Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies.

Epilepsy is a common finding in patients with chromosomal macro- and micro-rearrangements but only few aberrations show a constant pattern of seizures. DNA array-based studies have reported causative copy number variations (CNVs) in 5-30% of patients with epilepsy with or without co-morbidities. The interpretation of many of the detected CNVs remains challenging. In order to identify CNVs carrying epilepsy-related genes we investigated 43 children with various patterns of epileptic seizures, intellectual disability (ID), and minor dysmorphism, using the Illumina® Infinium Human1M-DuoV1 array. In three patients we found likely causative de novo CNVs, i.e. deletions in 1q41q42.12 (3.4 Mb) and 19p13.2 (834 kb), and a mosaic two-segment duplication in 17p13.2 (218 kb) and 17p13.1 (422 kb). In six additional patients there were aberrations (a deletion in one and duplications in five patients) with uncertain clinical consequences. In total, the finding of causative chromosomal micro-rearrangements in 3 out of 43 patients (7%) and potentially causative CNVs in 6 additional patients (14%) with epilepsy and ID but without major malformations confirms the power of DNA arrays for the detection of new disease-related genetic regions.

Long-term outcome of children with acute cerebellitis.

BACKGROUND: Acute cerebellitis (AC) is characterized by cerebellar symptoms and magnetic resonance imaging (MRI) changes primarily confined to the cerebellum. OBJECTIVE: To analyze the neurological and cognitive long-term outcome of children with AC. METHODS: Children with AC diagnosed by typical clinical features and MRI findings were included in this retrospective study. Medical charts were reviewed and neurological deficits were assessed by neurological examination or by the expanded disability status scale telephone interview. Cognitive outcome was evaluated with a parental questionnaire (Kognitive Probleme bei Kindern und Jugendlichen). RESULTS: A total of 11 children (6 boys, 5 girls; age range: 3 years to 14 years and 10 months) were included. Of them, six children had a severe disease manifestation including mental status changes and neurological symptoms. Of the rest, two children had a moderate and three children had a mild form of AC. MRI of the cerebellum was obtained in the acute phase revealing signal alterations with different patterns. The average follow-up period was 4 years and 4 months. A complete recovery was observed in five children. Neurological sequelae were reported in five children ranging from ataxia to mild tremor. Cognitive deficits were found in six patients. The affected areas of cognition did include spatial visualization ability, language skills, and concentration. CONCLUSION: Neurological and cognitive sequelae are common in children with AC and underline the role of the cerebellum in cognition.

Clinical outcome of children presenting with a severe manifestation of acute disseminated encephalomyelitis.

BACKGROUND: Acute disseminated encephalomyelitis (ADEM) is an acute, inflammatory-demyelinating disorder of the CNS with a favourable outcome in the majority of cases. OBJECTIVE: The aim of this study was to examine the long-term outcome of children with an initially severe form of ADEM. METHODS: Children with ADEM according to the criteria of the International Pediatric MS Study Group (IPMSSG) referred to the rehabilitation centre Vogtareuth were included. Neurological impairment was evaluated with a standardized telephone-based interview assessing the EDSS score. Neuropsychological outcome was assessed with review of the medical records and a standardized parental questionnaire (KOPKIJ). RESULTS: Twelve children (1 year 9 months to 13 years of age) were included. All children had focal-neurological signs and changes in mental status at presentation and an MRI of the brain showing a range of white and gray matter lesions. 11/12 patients with a mean follow-up of 6.2 years (2-13.6 years) had a monophasic course of the disease. One child had a multiphasic ADEM. Two children had an EDSS score of 0, three an EDSS of 2, five an EDSS between 3 and 5 and two children had an EDSS score of 6 and 9. Results of a standardized parental questionnaire (KOPKIJ) revealed that 7 children had deficits in the categories alertness, memory, school performance, visual-spatial skills and/or impulse control. CONCLUSION: The results of our study indicate that children with an initially severe manifestation of ADEM continue to have in the majority of cases neurological and neuropsychological handicaps.

Natural antibody in mammary tumor virus-infected mice that reacts with intracytoplasmic A particles of mouse mammary tumors.

