John M. Skinner

Development and characterization of primary cultures of smooth muscle cells from the fibromuscular stroma of the guinea pig prostate

SummaryPrimary cultures of smooth muscle cells (SMCs) were obtained by a two-step enzymatic digestion of guinea pig prostatic stroma. Ultrastructural morphology and growth characteristics of these cells conformed to those reported for SMCs isolated from vascular and visceral tissue sources. Electron microscopic examination indicated that the cells assumed modified myofibroblastoid features in culture. Microfilaments with associated dense bodies were markedly depleted in cultured smooth muscle cells, in comparison with those of the parent tissue. Cultured cells also possessed increased content of rough endoplasmic reticulum indicating the increased secretory or protein-synthetic capacity of the cells. Immunoperoxidase staining for cytoskeletal markers using monoclonal antibodies to desmin and vimentin supported the ultrastructural observations, suggesting a decline in desmin-staining intermediate filaments during “modulation” to the myofibroblastoid form. Despite this depletion of smooth muscle-specific differentiation markers and reversion to more general mesenchymal properties, the cells retained the ability to contract on challenge with norepinephrine, and grew in the characteristic “hill and valley” pattern on attaining confluence. Inasmuch as the estrogen and androgen receptor expression of the parent stromal tissue is also retained, these primary cell cultures should provide a useful model to study regulation of prostatic development.

Monoclonal antibodies against antigens of the human platelet surface: preparation and properties

Summary Three monoclonal antibodies, FMC24, 25 and 27, reactive with human platelets, are described. In normal blood the 3 antibodies are specific for platelets, but FMC27 reacts with leukemic blast cells in a proportion of acute leukemias. FMC25 precipitates the platelet membrane glycoprotein lb and a glycoprotein of molecular weight 22,000. The antibodies show different effects on platelet aggregation.

The typing of non-hodgkins lymphomas using fine needle aspiration cytology

Summary Fine needle aspiration cytology smears and histology sections from 53 cases of non‐Hodgkins lymphoma (NHL) were reviewed blindly and independently to determine the accuracy of cytological subtyping of NHL. It was decided to use the Kiel classification as it is based on cytological criteria. There was full agreement between the cytological and the histological classification in 44 of 53 cases. the cases were correctly assigned to a good or bad prognostic group in 48 of 49 instances in which a cytological diagnosis was made. It is felt that fine needle aspiration biopsy for cytology is sufficiently accurate to be of practical value in the following circumstances: 1. As a sole morphological diagnosis so as to avoid major surgery in cases of advanced disease without superficial node invoIvement. 2. To assess the extent of node involvement following histological diagnosis without further surgery. 3. in the selection of a representative node for surgical biopsy. 4. in the investigation of recurrent disease.

Crystalloidal Paraprotein Deposits in the Cornea: An Ultrastructural Study of Two New Cases with Tubular Crystalloids that Contain IgGk Light Chains and IgGγ Heavy Chains

The fine structure and immunoprotein content of the crystalloids are described in two cases of paraproteinemic crystalloidal keratopathy, both of which had clinical features thought by the referring ophthalmologists to be those of atypical lattice-type corneal dystrophy (presumably because of lattice-like lines). Most keratocytes in one case were surrounded by a mantle of densely packed tubular crystalloids. Individual tubules were annular in cross section with mean dimensions as follows: overall diameter, 29.32 nm (SD 1.26); internal diameter (core), 8.53 nm (SD 1.12); wall thickness, 10.39 nm (SD 0.85) (n = 10). Crystalloids were extracellular and found only in the corneal stroma, with none in Bowmans layer or Desce-mets membrane. In the second case, the tubules had a similar distribution but formed geometric arrays with no clear relationship to, or envelopment of, the keratocytes. The tubules were thin-walled, with mean dimensions as follows: overall diameter, 26.12 nm (SD 1.12); internal diameter (c...

