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Aims:   Follicular lymphoma (FL) is associated with bone marrow (BM) involvement in approximately 40-70% of cases. Previous studies have suggested that the immune-microenvironment of FL plays an important role in the clinical behaviour of the disease. To investigate the role of the microenvironment in BM involvement of FL, we performed immunophenotypical analysis of the reactive cell populations in lymph nodes (LN) and corresponding BM of 35 patients with FL. Microenvironment patterns of BM infiltrates were compared to the corresponding features of the LN in cases with BM manifestation, and the LN microenvironment was compared in FL cases with and without BM involvement., Methods and Results:   Automated image-segmentation-based quantitation was performed in whole digital slides of tissue microarrays of formalin-fixed paraffin-embedded tissue biopsies. We found significantly more CD8(+) T lymphocytes, forkhead box protein 3 (FoxP3)(+) T lymphocytes and CD68(+) macrophages and fewer PD1(+) T lymphocytes in the BM than in the matching LN samples. Furthermore, we observed significantly fewer CD8(+) T cells and CD68(+) macrophages in cases involving the BM compared to those localized only to the LNs., Conclusions:   Our study showed that different tumour cell growth in the LN and BM may generate different microenvironments, and suggested that the reduced number of cytotoxic T lymphocytes and macrophages in LNs favours BM infiltration of neoplastic cells in FL., (© 2012 Blackwell Publishing Ltd.)

Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

Background: Robust hardware and software tools have been developed in digital microscopy during the past years for pathologists. Reports have been advocated the reliability of digital slides in routine diagnostics. We have designed a retrospective, comparative study to evaluate the scanning properties and digital slide based diagnostic accuracy., Methods: 8 pathologists reevaluated 306 randomly selected cases from our archives. The slides were scanned with a 20× Plan-Apochromat objective, using a 3-chip Hitachi camera, resulting 0.465 μm/pixel resolution. Slide management was supported with dedicated Data Base and Viewer software tools. Pathologists used their office PCs for evaluation and reached the digital slides via intranet connection. The diagnostic coherency and uncertainty related to digital slides and scanning quality were analyzed., Results: Good to excellent image quality of slides was recorded in 96%. In half of the critical 61 digital slides, poor image quality was related to section folds or floatings. In 88.2% of the studied cases the digital diagnoses were in full agreement with the consensus. Out of the overall 36 incoherent cases, 7 (2.3%) were graded relevant without any recorded uncertainty by the pathologist. Excluding the non-field specific cases from each pathologist's record this ratio was 1.76% of all cases., Conclusions: Our results revealed that: 1) digital slide based histopathological diagnoses can be highly coherent with those using optical microscopy; 2) the competency of pathologists is a factor more important than the quality of digital slide; 3) poor digital slide quality do not endanger patient safety as these errors are recognizable by the pathologist and further actions for correction could be taken., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1913324336747310.

Background: The early molecular detection of the dysplasia-carcinoma transition may enhance the strength of diagnosis in the case of colonic biopsies. Our aims were to identify characteristic transcript sets in order to develop diagnostic mRNA expression patterns for objective classification of benign and malignant colorectal diseases and to test the classificatory power of these markers on an independent sample set., Methodology/principal Findings: Colorectal cancer (CRC) and adenoma specific transcript sets were identified using HGU133plus2 microarrays and 53 biopsies (22 CRC, 20 adenoma and 11 normal). Ninety-four independent biopsies (27 CRC, 29 adenoma and 38 normal) were analyzed on microarrays for testing the classificatory power of the discriminatory genes. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma and 20 normal). A set of 11 transcripts (including CXCL1, CHI3L1 and GREM1) was determined which could correctly discriminate between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples was correctly classified in discriminant analysis., Conclusions/significance: The identified transcripts could correctly characterize the dysplasia-carcinoma transition in biopsy samples, also on a large independent sample set. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic RT-PCR cards can become part of the automated routine procedure.

