Immunohistologic parameters in minimal morphologic change duodenal biopsies from patients with clinically suspected gluten-sensitive enteropathy.

Subclinical or latent cases of gluten-sensitive enteropathy (GSE) are difficult to diagnose, and serology-positive, histology-negative (minimal morphologic change) and serology-negative, histology-positive patients have been identified. Both, particularly the histology-negative group, require the correct diagnosis for proper management, especially because the concept of minimal histologic change GSE has escaped attention in standard textbooks. We assessed the numbers and distribution of intraepithelial T cells and their subsets with CD3, CD8, and CD4 immunostaining and examined for crypt hyperplasia with mitotic and Ki-67 proliferative indices with the aim of refining the criteria for the diagnosis of minimal change GSE. Duodenal biopsies from 46 clinically suspected cases of GSE tested for antigliadin, antiendomysium, and antitissue transglutaminase antibodies were divided into four groups: serology-positive, histology-positive (S+H+, n = 20); serology-positive, histology-negative (S+H-, n = 22), representing the minimal morphologic change group; serology-negative, histology-positive (S-H+, n = 4); and serology-negative, histology-negative (S-H-, n = 28), controls with histologically normal duodenal biopsies obtained for unrelated reasons. The numbers of CD3+ and CD8+ intraepithelial T cells (IETCs) were significantly higher in histology-positive biopsies with (mean, 40.3/100 and 39.3/100 enterocytes, respectively) and without positive serology (mean, 33.3/100 and 35/100 enterocytes, respectively) compared with all other groups (S+H-, mean, 26.5/100 and 24.3/100 enterocytes, respectively; S-H-, mean, 23.3/100 and 17.9/100 enterocytes, respectively). Values for Ki-67 index in crypt enterocytes were also significantly different between histology-positive and histology-negative groups (P = 0.000). The distribution of CD3+ and CD8+ IETCs was mostly even along the surface enterocytes in histology-positive cases compared with the controls, which showed an uneven distribution. The 2 parameters that significantly discriminated between minimal morphologic change GSE (S+H-) and controls (S-H-) were Ki-67 index (P = 0.007) and the distribution pattern of CD8+ IETCs (P = 0.049). CD4 IETC counts were generally low, with no significant difference between all groups. The few S-H+ cases seen most likely represented false-negative serology, because the assessed parameters of this group and S+H+ cases were indistinguishable.     

Tumor genes and their proteins in cytologic and surgical specimens: Relevance and detection systems

Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases. Tumor-related genes can be classified into functional categories. Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation. More recently, three other groups of tumor-related genes have been recognized. They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes. Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response. The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques. These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist. © 1995 Wiley-Liss, Inc.     

Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications

AIMS: Nodal expression of the carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and guanylyl cyclase C (GCC) genes was measured in tandem in patients with colorectal cancer (CRC) to assess whether there would be sufficient agreement between these markers in their ability to detect micrometastasis to qualify one of them as a universal marker, and whether frozen and paraffin wax embedded tissues would yield similar results. METHODS: One hundred and seventy five frozen lymph nodes (FT) and 158 formalin fixed, paraffin wax embedded lymph nodes (PET) from 28 CRC cases were analysed using gene specific quantitative real time polymerase chain reaction, carried out on the LightCycler system with SYBR Green chemistry. RESULTS: There was significant disparity in positive detection of the three biomarkers in FT versus PET, with notable agreement achieved only for CEA (66.6%) in FT versus PET in Dukes' B disease, and between CK20 and GCC (44.6%) in FT, also in Dukes' B disease. One patient with full concordance in all three tumour markers with both tissue types suffered a relapse and died within two years of follow up. CONCLUSIONS: There was considerable discordance in the positive detection of the three tumour markers in both tissue types (FT versus PET). This brings into question whether using a single tumour marker to detect micrometastasis in one tissue type (FT or PET) is adequately representative, and challenges the concept of universal markers for molecular CRC metastatic detection. Multiple tumour markers would predict more accurately the metastatic potential of Dukes' B CRCs.     

High density of immobilized galactose ligand enhances hepatocyte attachment and function

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.     

Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.     

Translocation (X;12)(q27;q14) in a lipoma

Cytogenetic analysis by short-term culture of a lipoma from the posterior neck region of a 63-year-old man showed a t(X;12)(q27;q14) as the sole chromosomal abnormality. Rearrangement of band 12q14 is nonrandom in lipomas, although its involvement with a sex chromosome has not been reported earlier.     

B1b lymphocytes confer T cell-independent long-lasting immunity

Many microbial pathogens employ antigenic variation as a strategy to evade the immune system, posing a challenge in vaccine development. To understand the requirements for immunity against such pathogens, we studied Borrelia hermsii, a relapsing fever bacterium. We found that mice deficient in T, follicular B, marginal zone B, or B1a lymphocytes resolved B. hersmii bacteremia and became resistant to reinfection. The resolution of bacteremia coincided with an expansion and persistence of B1b lymphocytes, and purified B1b lymphocytes from convalescent wild-type or TCR-betaxdelta-/- mice conferred immunity to Rag1-/- mice. The B1b lymphocytes in the reconstituted Rag1-/- mice provided long-lasting immunity by rapidly generating B. hermsii-specific IgM but not IgG upon bacterial challenge. Unmutated IgM is sufficient to eliminate B. hermsii, because AID-/- mice deficient in somatic hypermutation and class switch recombination efficiently resolved all bacteremic episodes. These data demonstrate that B1b lymphocytes can provide long-lasting T cell-independent IgM memory.