P. G. Natali

Expression of Ia-like antigens in normal human nonlymphoid tissues.

Recent results have demonstrated that Ia-like antigen expression in animal tissues is not restricted to cells associated with immune functions. Whether this phenomenon occurs in normal human tissues has been investigated in this study by indirect immunofluorescence on cryostat sections using monoclonal antibodies to framework determinants of Ia-like antigens. The results of this study show that la-like antigens are expressed on several normal human tissues of different embryonic origin. These include the epithelium of the gastrointestinal tract, urinary bladder, bronchial glands, thymic reticuloepithelial cells (entodermie origin), epithelium of mammary gland, acinar cells of parotid, astrocytes (ectodermic origin), alveolar macrophages, Kupffer cells, glomerular and peritubular renal endothelium, endometrium, and Langerhans cells (mesodermic origin).

Morphological, immunochemical, and biochemical study of rabbit achilles tendon at various ages.

UNLABELLED With aging, rabbit tendon tissue undergoes a series of morphological and biochemical changes which involve both the cells and the extrace-lular matrix. The extracellular matrix increases in volume, causing a relative decrease of the number of cells per unit of tissue surface. The tenoblasts become longer and more slender, while their cytoplasmic processes increase in number and become thinner and more elongated, forming a dense network. In addition, tendon cells show a marked decrease in the intracytoplasmic organelles responsible for protein synthesis, while their intracellular content of contractile proteins does not change. With aging collagen fibers increase in diameter and vary more in thickness. These morphological changes correspond to biochemical changes that include an increase in collagen, a decrease in mucopolysaccharides, and a decrease in water content. During aging parallel changes occur in the elastic fibers, which decrease in number and show structural alterations. CLINICAL RELEVANCE Ultrastructural and biochemical studies of tendon diseases need a normal comparison. Out ultrastructural and biochemical findings in aging tendon may be useful in that regard. The presence of actin and myosin in tendon cells could be related to some aspects of tendon physiology and pathology.

Changes in expression of alpha 6/beta 4 integrin heterodimer in primary and metastatic breast cancer.

The alpha 6/beta 4 integrin complex has been shown to be expressed in murine tissues at the basolateral aspect of most epithelial cells including the mammary epithelium, thus suggesting that this heterodimer may interact with components of the basement membrane. Because transformation of mammary epithelium frequently results in disappearance of basement membranes and loss of cell polarisation we have analysed in the present study whether expression of the alpha 6/beta 4 complex is altered in human breast tumours. The results of the present study confirm that in human mammary gland alpha 6 and beta 4 subunits colocalise at the basolateral aspect of the epithelium. While in benign breast lesions this distribution pattern remains mostly unchanged, in primary carcinomas the expression of both chains is either redistributed over the cells surface or significantly reduced. This altered pattern of expression is paralleled by the lack of detection of basement membrane laminin and collagen type IV. In metastatic lesions the expression of the heterodimer is maintained in most of the lymphnodal foci, but less frequently detected in metastasis localised in the pleural cavity and in parenchymal tissues. These findings indicate that in breast epithelium expression of the alpha 6/beta 4 heterodimer is modulated by the presence of basement membrane and is possibly influenced by microenvironmental factors as suggested by the different pattern of alpha 6/beta 4 expression in nodal and extranodal metastatic foci.

CXCR6, a Newly Defined Biomarker of Tissue-Specific Stem Cell Asymmetric Self-Renewal, Identifies More Aggressive Human Melanoma Cancer Stem Cells

Background A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+. Methods/Findings We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone. Conclusions/Significance The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Differential tissue distribution and ontogeny of DC-1 and HLA-DR antigens

The tissue distribution and the ontogeny of DC-1 antigens have been investigated and compared with those of HLA-DR antigens. Indirect immunofluorescence (IIF) staining of surgically removed normal tissues from adults with the monoclonal antibody (MoAb) BT3.4 has detected DC-1 antigens in tissues of various embryologic origin. The tissue distribution of DC-1 antigens is more restricted than that of HLA-DR antigens, as the former are not detected in duodenal epithelium, colon mucosa, and ductal mammary gland epithelium. In fetuses up to 26 weeks of age, DC-1 antigens were detected only on cortical and medullary thymic dendritic cells with an anatomic distribution similar to that of reticuloepithelial cells and in endothelial cells of the small intestine. At this stage of intrauterine life, HLA-DR antigens have already reached their full tissue distribution. The tissue distribution and the ontogeny of DC-1 antigens resemble those of their murine counterparts, i. e., the I-A antigens

Regeneration of rabbit calcaneal tendon

SummaryThe regenerated tissue which fills the gap between the stumps of sectioned and unsutured rabbit calcaneal tendon was studied by immuno-fluorescence, light and electron microscopy from 2 days to 30 weeks after surgery. In the early stages, the newly formed tissue consisted of few connective tissue cells of variable shape dispersed in an abundant intercellular matrix. At 7 days after tenotomy most of the cells were spindle shaped and arranged along the major tendon axis. They showed a well developed rough endoplasmic reticulum, a prominent Golgi complex and bundles of thin and thick filaments. Moreover, they appeared intensely stained when treated with anti-actin and anti-myosin sera. The bulk of the intercellular matrix consisted of bundles of collagen fibers, mostly arranged parallel to the cells.In the subsequent stages the regenerating tissue became more compact, acquiring the morphological characteristics of tendon tissue. At 30 weeks after tenotomy, however, it did not show yet the typical texture of the normal adult tendon. The tenocytes were more numerous and less uniformly distributed, and contained a greater amount of ergastoplasm and contractile proteins. The collagen fibers were similar in size to those of the neonatal normal tendon and the elastic fibers appeared often immature.These findings suggest that, at least on the experimental conditions under which this study was performed, the regenerated tendon does not acquire the typical morphology of the normal adult tendon, possibly owing to the reduced mechanical stress acting on it.

