Automated microscopic image analysis and the prognosis of preneoplasms and carcinomas of the mammary gland

Studies of preneoplasias and carcinomas of the mammary gland have been conducted by means of automated microscopic image analysis, on the basis of previous results as well as with references to the international literature, for 2 purposes: 1. Determination in the context of a clinical follow-up study of the individual carcinoma risk for patients with proliferative fibrocystic breast disease (mastopathy) and 2. Preparation of an objective automated grading of ductal breast carcinomas for better assessment of prognosis. Against the background of the assumption that the majority of carcinomas and precancerous lesions of the breast originate from the terminal ductal lobular unit, an effort is made to determine, independent severity of the mastopathy, the biological valence of solid, cribriform, and papillary ductal epithelial proliferations (no, possible or inevitable preneoplasia). Proliferation patterns without and with atypical features are checked for their similarity with intraductal and invasive carcinomas (similarity principle) and are additionally examined for differences, depending on localization and distance from tumour (topological principle). Automated histological tumour grading is to distinguish with greater subtlety within the large heterogeneous group of moderately differentiated carcinomas and is to objectify and thus facilitate the difficult task of delimitation of moderately and poorly differentiated carcinomas. Nuclear grading will be the major basis for classification along these lines. Objectified and reproducible tumour grading is believed to be extremely helpful in better prognostication of breast cancer.     

The role of three distinct Ia-like antigen molecules in human T cell proliferative responses: effect of monoclonal anti-Ia-like antibodies

Murine monoclonal antibodies (MoAb) to three distinct Ia-like molecules were studied for their inhibitory effects on antigen- and alloantigen-induced T cell proliferations. The MoAb were classified into three groups according to the molecules they recognized. Both the group I MoAb reacting with DR molecules and the group III MoAb were capable of inhibiting T cell proliferative responses to PPD- and HSV-Ag-pulsed APC, autologous B-LCL, and alloantigens. On th other hand, the group II MoAb, which reacted with a determinant on the molecule carrying MB1 determinants, was only capable of inhibiting T cell responses to alloantigens. These results suggest that the structure of the molecules correlates with the functional repertoire of the human Ia-like antigens.     

Are there differences in the cell cycle of normal and malignant cells? An approach to the analysis of the clonal expansion of cells in malignant epithelial proliferations in situ

The Williams-Bjerknes model for clonal expansion of transformed cells in epithelia was used to examine a series of related questions about the growth of in situ proliferations in epidermis. Bowens disease, actinic keratosis and lentigo maligna lesions were reconstructed on a three-dimensional basis, and all were found to behave differently: the evidence supported the proposal that the 'carcinogenic advantage' enjoyed by transformed cells was proliferative in nature, and indeed varied between the three lesions. We conclude that the clonal expansion of a single cell, with different proliferative carcinogenic advantage, can explain the three-dimensional appearances of these in situ proliferations; furthermore, the well-known 'multifocality' of in situ epidermal proliferations can also be explained by the clonal expansion of a single transformed cell.     

TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

The class III β-tubulin isotype (β_III) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III β-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-β (TGF-β) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

Immunohistochemically demonstrated increase in glutathione S-transferase species in propylnitrosamine-induced focal proliferative and neoplastic Syrian hamster pancreatic lesions

Immunohistochemical investigation of focal proliferative and neoplastic Syrian hamster pancreatic lesions induced by propylnitrosamine administration revealed a distinct pattern of expression of different molecular forms of glutathione S-transferase (GST) during neoplastic development. Initial increase in levels of GST placental (P) and B forms in early ductal/ductular proliferations and atypical (dysplastic) lesions was followed by a drop in the latter during transition to carcinoma. Unequivocal acinar cells observed within so-called 'pseudoductules' did not share the altered phenotype evident in ductular elements, suggesting their non-involvement in the generation of early lesions. However, the fact that component cells were occasionally of abnormal morphology did not allow the exclusion of acinar cell participation in histogenesis. Elevation of GST-A and C forms was limited to stromal elements surrounding the epithelial lesions and since they were associated with benign, cystic as well as atypical lesions and a similar increase was observed after common duct ligation, they appeared to be non-specific. The results indicated independent control of expression of individual GST forms and suggest that biochemical similarities exist between early, putative preneoplastic lesions induced by propylnitrosamines in the hamster lung, liver (both hepatocellular and intrahepatic bile duct) and pancreas.     

