E/CDK2 are, however, not well defined. We screened for cyclin E/CDK2 substrates in nuclear extracts of breast cancer cells as well as using a spotted-array protein library with purified active cyclin E/CDK2 complexes that were expressed in and purified from insect cells. Using this technique several potential cyclin E/CDK2 substrates were isolated. Further work is presently underway to identify these substrates and verify their authenticity as in vivo substrates of cyclin E/CDK2. These potential new substrates will help to unravel the highly complex mechanisms of cell cycle control and perhaps offer new targets for the diagnosis and/or treatment of breast cancer patients.

Murine and human TSPYL genes: novel members of the TSPY-SET-NAP1L1 family.

Using expressed sequence tag (EST) sequence information, novel genes related to the Y chromosome gene family TSPY were isolated and characterized. Because of a significant homology to TSPY, the novel genes were designated TSPY-Like (TSPYL) and were assigned as new members of the TSPY-SET-NAP1L1 family. A human TSPYL gene was localized on chromosome 6 and a murine Tspyl gene was localized on chromosome 10. Expression of TSPYL was observed in all tissues investigated, as well as early onset during development. Both the human and murine Tspyl homologs lack introns over the entire region so far investigated and are thought to have arisen by an ancient retroposition event. Retroposition of Tspyl genes is supported by the isolation of a murine Tspyl pseudogene on chromosome 12 which also lacks intronic sequences, and by its observed proximity to an R element, a family of dispersed repetitive DNA.

Microdeletion 22q11 in complex cardiovascular malformations.

Besides DiGeorge, velocardiofacial and conotruncal anomaly face syndromes, some of the isolated congenital heart diseases have also been associated with a chromosomal deletion in 22q11. These disease entities, which had originally been considered to have a different genetic background, are now included in the CATCH-22 microdeletion complex. CATCH 22 is an acronym for cardiac defect, abnormal facies, thymic hypoplasia or aplasia and T-cell deficiency, cleft palate, hypoparathyroidism, and hypocalcemia. In the present study, we focused on the complex cardiovascular defects (CCVD) and screened 40 patients for a microdeletion of 22q11 by fluorescence in situ hybridization using the D22S75 DNA probe and for associated CATCH features. The patients were from genetic counseling (n = 15) or fetopathology (n = 3) of the Clinical Genetics Department in Marburg and from the Pediatric Cardiology Department (n = 22) in Mainz. Monosomy 22q11 was detected in 9 cases (= 22.5%). Familial transmission with one mildly affected parent and one affected sib each was proven in two cases. The CCVDs comprised complex conotruncal defects such as tetralogy of Fallot, double outlet right ventricle, transposition of great arteries and truncus arteriosus communis, or anomalies of the derivatives of the branchial arch arteries in association with a ventricular septal defect, including one case of atresia of the ductus arteriosus with pulmonary artery aneurysm and resulting in fetal hydrops. All 13 patients with a deletion of 22q11 showed at least one additional CATCH symptom. Most consistently, facial dysmorphy was apparent (92%), while hypocalcemia, mostly at threshold values, was present in 62% and thymic hypoplasia including borderline low T-lymphocyte numbers was observed in 41%. None of the patients presented with a cleft palate. A high intrafamilial variability in expression was also evident with respect to the CCVD. Our findings indicate that seemingly isolated complex cardiovascular defects associated with a 22q11 microdeletion most probably do not represent a distinct subgroup within the CATCH-22 complex but are syndromal in nature with extracardiac features that are often overlooked.

Chronic eosinophilic leukaemia (CEL): a distinct myeloproliferative disease.

