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We report a constitutional point mutation of codon 278 in exon 8 of the TP53 gene that has not yet been described as a germ-line mutation. A 52-year-old female developed multiple primary malignancies (liposarcoma, breast cancer, malignant histiocytoma, occult adenocarcinoma). The mutation found in her tumour and peripheral blood lymphocyte DNA is a cytosine to thymine transition at the second position of codon 278 resulting in an amino acid exchange from proline to leucine in the DNA-binding domain. Evaluation of the patient's family revealed that both of her sons were affected by the same mutation. Although the patient's mother had died already, we were able to demonstrate by polymorphic microsatellite analysis that the defective allele originated from the maternal side. As four brothers and one sister had inherited the same allele, which however was wild type, we were able to show that the mutation must have occurred in the germ cells of the patient's mother and that it may therefore be called de novo. This explains the lack of a high cancer incidence in the family history. All tumours tested showed positive immunohistochemical staining for p53. Loss of heterozygosity was found in five of seven tumours, one showing chromosome 17 monosomy. Images Figure 1

We analysed antibodies specific for human p53 in sera from primary breast cancer patients using three different immunoassays and we related these results to the p53 level in tumour tissue detected by immunohistochemistry. Only 44% (11/25) of apparently enzyme-linked immunosorbent assay (ELISA)-positive sera were from patients with a high level of p53 protein in more than 50% of their tumour cells. Surprisingly, 36% (9/25) of the sera originated from patients with no detectable p53 protein at all. Immunoprecipitation data suggested that the reason for this discrepancy is that at least some of the antibodies detected as positive in the ELISA in these sera from patients with clinical stage I and stage II breast cancers may be induced by immunogens other than p53 protein. Many of these reactions give apparently positive signals in a variety of p53 assays, and very stringent analysis is required to avoid possible misinterpretation of these responses as a p53-specific B-cell response in human cancer patients. Images Figure 1

The staining pattern of a monoclonal antibody directed to the monomorphic determinant of HLA-DR antigens was examined on sections of human mammary gland tissues at various stages of differentiation as well as on 50 benign and 72 malignant breast lesions. Normal resting breast epithelium lacked HLA-DR, whereas late-pregnant and lactating epithelia expressed high levels of HLA-DR antigens, followed by a decline in the post-weaning regression period. Most benign breast lesions revealed heterogeneous staining ranging from very few up to 20-25% positive epithelial Greater variability was observed among carcinomas, where a small group (approximately 7%) of cases showing 40-95% positive tumour cells was found, in addition to negative tumours and those with the minority of HLA-DR expressing carcinoma cells. The density of the leukocytic infiltrate was higher in carcinomas than in either normal breast tissue or benign lesions, the HLA-DR phenotype of the mononuclear infiltrating cells lacking any obvious correlation with the HLA-DR status of the epithelial component. Immunoblotting analyses of whole-tissue lysates separated by SDS-PAGE confirmed the immunohistochemical data and demonstrated the reactivity with only one protein band predicted for HLA-DR alpha-chain. The combination of immunohistochemistry and autoradiography on sections of human reduction mammoplasty organoids cultured in collagen gels and labelled with tritiated thymidine revealed a lack of HLA-DR expression on proliferating breast epithelial cells suggesting factors other than cell kinetics must be responsible for induction of HLA-DR antigens seen in pregnant and lactating breast epithelium and some tumours. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

1. Bei 19 Patienten mit funikularer Spinalerkrankung wurden insgesamt 35 Serumeiweis-Elektrophoresen durchgefuhrt. Bei 13 dieser Patienten wurden auserdem Liquoreiweis-elektrophoretische Untersuchungen vorgenommen. 2. Auser einer signifikanten Gesamteiweiserhohung im Liquor konnten in den einzelnen Eiweisfraktionen keine Normabweichungen beobachtet werden. 3. Der Gesamteiweisgehalt im Serum war nicht verandert. Doch zeigte sich bei der Serumelektrophorese eine signifikante Erhohung der Albumine bei gleichzeitig signifikanter Erniedrigung der α1-Globuline.

Skin tests with autologous irradiated tumour cells were performed in 20 malignant melanoma, 7 breast and 6 ovarian cancer patients. In the majority of cases evident reaction was noted with cholesteryl hemisuccinate (CHS)-treated cells while the reaction with untreated cells was mostly negative. No correlation was found between this reactivity and the ability of patients to be sensitized to DNCB and to their reactivity to PPD. No correlation was found between reactivity to CHS-treated tumour cells and the stage and course of the disease.