Differences between standard and high-sensitivity immunohistology in tissue sections — comparison of immunoperoxidase staining methods using computerized video image analysis techniques

&NA; High‐sensitivity immunoperoxidase labelling can be achieved using heavy metal enhancement of the di‐amino (DAB) reaction product.1,2 For example nickel chloride combined with DAB improves the sensitivity of the method approximately 7–10 fold. This allows detection of approximately 100–200 molecules on cell surfaces. This has an obvious advantage over standard non‐enhanced DAB methods which detect 1000–2000 molecules under similar conditions.3 This study compares standard and nickel enhanced DAB immunoperoxidase staining of cells in tissue sections using video‐image analysis (VIA) measurement techniques. VIA is an objective method of evaluation of immunoperoxidase staining of separated cells and for immuno‐stained cells in tissue sections. Nickel enhancement of the DAB reaction product reveals more positively stained cells, with higher contrast over background, giving superior qualities for VIA analysis providing continuous, reproducible, and objective data for statistical analysis.

The use of computer-assisted video image analysis in the enumeration of immuno-stained cells in tissue sections

A novel method of computer-assisted video image analysis (VIA) was used to determine the number of immunostained cells in tissue sections. This method permitted an accurate and objective quantification of cells of a particular phenotype. This enumeration was achieved by measuring the area stained by a test monoclonal antibody (such as the T cell marker, CD3) and comparing it with the area stained by a leukocyte common (LCA) monoclonal antibody (CD45). The proportion of T cells within the total leukocyte population in a particular tissue was then calculated. The differentiation of positive (stained) and negative (unstained) cells was uniformly maintained by setting the computer to detect a threshold for staining intensity. This enabled consistency to be maintained within a tissue section as well as between sections stained with the same antibody. In the present study, we determined the phenotype of leukocytes in colonic carcinomas by VIA and compared this with results obtained by normal visual analysis. The VIA method showed distinct advantages over normal visual analysis especially in sections which contained moderate numbers of stained cells.

A comparison of the sensitivity of immunoperoxidase staining methods with high-sensitivity fluorescence flow cytometry-antibody quantitation on the cell surface.

Surface molecules present in low copy numbers can be detected with high-sensitivity fluorescence flow cytometry. Many cells previously thought not to express certain molecules on their surface can now be shown to have these molecules in very low copy numbers by high-sensitivity fluorescent cytometric methods. Detection of molecules by immunoperoxidase staining methods has not previously been compared with high-sensitivity flow cytometry techniques. Computerized video image analysis (VIA) is a method that allows measurement of area and density of the immunostain chromogen reaction product in a standardized fashion analogous to flow cytometry. In this study, we compared immunoperoxidase reaction products measured by VIA methods with high-sensitivity flow cytometric measurements for cells with 10,000 down to 50 antibody molecules bound to their surfaces. Detection of 100-200 surface molecules was possible with heavy metal-enhanced immunoperoxidase methods, whereas standard immunoperoxidase methods were not as sensitive. The sensitivity of the nickel-enhanced immunoperoxidase staining method was confirmed for detection of an epitope (Tac-IL2 receptor alpha-chain) present in low numbers on the surface of peripheral blood lymphocytes.

Expression of MHC Class I and Class II Antigens in Colonic Carcinomas

&NA; Malignant and non‐malignant (‘normal’) colonic tissues from patients with colonic carcinoma were examined for the expression of MHC class I and class II antigens by immunoenzymatic staining using monoclonal antibodies. The amount of class I antigen as detected by 2 monoclonal antibodies, FMC 16 or W6/32 was clearly diminished in 11 of 14 tumours when compared to the amount present on ‘normal’ colonic tissue from the same individual. The loss of class I antigen did not correlate with tumour stage or differentiation. The reactivities of FMC 16 and W6/32 with these tissues were not identical, which indicates that the 2 monoclonal antibodies may recognize different epitopes on the HLA class I molecule. Class II antigens were absent from ‘normal’ colonic epithelium but were present on 20 of 28 tumours, with DR being detected more often than DP, and DQ found only on 4 of 28 tumours. When present, staining for class II antigens was heterogeneous within the tumour, in that all tumour cells did not stain equally. DR and DP antigens were found more often on moderately or poorly differentiated adenocarcinomas and on stage 6, C and D tumours in that order of frequency. Thus tumours with a better prognosis were less likely to express DR and DP. The expression of DQ was unrelated to staging or differentiation.