We tested the expression of known (p16(ink4), Ki67, p53, EGFR) and a new immunohistochemical (collagen XVII/BP180) biomarker in head and neck squamous cell carcinomas (SCC) of diverse anatomical localization. Tissue microarrays (TMA) of 124 SCC were created, immunostained, and analyzed following whole slide digitalization using the Pannoramic Scan and the TMA Module software (3DHISTECH Kft, Budapest, Hungary). Statistical analysis of scoring results was carried out using Pearson's chi-square test. We observed the significant elevation of p16(ink4) and Ki67 expression in supraglottic, tonsillar and tonsillo-lingual SCCs compared to those affecting the oral cavity, oropharynx without tonsils, larynx without supraglottis and the hypopharynx. This differential antigen expression may reflect the diverse route of embryologic differentiation followed by the affected regions except those of the tonsils and the supraglottis which show similar antigenic pattern but diverse developmental path. All the other biomarkers tested including p53, collagen XVII and EGFR were detected in the majority of cancers including high grade cases, but did not reveal any significant regional difference. Based on our results oropharyngeal squamous cell carcinomas may not be regarded as one entity. Concerning the oral cavity and the oropharynx, cancers affecting the tonsils (palatine and lingual) show significantly elevated p16(ink4) and Ki67 expression; so as the cancers of the supraglottis compared to the rest of larynx. Consequently, tonsillar and supraglottic cancers show similar biomarker profiles. Correlation of differential biomarker expression with diverse biological behavior in head and neck cancers need further investigations.

Aims: Colonic inflammation is followed by regeneration supported by bone marrow-derived cells (BMDCs) including multipotent cells. They migrate to the colonic epithelial layer and may transdifferentiate into epithelial-like cells or keep their stem cell characteristics and produce progenies. The aim was to study the role of lymphoid aggregates in the migration and transition of BMDCs in both healthy colons and non-specific colitis (NSC)., Methods: Samples of normal colon (n=5) and NSC (n=5) from female patients who were initially transplanted with male bone marrow were studied. After detecting XY chromosomes using fluorescent in situ hybridisation, tissue sections were digitalised, the coverslips were eliminated and the samples were double stained for CD45 and cytokeratin with immunofluorescence. Then CDX2 expression, as a sign of intestinal epithelial commitment of Musashi-1+ stromal BMDCs, was also tested with both immunoperoxidase and parallel immunofluorescence stainings. The slides were digitalised again and analysed simultaneously., Results: A significant increase in intraepithelial CD45-BMDCs was found in regions adjacent to lymphoid aggregates (median: 1.01) compared with healthy epithelial regions (median: 0.0175) or NSC (median: 0.04) samples. The stromal Musashi-1+ cells were positive for CDX2 as well, as a sign of epithelial differentiation. The CDX2+ cells bearing the Y chromosome proved the epithelial commitment of several stromal BMDCs., Conclusion: Elevated number of intraepithelial CD45-BMDCs at lymphoid aggregates suggests that BMDCs play a role in epithelial regeneration and that lymphoid aggregates serve as their migration route.

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (RA). The underlying mechanism that explains the connection of smoking, citrullination [catalyzed by peptidyl arginine deiminases (PADs)] and ACPAs is still unclarified in RA. Thus, we searched for a non-arthritic model in which an increased cell death allows the formation of autoantibodies. Data supporting that lung cancer might be a good candidate are as follows: (i) smoking plays a role in its pathogenesis, (ii) the disease is frequently accompanied by paraneoplastic syndrome, (iii) smoking increases citrullination in the lung, (iv) various types of malignancies are associated with increased citrullination and (v) lung cancer tissue shows similarities with RA synovium. Serum PAD4, rheumatoid factor (RF) and ACPA levels were measured in 42 lung cancer patients; expression of cytokeratin 7 (CK7), PAD4 and citrullinated proteins was visualized in 113 lung cancer tissues. All parameters were analyzed in correlation with smoking history. None of the patients had polyarthritis or autoimmune disease. Significantly increased RF levels were associated with higher PAD4 levels in smoker lung cancer patients compared with non-smokers. Both PAD4 and citrullination immunostaining strongly correlated with that of CK7 in lung cancer, however, did not differ according to smoking history. Two of 30 smoker lung cancer patients had high anti-cyclic citrullinated peptide levels. In conclusion, PAD4 and citrullination may be helpful in distinguishing lung cancer from healthy tissue. Smoking, abnormal serum PAD4 and RF levels may not be sufficient for the production of ACPAs and development of autoimmunity.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