Chronic eosinophilic leukaemia has not yet been clearly defined, mainly due to the fact that it has not been conclusively shown as a monoclonal disease which should be separated from chronic myelogenous leukaemia, acute myelogenous leukaemia with eosinophilia (AML, FAB M4Eo), and the idiopathic hypereosinophilic syndrome. We report a patient with a white blood cell count of 17.6 x 10(9)/l with 74% eosinophils, normal platelet count and haemoglobin. No blasts were seen in the peripheral blood and the percentage of blasts in the bone marrow was < 3%. A diagnosis of chronic eosinophilic leukaemia was made. Chromosome analysis of a bone marrow aspirate disclosed a trisomy 15 together with loss of the Y chromosome. Moreover, FISH analysis on May-Grünwald-Giemsa-stained peripheral blood smears demonstrated trisomy 15 in the eosinophils. 3 months after initial diagnosis the patient went into blast crisis and died. The blast cells exhibited trisomy 15 and loss of the Y chromosome in a complex, aberrant karyotype. In conclusion, the case shows that chronic eosinophilic leukaemia is a monoclonal, myeloproliferative disease with eosinophils as part of the malignant clone. Clinically, chronic eosinophilic leukaemia can be separated into a chronic phase and a blast crisis.

Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization.

Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of approximately 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of approximately 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring.

[Hydatidiform mole of the placenta and coexisting, living premature infant]. / Herdförmige Blasenmole der Plazenta und koexistentes, lebendes Frühgeborenes.

A pregnancy with a partial or complete hydatidiform mole and a coexistent living fetus is a rare condition. Most cases are consistent with a twin pregnancy, with distinct borders between placenta and mole on sonography. We report a case with a single placenta with cystic structures resembling histologically a complete hydatidiform mole. The pregnancy resulted in a diploid female fetus in the 34th week of gestation with marked hypotrophy. The non-molar part of the placenta showed a complex alteration of villous maturity resulting in intrauterine growth retardation but a living fetus. No malignancy occurred post partum. The clinical and pathogenetic aspects of this rare entity are discussed.

[Simultaneous coexistence of a hydatidiform mole and a live premature infant]. / Gleichzeitiges Nebeneinander von Blasenmole und einem lebenden Frühgeborenen.

Moles and coexisting living fetus are rare. Most cases are twin gestations. We report a case of a single placenta with focal hydatidiform mole of the placenta and a coexisting living fetus. The fetus with hypotrophy was born in the 34th weeks of gestation with a normal diploid female karyotype. The placenta showed a complex alteration of the non molar part with consecutive vascular hyperplasia. The differential diagnosis of molar pregnancies and coexistent living fetuses are discussed. Postpartal are cytogenetic examination of fetal tissue (skin biopsy) and both parts of the placenta, the molar and non molar, necessary. In each case should be a postpartal measurement of beta-HCG in serum of the mother to rule out a gestational trophoblastic disease.

[Detection or exclusion of fetal trisomy 18 by in-situ-hybridization of the histologic section]. / Nachweis oder Ausschluss einer fetalen Trisomie 18 durch In-situ-Hybridisierung am histologischen Schnitt.

In-situ-hybridization using a chromosome-specific centromeric DNA probe on paraffin embedded tissue of placental and foetal origin allows the detection of numerical chromosome anomalies, even after several years. This may facilitate the diagnosis of late abortions in those cases in which a conventional cytogenetic examination was not performed, but the clinical picture suggests a chromosomal syndrome. We demonstrate chromosome DNA fluorescence in-situ-hybridisation (FISH) with a chromosome 18 specific (peri) centromeric repeat probe, on paraffin sections of one case with a cytogenetically proven and of three cases with a questionable Edwards-syndrome, thus confirming or excluding trisomy 18 in two of the cases.

[Diagnosis of fetal virus infections by in situ hybridization]. / Die Diagnostik fetaler Virusinfektionen durch In-situ-Hybridisierung.

Non-radioactive in situ hybridization of formalin-fixed paraffin-embedded placental and foetal tissue, using virus-specific DNA or RNA probes, may be helpful for the diagnosis of foetal virus infection causing foetal hydrops, granulomatous placentitis and abortion. We present four cases of intrauterine CMV-, Parvo-B19- and Varicella-Zoster virus infection, in which this diagnostic method established detection of the virus.