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region. HLA-DR antigens appear more frequently than the other two types of HLA class II antigens and HLA-DP antigens more frequently than HLA-DQ antigens. Differential expression of the gene products of the HLA-D region was also found in autologous metastases removed from different anatomic sites from patients with melanoma. The HLA class II phenotype of surgically removed malignant lesions did not correlate with the degree of differentiation of tumor cells and/or with the expression and/or cellular distribution of HLA class I antigens. Furthermore, in melanoma lesions, no relationship was found between the HLA class II phenotype and the expression of 3 membrane bound and 1 cytoplasmic melanoma associated antigen recognized by monoclonal antibodies. The functional significance and the practical implications of the differential expression of the gene products of the HLA-D region by tumor cells are discussed.

Changes in Ia-Like Antigen Expression on Malignant Human Cells

In human and in animal model tumor systems malignant cells may express allospecificities or types of histocompatibility antigens that are not detectable on their normal counterparts (Invernizzi and Parmiani 1975, Garrido et al. 1976, Pellegrino et al. 1976, Martin et al. 1977, Winchester et al. 1978, Wilson et al. 1979 a). Among them the appearance of Ia-like antigens on human melanoma cells (Winchester et al. 1978, Wilson et al. 1979 a) is of particular interest because of the possible role of these antigens in the interaction between hosts immune system and tumor cells (Forni et al. 1975). In view of this finding we have investigated whether variations in expression of Ia-like antigens occur in other human tumors of nonlymphoid origin by testing surgically removed tumor specimens with monoclonal antibodies to Ia-like antigens in indirect immunofluorescence (IIF). Monoclonal antibodies Q 5/13 (MoAb Q S/13) to framework determinants of Ialike antigens were prepared and characterized as described (Quaranta et al. 1980). Tumor specimens, which were obtained from patients undergoing surgery in the Regina Elena Cancer Institute, Surgery Division, were immediately frozen in liquid nitrogen. Two consecutive 4 g cryostat sections from each tumor sample were fixed for 5 minutes in absolute acetone, which was shown by preliminary experiments not to effect the antigenicity of Ia-like antigens in tissue. One section was stained with Toluidine blue (0.1~o in phosphate-buffered saline) and the other was used as substrate in an IIF test with the anti-Ia-like antigen MoAb Q5/13 with an average concentration of 5 gg/ml and a fluorescein labeled (FITC) rabbit anti-mouse Ig antiserum (Meloy Laboratories, Springfield, Virginia) as second antibody. The FITC anti-mouse Ig xenoantiserum, which had fluorescein/protein ration of 3 and a protein concentration of 1.5 mg/ml, was preabsorbed with ABRH + red blood cells and with peripheral blood lymphocytes (PBL) of chronic lymphocytic leukemia (CLL) patients, which express high levels of Ia-like antigens. The specificity of the strain observed on different tumor-tissue substrates was assessed by direct staining with the FITC-labeled antiserum and by indirect staining with MoAb Q5/13 preabsorbed extensively with an equal volume of PBL from CLL patients or

The impact of monoclonal antibodies on the study of human malignant melanoma

Monoclonal antibodies, directed against human melanoma-associated antigens (MAA), have been developed for more than 5 years now, using hybridoma technology. After the first report by Koprowski and his colleagues (1978), monoclonal antibodies to MAA have been described by several investigators (Yeh et al. 1979, Carrel et al. 1980, Dippold et al. 1980, Imai et al. 1980, Woodbury et al. 1980, Johnson et al. 1981, Liao et al. 1981, Loop et al. 1981, Natali et al. 1981, Wilson et al. 1981, Bumol & Reisfeld 1982, Chee et al. 1982, Houghtonetal. 1982, Imai et al. 1982a, 1982b, Liao et al. 1982, Natali ct al. 1982, Nudelman et al. 1982, Saxton et al. 1982, Hellstrom et al. 1983). Exchange of monoclonal antibodies at a workshop under the auspices of the National Cancer Institute has enabled identifieation of MAA with distinct tissue distributions and molecular profiles as described in the report on this workshop (Reisfeld 1982). The aim of this review is to discuss the new information generated by studies using monoclonal antibodies as compared to conventional antisera and the new directions of investigation which are now possible as a result of techniques using monoclonal antibodies. Because of lack of space, this chapter is not intended to be an exhaustive review of the literature, but will foeus mainly on areas of study in our laboratory.

Postnatal development of epididymis and ductus deferens in the rat

SummaryThe postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.