Reproducibility of immunostaining quantification and description of a new digital image processing procedure for quantitative evaluation of immunohistochemistry in pathology.

Quantification of immunostaining is a widely used technique in pathology. Nonetheless, techniques that rely on human vision are prone to inter- and intraobserver variability, and they are tedious and time consuming. Digital image analysis (DIA), now available in a variety of platforms, improves quantification performance: however, the stability of these different DIA systems is largely unknown. Here, we describe a method to measure the reproducibility of DIA systems. In addition, we describe a new image-processing strategy for quantitative evaluation of immunostained tissue sections using DAB/hematoxylin-stained slides. This approach is based on image subtraction, using a blue low pass filter in the optical train, followed by digital contrast and brightness enhancement. Results showed that our DIA system yields stable counts, and that this method can be used to evaluate the performance of DIA systems. The new image-processing approach creates an image that aids both human visual observation and DIA systems in assessing immunostained slides, delivers a quantitative performance similar to that of bright field imaging, gives thresholds with smaller ranges, and allows the segmentation of strongly immunostained areas, all resulting in a higher probability of representing specific staining. We believe that our approach offers important advantages to immunostaining quantification in pathology.

Testosterone stimulates growth and secretory activity of the female prostate in the adult gerbil (Meriones unguiculatus).

The prostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.

Patterns of adherence to HEp-2 cells and actin polymerisation by toxigenic Corynebacterium diphtheriae strains.

Corynebacterium diphtheriae strains displayed different degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns, termed localised (LA) and diffuse (DA). The LA phenotype predominated over the DA phenotype. The non-sucrose fermenting strains expressing DA pattern adhered mostly with high index values (> or =10bact/cell). Low adhesion index (<10bact/cell) was mainly observed among sucrose fermenting strains. The fluorescein isothiocyanate (FITC)-labelled phalloidin assay (fluorescent-actin staining test) showed positive results for microorganisms of both LA and DA phenotypes. The FITC-labelled C. diphtheriae non-fimbrial surface proteins 67-72p interacted directly with HEp-2 cell membranes. Therefore, toxigenic C. diphtheriae exhibited LA and DA adherence patterns and ability to induce actin polymerisation. The experimental evidences also pointed to 67-72p as putative adhesins of C. diphtheriae to HEp-2 cells.

Creating a histology-embryology free digital image database using high-end microscopy and computer techniques for on-line biomedical education.

The development of new technology and the possibility of fast information delivery by either Internet or Intranet connections are changing education. Microanatomy education depends basically on the correct interpretation of microscopy images by students. Modern microscopes coupled to computers enable the presentation of these images in a digital form by creating image databases. However, the access to this new technology is restricted entirely to those living in cities and towns with an Information Technology (IT) infrastructure. This study describes the creation of a free Internet histology database composed by high-quality images and also presents an inexpensive way to supply it to a greater number of students through Internet/Intranet connections. By using state-of-the-art scientific instruments, we developed a Web page (http://www2.uerj.br/~micron/atlas/atlasenglish/index.htm) that, in association with a multimedia microscopy laboratory, intends to help in the reduction of the IT educational gap between developed and underdeveloped regions.

Influence of polarisation and differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human enterocyte-like Caco-2 cell line.

It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.

Isolation and biochemical characterization of the plasma membrane of Tritrichomonas foetus

A fraction containing plasma membrane-enriched vesicles has been prepared from Tritrichomonas foetus. Cells were ruptured using a Potter type homogenizer, under well controlled conditions, and membranes were isolated by differential centrifugation and in discontinuous sucrose gradient. This fraction was enriched 8 and 10-fold in the plasma membrane marker enzymes 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase, respectively. Determination of Glucose-6-phosphatase and NADPH cytochrome c reductase activities in this fraction, indicates a minimal contamination with endoplasmic reticulum membranes. Analysis by Sodium Dodecyl Sulfate-Polyacrylamide (SDS-PAGE) gradient gel showed that the plasma membrane fraction contains several proteins with major bands corresponding to apparent molecular weights of 48, 45, 39, 37, 32, 30, 27, 23, 20, 19, 17, and 15 kDa.

Isolation and biochemical characterization of the plasma membrane of Tritrichomonas foetus

A fraction containing plasma membrane-enriched vesicles has been prepared from Tritrichomonas foetus. Cells were ruptured using a Potter type homogenizer, under well controlled conditions, and membranes were isolated by differential centrifugation and in discontinuous sucrose gradient. This fraction was enriched 8 and 10-fold in the plasma membrane marker enzymes 5-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase, respectively. Determination of Glucose-6-phosphatase and NADPH cytochrome c reductase activities in this fraction, indicates a minimal contamination with endoplasmic reticulum membranes. Analysis by Sodium Dodecyl Sulfate-Polyacrylamide (SDS-PAGE) gradient gel showed that the plasma membrane fraction contains several proteins with major bands corresponding to apparent molecular weights of 48, 45, 39, 37, 32, 30, 27, 23, 20, 19, 17, and 15 kDa.(AU)