By an indirect immunofluorescence technique with prolonged serum incubation on murine mammary tumor (MT) slices, 179 of 424 mice examined were found to possess natural serum antibody (antibodies) that reacted with intracytoplasmic A particles (iAp) of MT cells. The immunologic specificity of this antibody was supported by absorption and blocking experiments. Furthermore, a strong similarity was seen between the mouse antibody reaction on various MT and the fluorescence pattern of rabbit anti-iAp antiserum on these tumors. In female mice, incidence and geometric mean titers of the antibody in part were correlated to the spontaneous MT frequency of the mouse strains examined. Some mice of the strains XVII/Bin and CBA/BinfXVII/Bin, hitherto regarded as "free" of the mouse mammary tumor virus (MuMTV), also contained anti-iAp antibody in their sera. In contrast to MuMTV)-producing CBA/Bin micethese animals did not possess detectable spontaneous antibody reacting with MuMTV-B particles. Therefore, hypothetically, the antibody response in these mice might be induced by incomplete MuMTV expression. In the strain CBA/Bin, females 4 months old and older possessed the antibody in significantly higher geometric mean titers when compared to 4-week-old female mice. The history of lactation seemed to have no influence on the titer of antibody. In the comparatively high MT strains CBA/Bin and C3H/Bin, adult (4-month-old) females had the antibody in significantly higher levels when compared to age-matched males.

Specificity of human antibodies to intracytoplasmic type-A particles of the murine mammary tumor virus.

Large-scale studies showed that antibodies previously detectable in women with proliferating mastopathy or breast cancer were directed to intracytoplasmic type-A particles (iAp) of mouse mammary tumor virus. Immunofluorescence revealed the human antibodies to be bound only by those tumors producing a certain amount of iAp clusters visible by light microscopy. The intensity of the reaction corresponded to the iAp content of every tumor tested as revealed by electron microscopy and rabbit antisera to iAp. The fluorescence patterns obtained with positive human sera were similar to those obtained with rabbit antisera specific for iAp and resembled the tissue distribution patterns of iAp inclusions stained by acid fuchsin. The reaction with human sera was entirely blocked by rabbit antisera to iAp and, less so, by rabbit or mouse antisera to B particles. The human antibody activity was exhaustively absorbed by purified iAp or purified and disrupted B particles, which indicated that the human antibodies were directed to antigenic components shared by iAp and B particles. Preliminary immunoperoxidase studies supported the assumption that the human antibodies were bound to the iAp membrane; technical details might have accounted for the finding that the human antibodies reacted with the iAp but not with B particles in situ.

Immunohistochemical demonstration of MAM-3 and MAM-6 antigens in normal human skin appendages and their tumors.

The expression of MAM-3 and MAM-6 antigens was immunohistochemically investigated on 110 tumors of human skin appendages. Forty-two samples from tumor-adjacent normal skin appendages were also studied. MAM-3 antigens, as detectable by monoclonal antibodies (MoAbs) 67D11, 115G3, and 115H10 were present in the inner layer cells but not in the outer layer cells of normal eccrine excretory ducts. Sporadic positivity was also found in cytoplasm of apocrine acini with 115G3, while 67D11 and 115H10 were negative. MAM-6 antigens, as detectable by the MoAbs 115D8, 115F5, 139H2, 140C1, and 126E7 were found in the secretory canaliculi of normal eccrine acini and within the apical lumina at the terminal portion of ducts. Apocrine acinar cells mainly exhibited an apical staining, but a focal supranuclear dot-like staining could also be observed. A foamy reaction pattern for MAM-6 was noted in mature sebocytes. However, none of the antigenic epitopes was expressed in normal squamous epithelium or hair follicles. In benign tumors, the staining patterns for both antigens, in general, resembled their distribution in the corresponding normal tissues. However, carcinomas originating from sweat glands, sebaceous glands, and the pilar apparatus expressed both antigens in a more irregular and heterogeneous pattern. This might preferably be explained by the loss of those mechanisms controlling the antigen expression in mature, functional tissues. Conclusions from these immunohistochemical studies with regard to the histogenesis mainly of the malignant skin appendage tumors should be drawn with caution.

Widespread occurrence in mammals of antibodies reactive to intracytoplasmic A particles of the mouse mammary tumor virus.