Bone marrow derived mesenchymal stem cells (BM-MSCs) take part in the colonic mucosal regeneration. They are multipotent cells, which can be identified with both negative (i.e. CD13, CD 14, CD45, c-Kit, major histocompatibility complex /MHC class I and II) and positive (i.e. CD54 (ICAM1), CD133, CD146 (MCAM), CD166, Flk-1, Sca-1, Thy-1, stage-specific antigen I /SSEA-I and Musashi-1, HLA class I) markers. These cells can repopulate the gastrointestinal mucosa as they may differentiate into stromal- (i.e. myofi-broblast) or epithelial-like (Paneth-, epithel-, goblet or enteroendocrin) cells without proliferation. During the mesenchymal to epithelial transition (MET) stem cells enter the epithelial layer and take up epithelial cell-like properties. Rarely BM-MSCs may retain their stem cell characteristics and are capable of producing progeny. The isolated lymphoid aggregates may serve as a platform from where BM-MSCs migrate to the nearby crypts as mediated by several chemoattractant proteins, which are expressed in injured tissue. The number of BM-MSCs is influenced by the degree of inflammation. In this review we summarize the current information about the role of BM-MSCs in the repair progress of injured colonic epithelium and their potential clinical applications.

Background: The immunohistochemical detection of estrogen (ER) and progesterone (PR) receptors in breast cancer is routinely used for prognostic and predictive testing. Whole slide digitalization supported by dedicated software tools allows quantization of the image objects (e.g. cell membrane, nuclei) and an unbiased analysis of immunostaining results. Validation studies of image analysis applications for the detection of ER and PR in breast cancer specimens provided strong concordance between the pathologist's manual assessment of slides and scoring performed using different software applications., Methods: The effectiveness of two connected semi-automated image analysis software (NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14) for determination of ER and PR status in formalin-fixed paraffin embedded breast cancer specimens immunostained with the automated Leica Bond Max system was studied. First the detection algorithm was calibrated to the scores provided an independent assessors (pathologist), using selected areas from 38 small digital slides (created from 16 cases) containing a mean number of 195 cells. Each cell was manually marked and scored according to the Allred-system combining frequency and intensity scores. The performance of the calibrated algorithm was tested on 16 cases (14 invasive ductal carcinoma, 2 invasive lobular carcinoma) against the pathologist's manual scoring of digital slides., Results: The detection was calibrated to 87 percent object detection agreement and almost perfect Total Score agreement (Cohen's kappa 0.859, quadratic weighted kappa 0.986) from slight or moderate agreement at the start of the study, using the un-calibrated algorithm. The performance of the application was tested against the pathologist's manual scoring of digital slides on 53 regions of interest of 16 ER and PR slides covering all positivity ranges, and the quadratic weighted kappa provided almost perfect agreement (κ = 0.981) among the two scoring schemes., Conclusions: NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14 software application proved to be a reliable image analysis tool for pathologists testing ER and PR status in breast cancer.

Background: Ollier disease is a rare, non-hereditary disorder which is characterized by the presence of multiple enchondromas (ECs), benign cartilaginous neoplasms arising within the medulla of the bone, with an asymmetric distribution. The risk of malignant transformation towards central chondrosarcoma (CS) is increased up to 35%. The aetiology of Ollier disease is unknown., Methods: We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis using Affymetrix SNP 6.0 array on 37 tumours of 28 Ollier patients in combination with expression array using Illumina BeadArray v3.0 for 7 ECs of 6 patients., Results: Non-recurrent EC specific copy number alterations were found at FAM86D, PRKG1 and ANKS1B. LOH with copy number loss of chromosome 6 was found in two ECs from two unrelated Ollier patients. One of these patients also had LOH at chromosome 3. However, no common genomic alterations were found for all ECs. Using an integration approach of SNP and expression array we identified loss as well as down regulation of POU5F1 and gain as well as up regulation of NIPBL. None of these candidate regions were affected in more than two Ollier patients suggesting these changes to be random secondary events in EC development. An increased number of genetic alterations and LOH were found in Ollier CS which mainly involves chromosomes 9p, 6q, 5q and 3p., Conclusions: We present the first genome-wide analysis of the largest international series of Ollier ECs and CS reported so far and demonstrate that copy number alterations and LOH are rare and non-recurrent in Ollier ECs while secondary CS are genetically unstable. One could predict that instead small deletions, point mutations or epigenetic mechanisms play a role in the origin of ECs of Ollier disease.