Histochemistry of estrogen sulfatases in human breast diseases

Two estrogen sulfatases, arylsulfatase C-estrone sulfatase (ASC-ES) and d-equilenin sulfatase (EqS) were demonstrated histochemically in the normal human female breast, in benign breast diseases and in infiltrating mammary ductal carcinomas to study their significance in the pathogenesis of epithelial proliferations. By hydrolyzing estrone sulfate, the amount of which in female blood is about ten times greater than that of estradiol or estrone, estrogen sulfatases can produce a high local concentration of estrogens. A simultaneous azo-coupling method for histochemical demonstration of ASC-ES is described in the present study; EqS was demonstrated by a previously described method. Estrogen sulfatases were not found in the normal female breast. Both estrogen sulfatases were found in epithelial cells in some examples of mastopathic disease and in fibroadenomas, while ASC-ES was found in periductal fibroblasts. In some cases of infiltrating ductal carcinomas, estrogen sulfatases were present in carcinoma cells. In most of these tumors ASC-ES activity was observed in fibroblasts around infiltrative cell cords. There was no correlation between the presence of estrogen sulfatases and of hormone receptors in carcinomas. It is concluded that estrogen sulfatases play no role in the early stages of benign or malignant epithelial proliferations. However, the induction of estrogen sulfatases may promote epithelial proliferation in some cases if estrogen receptors are present in epithelial cells.     

The basalis of the primate endometrium: a bifunctional germinal compartment

Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.     

Action of ketotifen on the mitogen-evoked proliferative response of human mononuclear cells

Ketotifen at low concentrations (5 and 50 microns) potentiated and at high ones (250 and 500 microM) blocked the proliferative response of human peripheral blood mononuclear cells ( NNC ) induced by PHA. The proliferative response was evaluated by 3H-thymidine incorporation into the cells. At doses inhibiting proliferative response to PHA ketotifen blocked both Con A-induced and Pokeweed mitogen-induced proliferations of MNC. At tested concentrations ketotifen inhibited and at the highest concentration (250 microM) blocked PHA-induced increase of protein synthesis in MNC evaluated by incorporation of 14C-L-leucine into the cells. The presented data showed that ketotifen acted not on the inductive step of the proliferative response but on the steps preceding the cell shift into S-phase.     

Cellular and humoral autoreactivity in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a morbid, refractory lung disorder with an unknown pathogenesis. To investigate potential adaptive immune mechanisms in IPF, we compared phenotypes and effector functions of peripheral CD4 T cells, autoantibody production, and proliferative responses of pulmonary hilar lymph node CD4 T cells to autologous lung extracts from afflicted patients and normals. Our results show that greater proportions of peripheral CD4 T lymphocytes in IPF subjects expressed MHC class II and CD154 (CD40L), and they more frequently elaborated TGF-beta1, IL-10, and TNF-alpha. Abnormal CD4 T cell clonal expansions were found in all IPF patients, and 82% of these subjects also had IgG autoantibodies against cellular Ags. IPF lung extracts stimulated proliferations of autologous CD4 T cells, unlike preparations from normals or those with other lung diseases, and the IPF proliferative responses were enhanced by repeated cycles of stimulation. Thus, CD4 T cells from IPF patients have characteristics typical of cell-mediated pathologic responses, including augmented effector functions, provision of facultative help for autoantibody production, oligoclonal expansions, and proliferations driven by an Ag present in diseased tissues. Recognition that an autoreactive immune process is present in IPF can productively focus efforts toward identifying the responsible Ag, and implementing more effective therapies.     

The stimulation of bone marrow granulopoiesis of normal CBA mice in vitro and in vivo by serum obtained from leucophoretic rats

Abstract. The effect of leucophoretic serum (LS), obtained from rats with polyvinylpyrrolidone (PVP)-induced inflammation, on granulopoiesis in the bone marrow of normal CBA mice was studied. The following test systems were used: short term cultures (4 hr), diffusion chambers (8, 24, 48 and 72 hr) and in vivo assays (12, 24 and 48 hr). The results indicate that LS stimulates the proliferations of granulocytic cells by increasing the number of proliferative granulocytes in mitosis, as well as increasing the total number of proliferative granulocytes. LS did not appear to effect monocytes and other cell lines. It is concluded that a factor present in LS specifically stimulates the proliferation of granulocytic cells, both in vitro and in vivo .     

Pulmonary Multinucleate Giant Cells in Dermatosis Vegetans in Swine: Light microscopic and immunohistochemical investigations

Five pigs with dermatosis vegetans (DV), aged 1–28 days, were examined with the purpose of describing the pulmonary changes and to characterize the pulmonary multinucleate giant cells (MGC) and their possible cytogenesis. No pulmonary changes were present at birth. From 7 days of age, lung changes were characterized by proliferation of alveolar epithelial cells and formation of MGCs. Immunostaining for cytokeratin by a peroxydase–streptavidin method gave a positive reaction in MGCs, bronchial, bronchiolar and alveolar epithelium. MGCs seemed to be formed in the course of alveolar epithelial proliferations, and type-II pneumocytes were proposed as possible precursors.     