Immune profiling in human breast cancer using high-sensitivity detection and analysis techniques

Objectives Evaluation of immune profiles in human breast cancer using high-sensitivity detection and analysis methods. Design Cohort comparative analysis studies of breast tissue. Setting Human hospital and laboratory healthcare facilities. Participants Women over 18 years. Main outcome measures Evaluation of the comparative immunophenotype of human breast carcinoma and normal breast tissues. Results Leukocyte density and specific subgroups of lymphocytes and macrophages were generally higher in breast cancers compared to normal breast tissues. CD3, CD4, CD45RO, CD45RA(2H4), CD45 and HLA Class II (on TIL) were significantly expressed on breast tumour tissues compared with normal tissues (p < .01). Some 30% of T-cells were γδ-TCR positive, but the majority were αβ-TCR in type. CD19 (B-cell), CD14 (FMC32 and 33) and HLA Class I levels (epithelial and TIL) showed no significant differences. IL-2α receptor expression was low or absent on most TIL. Conclusions High-sensitivity and image analysis techniques permitted accurate characterisation of the TIL infiltrate for immune profiling. Breast carcinoma showed predominance of CD4 T-cells of mainly memory phenotype. Normal breast tissues showed low leukocyte infiltration. Further correlation of these findings with clinical outcome, including survival, is proceeding with encouraging results.

Tumour associated antigens in polyps and carcinoma of the human large bowel

Eight metaplastic polyps, 16 tubulo-villous adenomas and 20 adenocarcinomas, all from the colon, were examined for the presence of a number of tumour associated antigens. Specific antibodies, raised in rabbits, were used to detect the antigens in formalin fixed (pH 7.0) paraffin embedded material. Antisera were directed against carcino-embryonic antigen (CEA), colon specific antigen (M. Wt) (CSA), α-fetoprotein, pregnancy specific β-lipoprotein 1 (SP 1), human β-chorionic gonadotrophin (HCG), human placental lactogen (HPL), and placental alkaline phosphatase (P Alk P). Antisera against isoferritins, reported in some non-gastrointestinal tumours and transferrin, normally found in small intestinal epithelium were also used. Specificity of antibodies was checked in other systems (RIA and immunodiffusion plates) and using appropriate fixed substrate tissues in the P.A.P. technique. Detection and localization of antigens in tissues was made using the P.A.P. (peroxidase-antiperoxidase) enzyme bridge immunohistochemical method. Results are shown in Tables 1 and 2. Placental alkaline phosphatase was found in only 2 specimens and is not included in the tables. Table 1 Antigen No. cases Condition 0 1 2 3 4 5 6 7 16 TVA 1 3 1 3 1 1 1 6 20 Cancers 0 2 2 0 1 3 3 9 8 Metaplastic polyps 0 1 5 0 0 0 0 2 Table 2 Antigen No. cases Condition SPI HPL βHGG CEA CSA AFP Transfer Ferrit 16 TVA 6 11 10 16 14 9 9 9 20 Cancers 12 9 10 20 20 12 13 10 8 Metaplastic polyps 2 7 2 8 7 2 2 2 There appears to be no difference in either number of antigens present, or in the number of cases positive for each antigen, between cancers and tubulo-villous adenomas, but the majority of metaplastic polyps show only CEA and HPL positivity. The 2 metaplastic polyps showing a full range of positivity were atypical in that they were greater than 5 mm in diameter and had some features of adenomatous polyps, although they appeared as typical metaplastic polyps to an experienced gastrointestinal surgeon. The findings have shown a remarkable similarity between polyps and cancer which strengthens the concept of the relationship between adenomatous polyps and carcinoma of the colon, but have revealed no definite antigenic marker of malignant transformation. It would appear that the difference in behaviour is not related to antigenic profile. Despite the literature stressing the benign nature of metaplastic polyps, a more cautious appraisal of those above 5 mm diameter may be indicated.