A molecular diagnostic assay using easily accessible peripheral blood would greatly assist in the screening and diagnosis of ulcerative colitis (UC) and Crohn's disease (CD). Transcriptional profiles in blood/biopsy samples from 12 UC (6/12), 9 CD (5/9), 6 non-inflammatory bowel disease (non-IBD) colitis (6/0), and 11 healthy (11/11) patients were assessed by Affymetrix HGU133Plus2.0 microarrays. Prediction analysis of microarrays, discriminant and ROC analyses were performed, the results were validated by RT-PCR and immunohistochemistry using also an independent set of samples (15 blood samples, 45 biopsies). A set of 13 transcripts was differentially expressed in IBD, non-IBD controls and healthy blood samples (100% specificity and sensitivity). Validated difference was found in 16 transcripts between UC, non-IBD and normal blood, and 4 transcripts between CD, non-IBD and normal samples. UC and CD blood cases could be also distinguished by 5 genes with 100% specificity and sensitivity. Some disease associated alterations in blood transcripts were also detected in colonic tissue. IBD subtypes may be discriminated from non-IBD (diverticulitis, infective and ischemic colitis) in vitro from peripheral blood by screening for differential gene expression revealed in this study. Transcriptional profile alterations in peripheral blood can be located in diseased colon.

Colorectal cancer progression is characterized by altered epithelial proliferation and apoptosis and by changed expression of tumor development regulators. Our aims were to determine the proliferative/apoptotic epithelial cell ratio (PAR) in the adenoma-dysplasia-carcinoma sequence (ADCS), and to examine its association with osteopontin (OPN), a previously identified protein product related to cancer development. One mm diameter cores from 13 healthy colons, 13 adenomas and 13 colon carcinoma samples were included into a tissue microarray (TMA) block. TUNEL reaction and Ki-67 immunohistochemistry were applied to determine the PAR. The osteopontin protein was also immunodetected. Stained slides were semiquantitatively evaluated using digital microscope and statistically analyzed with logistic regression and Fisher's exact test. The PAR continuously increased along the ADCS. It was significantly (p < 0.001) higher in cancer epithelium (8.84 ± 7.01) than in adenomas (1.40 ± 0.78) and in normal controls (0.89 ± 0.21) (p < 0.001). Also, significant positive correlation was observed between elevated PAR and the expression of osteopontin. Cytoplasmic OPN expression was weak in healthy samples. In contrast, cytoplasmic immunoreaction was moderately intensive in adenomas, while in colon cancer strong, diffuse cytoplasmic immune staining was detected. Increasing PAR and OPN expression along ADCS may help monitoring colorectal cancer progression. The significantly elevated OPN protein levels we found during normal epithelium to carcinoma progression may contribute to the increased fibroblast-myofibroblast transition determining stem cell niche in colorectal cancer.

Objective: Mesenchymal-epithelial transition may have crucial role in mucosal regeneration, hence we assayed epithelial growth factor receptor (EGFR), insulin-like growth factor receptor-1 (IGF1R), hepatocyte-derived growth factor receptor (HGFR), CDX2 and cytokeratin (CK) expression in lymphoid aggregates (LA) of ulcerative colitis (UC)., Material and Methods: Tissue microarrays (TMAs) made of biopsy samples from 20 mildly, 20 moderately and 20 severely active UC, 12 non-specific colitis (NSC) and 20 healthy colon were prepared, and immunolabelled with anti-EGFR, -IGF1R, -HGFR, -CDX2, -CK antibodies. After virtual microscopic evaluation, one-way ANOVA and correlation analysis were performed. For validation, TaqMan real-time RT-PCR was performed by using RNA from laser microdissected LA from 10 healthy colon and 10 endoscopically active UC biopsies., Results: The number of LA was in tight positive correlation with the severity of inflammation (r=0.9). The number of EGFR/HGFR positive subepithelial cells was found to be significantly elevated in severe (21.6+/-2.1%/21.3+/-1.9%), moderate (14.3+/-1.7%/14.6+/-1.6%) and mild (7.2+/-1.6%/7.4+/-1.3%) inflammation compared to healthy colon mucosa (2.6+/-1.4%/2.4+/-1.03%) (p < 0.005). Some alterations were found between UC and NSC samples regarding EGFR and HGFR expression. IGF1R immunoreactive cells were only found in a trace number in all cases. Increasing trend of CDX2 and CK positive subepithelial cells was found in active UC, but it was not in significant correlation with the severity of inflammation., Conclusion: EGFR and HGFR positive subepithelial cells in LA may be involved in the induction of the regenerative mucosal processes. The presence of CDX2/CK positive subepithelial cells suggests that mesenchymal-to-epithelial transition may be located to lymphoid aggregates.