Serum antibodies reactive to clusters of intracytoplasmic A particles in mouse mammary tumors and leukemia cells by means of an indirect immunofluorescence assay were detected in 11 mammalian species (ranging in frequency from 5.1 to 29.3%). These antibodies could not be found in sera from chicken, ducks and geese. Their presence in so many mammalian species, not only in mammary tumor virus-infected mice, sheds new light on similar antibodies in man detected 10 years ago.

Demonstration of mouse mammary tumour virus (MuMTV) antigenicity in human milk by means of immunodiffusion technique.

Using the micro-Ouchterlony immunodiffusion technique, we were able to demonstrate mouse mammary tumour virus (MuMTV) antigenicity in the 1.26-1.28 g/ml density fraction prepared from pooled human milk samples after treatment with detergents and ether. The cross-reacting antigen(s) was precipitated by rabbit antisera to MuMTV-B particles prepared from murine milk and intracytoplasmic A particles (iAp) isolated from mouse mammary tumour tissue. By confluence of precipitin lines, the human milk "core" antigen(s) was shown to be identical with the main antigen(s) of iAp which are known to share antigenicity with the MuMTV-B particle cores. Electron microscopy of the human "core" fraction revealed abundant particulate structures of 40-70 nm in diameter. However, the structural entities bearing the cross-reactive antigen(s) remain to be identified.

[S phase fraction and immunohistologic demonstration of the epithelial marker MAM-6 in orofacial tumors]. / S-Phasenfraktion und immunhistologischer Nachweis des Epithelmarkers MAM-6 in orofazialen Tumoren.

Tissue samples of 9 leukoplakias, 19 oral squamous cell carcinomas, 9 basaliomas and 6 specimens of normal oral mucosa were labeled with 3H-thymidine using an in vitro technique; the size of the S-phase fraction was determined histoautoradiographically. Whereas leukoplakias (with or without dysplasia) showed an homogeneous growth pattern, carcinomas and basaliomas demonstrated significantly higher proliferative activity in the tumor periphery. In the basaliomas the pallisade cells showed significantly higher indices than the polygonal cells. The proliferative potential of the oral squamous cell carcinomas is high compared to other neoplasias and depends on the grade and type of growth. The epithelial marker MAM-6 detected immunohistologically on the same tissue, showed a stronger expression in oral carcinomas than in leukoplakias. A direct relationship between the intensity of antigen expression and the proliferative behaviour (size of the S-phase fraction) was not detectable.

Electron microscopic investigations of intracytoplasmic A particles in mouse mammary tumours.

Mouse mammary tumour cells were investigated with special regard to intracytoplasmic A particles using ultrahistochemical techniques, especially for the detection of DNA. The findings are discussed in connection with biochemical and immunological results, previously reported by several groups.

RNA-dependent DNA polymerase activity in intracytoplasmic type-a particles of mouse mammary tumors.

Intracytoplasmic type-A particles isolated from mouse mammary tumors and purified by repeated gradient centrifugations exhibit both endogenous and exogenous (template-stimulated) RNA-dependent DNA polymerase activities. The enzyme has a preference for magnesium ions.

[Correlation between the production of mature mammary tumour virus particles and the histomorphological differentiation of murine mammary tumours]. / Korrelation zwischen der Produktion reifer Viruspartikel und der histo-morphologischen Differenzierung virusinduzierter muriner Mammakarzinome

The mammary tumour virus (MTV) contents of mammary tumours (MT's) of various mouse strains were investigated by means of the indirect immunofluorescence technique and the electron microscopy. Only in acinus-forming, well differentiated MT's MTV-B particles could be observed in high quantity. The release of B particles seems to be correlated with the degree of structural ripeness of the tumor tissue. As to intracytoplasmic A particles such a strong relationship could not be found out. Many A particles were seen in mature adenocarcinomas as well as in acinus-free tumours. Some mammary carcinomas of an extreme low degree of histomorphological differentiation were free of any detectable virus production. The indirect immunofluorescence test was confirmed as a sensitive method for the detection of MTV antigens in MT slices. Using polyvalent anti-MTV sera, obtained after immunization of rabbits with ether-disrupted B particles from tumour tissue or milk, the test system allowed the distinction of A and B particle antigens. Accumulations of B particles were seen as an intercellular fluorescence reaction while clusters of A particles were represented by a granular paranuclear reaction within the tumor cells.

