Dagmar Ivanyi

Expression of class III beta-tubulin in normal and neoplastic human tissues.

Abstract The class III β-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. Using a newly described monoclonal antibody we compared the distribution of class III β-tubulin in normal and neoplastic tissues. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III β-tubulin isotype, and its specificity was confirmed by immunoblotting, by competitive enzyme-linked immunosorbent assay and by immunofluorescence microscopy on cultured cells. In different cell lines of various origins the antibody reacted only with neuroblastoma Neuro-2a cells and with embryonal carcinoma P19 cells stimulated to neuronal differentiation by retinoic acid. Immunohistochemistry on formaldehyde-fixed paraffin-embedded normal human tissues revealed the presence of the class III β-tubulin isotype in cell bodies and processes of neuronal cells in the peripheral and central nervous systems. In other tissues, this β-tubulin isotype was not immunodetected. Class III β-tubulin was found in all cases of ganglioneuroblastoma, ganglioneuroma, medulloblastoma, neuroblastoma, sympathoblastoma and in one case of teratoma. In contrast, no reactivity was detected in tumours of non-neuronal origin, including 32 cases of squamous cell carcinoma. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III β-tubulin functions in neurons as well as for immunohistochemical characterisation of tumours of neuronal origin.

Molecular Heterogeneity of H-2 Antigens

Since the discovery of the H-2Ld molecule (Lemonnier et al. 1975) we have demonstrated that several K and D region alleles produce more than one type of H-2 molecules. Two of four different molecules were distinguished in the products of different alleles. Some of these molecules are products of different genes (H-2D, H-2L), in other instances the evidence for distinct genes is not available. Some of the different molecules produced by the same region might be modified products of the same gene. In the instances where no information implicating different genes is available, we use a neutral terminology which does not presume a genetic difference: H-2K1d and H-2K2d, H-2D1k, H-2D2k, H-2D1dx, H-2D2dx, H-2L1d, H-2L2d, etc. Immunoprecipitation experiments with some anti-H-2L and anti-Qa-2 sera revealed proteins with the apparent molecular weight of 41,000. We designate these antigens provisionally Lq and Qx, respectively. The Lq protein is polymorphic and it is at least partly under the control of H-2L-linked genes since it is absent from BALB/c-H-2dm2 cells. Since we have never seen the 41,000 proteins in precipitates of H-2K or H-2D antigens, it appears that whatever the origin of these molecules, they reveal some features common to products of L and Qa region. The basic relationship of H-2 K, D, L antigens is revealed also by the shared antigenic specificities between these H-2 molecules which we demonstrate using anti-H-2.28 sera. In summary, our results show that the class I antigens in each haplotype represent a family of several distinct but antigenically related molecules. The specificities of the H-2.28 family are the strongest allotype common to different H-2 K, D, and L molecules. Recent direct demonstration of several different genes in the Dd region (Steinmetz et al. 1981) provides evidence for the genetic complexity of H-2 genes which may be underlying basis of the molecular heterogeneity of H-2 antigens discussed here.

Complex genetic effect of B10.D2 (M504) (H-2dm1) mutation

Serological and capping experiments show that the strain B10.D2 (M504) carrying the mutant haplotypeH-2dm1 has two molecules in the products of theD region: H-2Ddm1 and H-2Ldm1 which are detectable by anti-H-2.4 and by anti-H-2.28 sera, respectively. Both these molecules differ serologically from the H-2Dd and H-2Ld molecules of the original (nonmutant) strain B10.D2. A third molecule, different from H-2D and H-2L, was detected inH-2d,H-2dm2 but not inH-2dm1 products.

A new H-2.1-PositiveD region allele,D dx , controlling two molecules, H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2dx, which had earlier been shown to have theKd,If,Sf, andGf alleles and a previously unknownD region allele,Ddx. We show here that theDdx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterDk) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the Ddx product comprises two molecules: H-2Ddx bearing the private specificity H-2.63, and H-2Ldx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated Ddx products shows that the H-2Ldx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).

Serological characterization of previously unknown h-2 molecules identified in the products of the kd and dk region.

The molecular relationship between H-2 private and some public specificities was studied in C3H.OH (H-202) mice using surface-antigen re-distribution methods. Besides the Kd- and Dk-region antigens, which can be capped by antisera against the private and public specificities characteristic for a given allele, a previously unknown type of molecule was found in the products of both theKd andDk regions. These can be capped by the respective anti-private serum but not by antisera against some public specificities. The two Kd-region molecules are provisionally named H-2K1d and H-2K2d. We detected them onH-202 (Kd,Id,Sd,Dk) and also onH-2dx (Kd,If,Sf,Ddx) T lymphocytes. Similarly, the two types of molecules detected on the products of theDk region are provisionally named H-2D1k and H-2D2k. The serological characteristics of these molecules are described. When compared with the products of theDd region, in which we previously described three different molecules (H-2Dd, H-2Md, and H-2Ld), the mutual relationship between H-2K1d and H-2K2d as well as between H-2D1k and H-2D2k appears to be similar to that between H-2Dd and H-2Md. In the absence of relevant recombinants or informative biochemical data, it is, however, difficult to establish homology between molecules produced by differentK- andD-region alleles.

Expression of cytokeratin 10, 13, and involucrin as prognostic factors in low stage squamous cell carcinoma of the uterine cervix.

Background. The identification of pretreatment markers with predictive significance for the presence of lymph node metastases and treatment outcome in low stage cancer of the uterine cervix is clinically important. Because the presence of differentiation‐related markers varies in this type of cancer, the authors investigated whether loss of these markers is related to a poor clinical course.