A comparative study of six mouse monoclonal antibodies against human 45 kDa keratin polypeptide (keratin No. 18) was undertaken using three experimental approaches: immunohistochemistry on normal human tissues, examination of interspecies cross-reactivity and identification of the target polypeptides in 1-D and 2-D immunoblots. The data suggest that at least five different antigenic sites of keratin 18 are recognized by this panel of reagents. The C-04 epitope is keratin 18-specific and widely conserved among mammalian species, while the antibodies DA7 and DC10 also react specifically with the 45 kDa keratin but stain simple epithelia of human origin only. Two antibodies, C-11 and C-66, decorate simple as well as stratified epithelia in human and in all seven animal species tested, but their respective target epitopes are shared by different groups of keratin polypeptides, which indicates their non-identity. In contrast to keratin specificity of the five above mentioned antibodies, the C-08 antibody cross-reacts with a 70 kDa nuclear lamina protein found in human and bovine tissues. The results of the present study provide the necessary basis for future applications of these antibodies in both routine immunodiagnostic work and as probes to study the biology of epithelial cells in general and the significance of keratin intermediate filaments in particular.

Three monospecific monoclonal antibodies (BA16, BA17 and A53-B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffin-embedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.

The diagnosis and treatment of congenital diaphragmatic hernia, CDH, is an interdisciplinary problem. The patient concentration with prenatally diagnosed CDH for comprehensive examination with subsequent decision on the type of delivery improves the prognosis of patients with CDH. Introduction of corticoid therapy in prenatally assessed CDH reduces dysfunction and surfactant deficiency and can reduce the degree of respiratory failure during delivery. In very early prenatally assessed CDH it is possible to consider the possibility of combined corticoid and TRH (thyroxin releasing hormone) treatment of CDH. The authors submit also contemporary possibilities of prenatal intervention treatment of CDH (indication criteria for reconstruction operations of foetal surgery, intrauterine closure of the trachea and artificial laparoschisis). The concentration of patients with postnatally diagnosed CDH and a severe grade of acute respiratory insufficiency in a department with conventional and non-conventional artificial pulmonary ventilation incl. inhalation of NO oe extracorporeal membrane oxygenation, ECMO, is a further step towards optimation of treatment. Continuous evaluation of parameters of pulmonary functions during the pre- and postoperative period may prove that a therapeutic protocol with delayed surgery is useful.

Reactivity patterns of seven mouse monoclonal antibodies to human keratin 7 were compared using immunoblotting and immunohistochemistry on cultured cells and normal human and animal tissues. Differences in keratin specificities as determined by two-dimensional immunoblots and interspecies cross-reactivity data on 8 mammalian species suggest that at least six nonidentical epitopes of the keratin 7 molecule are recognized by this panel of reagents. Immunohistochemical examination of a panel of various human neoplasms with monoclonal antibodies monospecific for keratins 7, 18 and 19 revealed potential value of keratin subtyping in differential diagnosis of tumors in general and in subclassification of carcinomas in particular.

Accumulation of the p53 protein was analysed in 212 human malignant lesions. Immunohistochemical staining with new polyclonal (CM-1) and monoclonal antibodies (BP 53-12 and BP53-24) to p53 on methacarn-fixed paraffin sections showed positive staining in 161 (76%). The positive tumours were found across a wide range of human malignancies including breast, colon, stomach, bladder and testis carcinomas, soft-tissue sarcomas and melanomas. The staining was always confined to the malignant lesion. Immunoprecipitation and quantitative ELISA assays established that the positive staining was associated with accumulation of the protein and that the protein was frequently in a mutant conformation. Accumulation of mutant p53 protein is therefore a common feature of human malignant disease.

We have analyzed p53 protein expression in 121 primary breast cancer biopsies by immunohistochemistry using the monoclonal antibody DO-1 and polyclonal serum CM-1. p53 protein overexpression has correlated in our study with mitotic activity (p=0.001), nuclear atypia (p=0.002), less favorable histological type of tumor and in a lesser extent with tumor size. The inverse, but highly significant, correlation (p=0.007) has been observed with lymph node involvement. There was also a trend for higher p53 positivity among DNA aneuploid tumors as compared with DNA diploid cases, but this was not significant. Our study suggests that p53, at least in some patients, may not be directly involved in the process of metastatic progression in breast cancer. Preliminary data would suggest that the detection of p53 protein overexpression could be a useful additional prognostic parameter in breast cancer.