The continuous reformation and rapid repair of the colonic mucosa is essential for avoiding the aggregation of pernicious mutations induced by bacterial, toxic, or mitogenic factors. Gut-associated lymphoid tissue is supposed to play a central role in the organization of the repair mechanisms. In inflammatory conditions, the number, the diameter and the density of isolated lymphoid follicles (ILFs) are increasing. They are involved not just in immune surveillance, but their presence is also indispensable in normal mucosal regeneration of the colon. The relation of ILFs to the components of mucosal renewal such as bone marrow derived stem cells, follicular dendritic cells, subepithelial myofibroblasts or crypt formation has not been directly studied, and data about their putative organizer role are scattered in scientific literature. Whether they act as a regenerative pool containing stem cells in case of mucosal damage, or they are responsible only for the optimal cytokine milieu for the differentiation of immigrating stem cells is a question under debate. Our aim is to review the relation of ILFs to the different elements of colonic mucosal repair.

Unlabelled: In the process of regeneration following colon inflammation mesenchymal stem cells (MSC) of bone marrow origin may also take part besides their local counterparts. These cells migrate in the colon epithelium where they may contribute to epithelial regeneration or form progeny for keeping up local stem cell pool. MSC cells probably leave circulation around lymphoid aggregates to then migrate into nearby crypts. During migration they change their phenotype upon the influence of local microenvironment., Aims: In this study epithelial migration and transition of bone marrow stem cells were examined. Samples from normal healthy individuals and from aspecific inflammation were used. The possible role of lymphoid aggregates in the epithelial regeneration was also studied., Materials and Methods: Samples of normal colon (2) and those showing mild aspecific colitis (3) from female patients who were initially transplanted with male bone marrow were studied. First we detected gender chromosomes with fluorescent in situ hybridization (FISH) and the samples were archived with digital scanning. Then CD45 and cytokeratin (CK) double immunofluorescent reactions (IF) were made followed by digitalization again. Digitalized samples were estimated simultaneously with virtual microscopy (Mirax Viewer)., Results: Significant elevation of CD45 negative/Y-FISH positive potential MSCs were found in crypts locating to the neighborhood of lymphoid aggregates (1,075%) compared with both normal (0,027%, p = 0,002) and mild colitis (0,045%, p = 0,004) samples., Conclusion: Local stem cells probably have enough regeneration capacity in case of minor inflammation. However, in aspecific inflammation the number of MSCs contributing to epithelial regeneration was elevated, suggesting their facilitated contribution to the repair process with less probable forming of local stem cell progeny.

Genetic instability in relation to clinical behavior was studied in 52 cases of giant cell tumor of bone (GCTB). Ploidy was determined in the mononuclear cell population by using native cell smears and image cytometry. A relocalization technique allowed fluorescent in situ hybridization (FISH) analysis of CD68-negative neoplastic cells for numerical changes of chromosomes X, 3, 4, 6, 11, and telomeric association on 11p. Genome-wide alterations were tested using array comparative genomic hybridization (array-CGH) on magnetically separated CD68-negative tumor cells. CTNNB1, TP53, and BCL2 protein expression was also analyzed in formol-paraffin sections to see if their pathways are involved in the development of chromosomal instability. CD68-positive histiocytes showed no significant numerical chromosome and telomeric alterations. Based on ploidy values and clinical outcome, we could distinguish five groups as follows: diploid nonrecurrent (n = 20), tetraploid nonrecurrent (n = 6), diploid recurrent (n = 5), tetraploid and/or aneuploid recurrent (n = 14), and malignant cases (n = 7). Random individual-cell aneusomy was significantly (P < 0.001) more frequent in the recurrent groups (36.01 +/- 11.94%) than in the benign nonrecurrent cases (10.65 +/- 3.66%). The diploid recurrent group showed significantly (P < 0.001) increased balanced aneusomy compared with the diploid nonrecurrent group and the tetraploid nonrecurrent group represented eusomic polysomy. Array-CGH and FISH showed clonal aberrations almost exclusively in the malignant group. None of the protein markers tested showed significant correlation with elevated aneuploidy/polysomy (P = 0.56). Our results show that ploidy determination combined with FISH analysis may help predicting recurrence potential of GCTB and suggest that chromosomal abnormalities superimposed on telomeric associations could be responsible for an aggressive clinical course.