Follicular epithelia and dermal papillae of mouse vibrissal follicles qualitatively change their hair-forming ability during anagen

We studied the hair-forming ability of epithelium and the relevant activity of dermal papilla (DP) in mouse vibrissal follicles during the hair cycle. Follicles were transversely cut into four pieces and each of them was associated with an isolated DP and grafted beneath the kidney capsule to induce hair formation. Various hair-cycle combinations of the fragments and DPs were examined. Hairs were generated not only in the follicle fragment containing the bulge (fragment III) but also in the fragment between the bulge and hair bulb (fragment II). The hair-forming frequencies were affected by the hair cycle stages of both the follicle fragments and DPs. Fragment III at late anagen (LA) and fragment II at catagen frequently generated hairs when associated with early anagen (EA)-DPs, but infrequently with mid-anagen (MA)-DPs. Oppositely, anagen fragment II produced hairs at a high frequency with MA-DPs and at a low frequency with EA-DPs. Hair generation in anagen fragment II is an unexpected finding because previous studies suggested that, during anagen, this region does not contain clonogenic epithelial cells that have been believed to be crucial for hair formation. Therefore, non-clonogenic epithelial cells would be able to generate hairs as well as clonogenic ones, and they should have a latent hair-forming ability that could be more effectively awakened by MA-DP than by EA-DP stimuli. Non-clonogenic epithelial cells might be a dormant phase of hair precursor cells. Proliferating follicular epithelial cells were detected in the middle and lower outer root sheath throughout the hair cycle but scarcely at LA. These findings suggest that the hair inductivity of DPs should be altered between EA and MA, and follicular epithelial cells would change their DP stimuli-directed hair-forming ability around LA, probably linked to the proliferative activity.     

Observer variation in histopathological diagnosis and grading of cervical intraepithelial neoplasia.

To assess the variability among histopathologists in diagnosing and grading cervical intraepithelial neoplasia eight experienced histopathologists based at different hospitals examined the same set of 100 consecutive colposcopic cervical biopsy specimens and assigned them into one of six diagnostic categories. These were normal squamous epithelium, non-neoplastic squamous proliferations, cervical intraepithelial neoplasia grades I, II, and III, and other. The histopathologists were given currently accepted criteria for diagnosing and grading cervical intraepithelial neoplasia and asked to mark their degree of confidence about their decision on a visual linear analogue scale provided. The degree of agreement between the histopathologists was characterised by kappa statistics, which showed an overall poor agreement (unweighted kappa 0.358). Agreement between observers was excellent for invasive lesions, moderately good for cervical intraepithelial neoplasia grade III, and poor for cervical intraepithelial neoplasia grades I and II (unweighted kappa 0.832, 0.496, 0.172, and 0.175, respectively); the kappa value for all grades of cervical intraepithelial neoplasia taken together was 0.660. The most important source of disagreement lay in the distinction of reactive squamous proliferations from cervical intraepithelial neoplasia grade I. The histopathologists were confident in diagnosing cervical intraepithelial neoplasia grade III and invasive carcinoma (other) but not as confident in diagnosing cervical intraepithelial neoplasia grades I and II and glandular atypia (other). Experienced histopathologists show considerable interobserver variability in grading cervical intraepithelial neoplasia and more importantly in distinguishing between reactive squamous proliferations and cervical intraepithelial neoplasia grade I. It is suggested that the three grade division of cervical intraepithelial neoplasia should be abandoned and a borderline category introduced that entails follow up without treatment.     

Immunohistochemical evidence for the overexpression of protein kinase C in proliferative diseases of human thyroid.

We report immunohistochemical evidence for the overexpression of protein kinase C in various proliferative diseases of human thyroid. Immunohistochemical characterization of various surgically removed thyroid tissues, viz., cancer tissues: papillary carcinoma and follicular carcinoma; adenoma tissues: tubular, trabecular and colloid adenomas; adenomatous goiter; and normal thyroid was done using the monospecific monoclonal antibodies MC-1a, MC-2a and MC-3a, each of which is specific for types I, II and III isozymes of protein kinase C, respectively. For protein kinase C type II, a remarkable difference in staining intensity was noted between the cancerous and normal tissues. The cytoplasm of papillary and follicular carcinoma cells stained more intensely than that of normal thyroid cells. In the benign tumor and adenomatous goiter tissues, stronger staining was noted in the papilliform-proliferating portion and cubic epithelial cells. In the normal thyroid tissues, epithelial cells of greater height were more strongly stained than simple squamous epithelial cells. These results indicated that protein kinase C type II isozyme is expressed in larger amounts in cancerous and proliferative tissues of the human thyroid.     

Feedback regulation of the proliferation of the undifferentiated spermatogonia in the Chinese hamster by the differentiating spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. The undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G1, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.     

Feedback Regulation of the Proliferation of the Undifferentiated Spermatogonia In the Chinese Hamster By the Differentiating Spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. the undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G₁, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

Formation of basement membrane in extracapillary proliferates in rapidly progressive glomerulonephritis.

In the extracapillary proliferations (crescents) of the glomeruli in glomerulonephritis, basement membranes appear and in addition "secretory bodies" are formed in the cisternae of the rough endoplasmatic reticulum. The findings permit the conclusion that proliferated visceral epithelial cells are involved in the crescent formation to a greater extent than previously assumed.