Qualitative and quantitative aspects of anti-H-2Ld sera.

Two anti-H-2Ld sera were analyzed, BALB/c-H-2dm2 anti-BALB/cBy and (C3H X BALB/c-H-2dm2)F1 anti-BALB/cHe, the latter also containing anti-Qa antibodies. Their reaction patterns were compared with an anti-Qa serum (C3H X BALB/cBy)F1 anti-BALB/cHe. Four H-2 specificities could be detected by the anti-H-2Ld sera, two already known (H-2.64, H-2.65) and two new specificities (H-2.81, H-2.82). According to their reaction pattern H-2.64, H-2.81, and H-2.82 can be regarded as members of the H-2.28 family of specificities. A quantitative difference in the expression of these H-2 specificities exists in different haplotypes. The cells of the strain against which the sera were made (BALB/cHe and BALB/cBy, respectively) did not give the highest titers with the antisera and had a relatively low absorbing capacity. The H-2dx haplotype carries two new specificities of the H-2.28 family, namely, H-2.81 and H-2.82. Lysostrip tests showed that the antibodies against those specificities cap the H-2.1-positive H-2Ddx molecules, suggesting that these molecules may react with both anti-H-2.1-like and anti-H-2.28-like antibodies. The H-2 specificities detected by the BALB/c-H-2dm2 anti-BALB/cBy serum were detected also in liver, kidney, spleen, heart, and lung tissue. New information on the strain distribution of Qa-2 was obtained from the experiments and a quantitative difference in Qa-2 antigens between H-2 congenic strains was observed as well. The H-2b strains react with these antibodies with higher titers than the strains carrying the H-2d haplotype.

Cytokeratin subtypes and involucrin in squamous cell carcinoma of the vulva. An immunohistochemical study of 41 cases

Background. Histologic grade seems to be of limited prognostic significance in patients with vulvar carcinoma. However, the study of cytokeratin expression is of potential interest because it allows a more precise evaluation of the degree of squamous differentiation. This study was was conducted to investigate whether differences in cytokeratin expression exist between normal vulvar epithelium and vulvar carcinoma and whether these differences are prognostically significant.

Modulation of mammary carcinoma cell phenotype and keratin expression patterns by retinoic acid.

Immunohistochemical and biochemical procedures were used to study the influence of retinoic acid (RA) on cellular expression and distribution of cytokeratins (CKs) in feline mammary carcinoma cells. These cells were grown in vitro as established cell lines (K248C and K266) and in vivo as xenografts in athymic mice. The results were compared with the distribution of CKs in normal feline mammary gland and in a series of invasive mammary carcinomas previously probed with a panel of monoclonal antibodies specific for individual CKs. Coexpression of CKs of both major mammary gland cell types (myoepithelial cells, MECs, CKs 5/14 positive, and luminal epithelial cells, LECs CKs8/18 positive) by K248C and K266 cells, suggested a stem cell-like character of both cell lines. RA increased CK19 expression in both cell lines and CK19 was also present in tumors developed in nude mice from both RA untreated (CK19 negative) and RA-treated (CK19 positive) K248C and K266 cells. In addition, RA had cell line specific effects as well. RA treatment induced differentiation of K248C cells to more mature LEC-like cells and this change was accompanied by the loss of the MEC keratins CKs 5/14. Under the same culture conditions however, RA treatment did not induce morphological changes in the K266 cell line and the expression of CKs 5/14 was not significantly reduced. These findings suggest that the modulation of CK19 and CKs 5/14 expression observed in mammary carcinoma cells upon RA treatment might be regulated through different pathways.

Functional heterogeneity of H-2Dk-region products.

The view that the D region codes for a single class I antigen was first challenged by the demonstration of a serologically distinct antigen, H-2L d (Lemonnier et al. 1975, D6mant et al. 1975), and by the discovery of a mutant lacking H-2L a (McKenzie et al. 1977, Hansen et al. 1977). Recent studies suggest that the D d region also encodes a third serologically distinct antigen, H-2M d (Ivfiny and D6mant 1979). Similarly, analysis of antigens encoded in the D k region have revealed that public specificities of some antisera distinguish separate molecules (provisionally designated H-2D1 k and H-2D2k), while antisera against the private specificity H-2.32 react with both (Ivfinyi and D6mant 1981, D~mant and Iv/mi 1981). Monoclonal antibodies used to inhibit allogeneic and H-2-restricted killing (Fischer Lindahl and Lemke 1979, Blanden et al. 1979) also suggested some heterogeneity among Dk-region products. Here we present evidence that the antibodies involved in the differential inhibition of Dk-restricted anti-H-Y, antiviral, and allogeneic anti-D k CTLs indeed distinguish H-2D1 k and H-2D2 k molecules. Thus, the molecular heterogeneity of the Dk-region antigens is manifest also in their functional heterogeneity. Table 1 details cocapping experiments on C3H.OH (H-2 °2, KdD k) cells with antisera defining the H-2D1 k and H-2D2 k molecules and antiserum D31 (antiH-2K d) serving as control. As reported (Ivfinyi and D6mant 1981) antiserum ASA-14 is one of the reagents which can distinguish between the two D k molecules, H-2D1 k and H-2D2 k. This antiserum reacts with the D k antigens since it can be capped by the anti-D k serum ASA-11 (H-2.32) and not by the anti-K d serum D-31. ASA-14, however, cannot cap all molecules reactive with the antiserum ASA-11. The molecules capped both by ASA-14 and ASA-11 are named H-2D1 k. The two monoclonal antibodies 27R9 and 30R3 react with antigens controlled by the D k