Stroma cells with the microenvironment around them have primary role in the regulation of inflammation processes, conformation of tumors and development of metastasis. Myofibroblasts have essential role in inflammation processes and in the regeneration of encompassed tissue. Molecules produced by them operate the cells of the immune system and effect the proliferation of epithelium. Tumors can activate myofibroblasts which can lead to uncontrolled epithelium proliferation across production of changed and increased regulation ligands (such as cytokines, chemokines, chemotactic and other growth factors) and activation of stem cell. This process could lead piling of uncontrolled epithelial cells and can impact later on conformation of tumors. In this study we present an overview about of myofibroblasts and their roles in inflammation and neoplastic processes.

Background: The effect of gonadotropin-releasing hormone (GnRH) analogs on prostate carcinoma is partly a result of breaking the pituitary-gonadal axis, and partly the direct action on tumor cells expressing the GnRH receptor (GnRHR). The aim of this study was to detect the extent of correlation between the expression of GnRHR and also the androgen receptor (AR) and the efficacy of total androgen blockade (TAB)., Patients and Methods: Needle biopsy samples of twenty advanced prostate carcinoma patients were investigated histologically regarding Gleason score, AR and GnRHR status of the tumor cells. An affinity purified polyclonal antibody reacting with GnRHR and a monoclonal antibody for AR were applied for immunoperoxidase reactions. TAB was started in each case. Pathological, radiological and laboratory-prostate-specific antigen (PSA) data obtained before the start of TAB and survival, PSA values, and radiological findings after three years of TAB were related to AR and GnRHR., Results: Regarding the clinical, radiological and laboratory findings before and after three years of TAB, 13 patients (group A) were considered to show a favourable' and 7 (group B) to show a 'poor' outcome. Twelve patients out of 14 with AR-positive tumor cells and all nine patients with GnRHR-positive tumor cells, as well as all eight patients with both AR- and GnRHR-positive tumor cells belonged to group A. The majority of AR-negative, GnRHR-negative or both AR- and GnRHR-negative cases belonged to group B., Conclusion: The presence of GnRHR and AR or both of these receptors indicates a more favourable outcome of advanced prostate carcinoma when treated with TAB, compared to GnRHR- and AR-negative cases. GnRHR and AR negativity may indicate a need for supplementary chemo- or radiotherapy.

Background: As most colorectal cancers (CRC) develop from villous adenomas, studying alterations in gene expression profiles across the colorectal adenoma-dysplasia-carcinoma sequence may yield potential biomarkers of disease progression., Methods: Total RNA was extracted, amplified, and biotinylated from colonic biopsies of 15 patients with CRC, 15 with villous adenoma and 8 normal controls. Gene expression profiles were evaluated using HGU133Plus2.0 microarrays and disease progression associated data were validated with RT-PCR. The potential biomarkers were also tested at the protein level using tissue microarray samples of 103 independent and 16 overlapping patients., Results: 17 genes were validated to show sequentially altered expression at mRNA level through the normal-adenoma-dysplasia-carcinoma progression. Prostaglandin-D2 receptor (PTGDR) and amnionless homolog (AMN) genes revealed gradually decreasing expression while the rest of 15 genes including osteonectin, osteopontin, collagen IV-alpha 1, biglycan, matrix GLAprotein, and von Willebrand factor demonstrated progressively increasing expression. Similar trends of expression were confirmed at protein level for PTGDR, AMN, osteopontin and osteonectin., Conclusions: Downregulated AMN and PTGDR and upregulated osteopontin and osteonectin were found as potential biomarkers of colorectal carcinogenesis and disease progression to be utilized for prospective biopsy screening both at mRNA and protein levels. Gene alterations identified here may also add to our understanding of CRC progression.