Mammary tumour virus (MTV)-specific immune complex deposites in renal glomeruli of MTV-infected tumour-free mice.

Using direct and indirect immunofluorescence tests MTV-specific immune complex deposits were found in tumour-free female mice of the strains C3H/Bln, CBA/Bln, XVII/BlnfCBA/Bln, and CBA/BlnfXVII/Bln. These deposits consist of immunoglobulin, complement, and MTV antigen(s). The immune complex deposition increases with age. Antibodies eluted from renal tissue homogenate react with both MTV-A and -B particles in immunofluorescence tests performed on mouse mammary tumour slices. By these results the earlier finding of age-dependent spontaneously occurring anti MTV antibodies in naturally MTV-infected mice is confirmed and completed.

[On clinical and differential diagnosis of primary ureteral tumors]. / Zur Klinik und Differentialdiagnostik primärer Uretertumoren.

Primary tumours of the ureter are to be regarded as rare tumours. Etiopathogenesis, pathology and clinic are discussed, taking into consideration own experiences. The authors cannot subscribe to the widespread view of operating primary solitary tumours of the ureter on principle only radically in form of the nephroureterectomy. A differentiated, individual approach with a primarily conservative-reconstructive orientation is recommended.

[Immunocytochemical studies of epithelial markers in pleural and peritoneal effusions as an adjunct to cytologic diagnosis]. / Immunzytologischer Epithelmarkernachweis in Pleura- und Aszitespunktaten als Ergänzung zur Zytodiagnostik.

Monoclonal antibodies were used in an indirect immunoperoxidase assay to investigate 83 smears of pleural or peritoneal effusions for expression of the epithelial markers MAM-6, MAM-3, and carcinoembryonic antigen (CEA). All but one smears containing tumor cells according to the evaluation of H&E preparations were positive for one, two or all three markers, the exception being a malignant melanoma. Three of 5 cases, suspicious by routine cytology, exhibited marker expression in a different number of cells and thus confirmed the cytological diagnosis. Five of 63 cytologically negative smears exhibited single cells or small cell clusters positive for up to three markers. Four of these patients were found to suffer from metastatic cancer, as established by clinical follow-up and subsequent biopsy or autopsy, respectively. Felty's syndrome and concomitant serositis were diagnosed and confirmed by autopsy in one of the MAM-6 positive cases with negative cytology. The comparatively strong MAM-6 expression in some mesothelial cells of this patient might have been induced by abnormal stimulation due to the rheumatic disease. The results of this study encourage wider use of immunocytochemistry as an adjunct to cytological diagnosis in effusions.

[Immunohistochemical studies of chordomas and choroid tumors]. / Immunhistochemische Untersuchungen an Chordomen und chondroiden Tumoren.

Chordomas are rare slow-growing but locally invasive tumors. They are thought to develop from residues of the notochord. Three chordomas and ten chondroid tumors were investigated by an indirect immunoperoxidase method for expression of the epithelial markers: MAM-6, keratin, tissue polypeptide antigen (TPA) and for carcinoembryonic antigen (CEA). In all the chordomas investigated the antigens MAM-6, keratin and TPA were detected using monoclonal antibodies and conventional antisera, respectively. These data provide further evidence of the epithelial nature and the ectodermal origin of chordomas. Moreover, the findings suggest that immunohistochemical methods may be useful for the differential diagnosis between chordomas and morphologically similar chondroid tumors.

[Immunohistochemical demonstration of tissue markers in histopathologic diagnosis]. / Immunhistochemischer Nachweis von Gewebsmarkern in der histopathologischen Diagnostik.

Our experience with the demonstration of tissue markers in histopathology supports their value in differential diagnosis of routine material. Microscopic diagnostic methods are genuinely enriched by the use of such monoclonal antibodies and conventional antisera whose specificity has been adequately demonstrated using indirect peroxidase techniques in cut sections and on cytologic preparations. On the basis of several epithelial markers (carcinoembryonic antigen, keratin, tissue polypeptide antigen, MAM-antigens) and other antigens (protein S-100, chromogranin) examples are given for their application to routine differential diagnosis problems. The detection of these markers has been adapted to standardized indirect immunoperoxidase techniques. More sensitive tissue markers (i.e. antigens which do not withstand formalin fixation and paraffin embedding) should best be dealt with by specialized working groups.