Purpose: Gene expression profile (GEP)-based classification of colonic diseases is a new method for diagnostic purposes. Our aim was to develop diagnostic mRNA expression patterns that may establish the basis of a new molecular biological diagnostic method., Experimental Design: Total RNA was extracted, amplified, and biotinylated from frozen colonic biopsies of patients with colorectal cancer (n=22), adenoma (n=20), hyperplastic polyp (n=11), inflammatory bowel disease (n=21), and healthy normal controls (n=11), as well as peripheral blood samples of 19 colorectal cancer and 11 healthy patients. Genome-wide gene expression profile was evaluated by HGU133plus2 microarrays. To identify the differentially expressed features, the significance analysis of microarrays and, for classification, the prediction analysis of microarrays were used. Expression patterns were validated by real-time PCR. Tissue microarray immunohistochemistries were done on tissue samples of 121 patients., Results: Adenoma samples could be distinguished from hyperplastic polyps by the expression levels of nine genes including ATP-binding cassette family A, member 8, insulin-like growth factor 1 and glucagon (sensitivity, 100%; specificity, 90.91%). Between low-grade and high-grade dysplastic adenomas, 65 classifier probesets such as aquaporin 1, CXCL10, and APOD (90.91/100) were identified; between colorectal cancer and adenoma, 61 classifier probesets including axin 2, von Willebrand factor, tensin 1, and gremlin 1 (90.91/100) were identified. Early- and advanced-stage colorectal carcinomas could be distinguished using 34 discriminatory transcripts (100/66.67)., Conclusions: Whole genomic microarray analysis using routine biopsy samples is suitable for the identification of discriminative signatures for differential diagnostic purposes. Our results may be the basis for new GEP-based diagnostic methods.

The aim of this study was to investigate the expression of p21(waf1/cip1), p27(kip1), p63 and androgen receptor proteins in relation to serum prostate specific antigen levels in low and high Gleason score prostate cancers. Biopsies of patients suffering from prostate adenocarcinoma of low (3 + 3 to 3 + 4) and high (5 + 4 to 5 + 5) Gleason scores (13 cases each group) were immunostained for positive regulators of cell cycle control (p21(waf1/cip1) and p27(kip1)), and essential markers of normal prostate gland ontogeny (p63) and growth (androgen receptor) to find differentially expressed markers of malignant progression. Serum prostate specific antigen levels were also monitored at the time of biopsy and following anti-androgen therapy. All cases except one in each group were androgen receptor positive. P63 and p21(waf1/cip1) proteins detected in normal basal cell nuclei were lost in all but one studied tumors respectively. P27(kip1) protein, however, was detected in all low Gleason score prostate cancers, but it was found in only 7/13 high score cases. Prostate specific antigen levels, either pre- or post-treatment, did not show strict correlation with the p27(kip1) results. The low to high grade dedifferentiation of prostate adenocarcinoma is accompanied with the down-regulation of p27(kip1) protein, which may be an important molecular sign of the lost cell cycle control.

Background and Aims: Chronic Helicobacter pylori infection affects approximately half of the world, leads to chronic gastritis and peptic ulceration, and is linked to gastric carcinoma. Our aims were to compare the gene expression profile (GEP) of H. pylori-positive and H. pylori-negative gastric erosions and adjacent mucosa to explain the possible role and response to H. pylori infection and to get erosion-related mRNA expression patterns., Methods: Total RNA was extracted, amplified, and biotinylated from gastric biopsies of patients with H. pylori-positive and H. pylori-negative antrum erosions (ER) (8/8) and adjacent macroscopically normal mucosae (8/8). The GEP was evaluated using HGU133plus2.0 microarrays. Two independent normalizations (MAS5.0, RMA), PAM feature selection, hierarchical cluster analysis, and discriminant analysis were done. The expression of 14 genes was also measured by real-time-polymerase chain reaction. VCAM-1 and CXCL13 immunohistochemistry (IHC) was done., Results: In H. pylori infection, significant overexpression of MHC class II antigen-presenting genes, interleukin-7 receptor, ubiquitin-D, CXCR4, lactoferrin immune response-related genes, CXCL-2 and -13, CCL18 chemokine ligand, and VCAM-1 genes were established. In erosive gastritis, increased proliferation (MET) and transport (UCP2, SCFD1, KPNA4) were found, while genes associated with adhesion (SIGLEC11), transcription regulation (ESRRG), and electron and ion transport (ACADM, CLIC6) were down-regulated. Discriminant analysis successfully classified all samples into four groups (HP+ER-, HP+ER+, HP-ER+, HP-ER-) using a reduced gene set (20). Significant overexpression of VCAM-1 and CXC13 protein was detected by IHC in HP+ samples (p < .05)., Conclusions: Whole genomic microarray analysis yielded new H. pylori infection and erosion-related gene expression changes. Discriminative genes can be used in mRNA-based diagnostic classification of gastric biopsies.

Whether a breast carcinoma is HER2-positive or -negative has a significant impact on its treatment. The most common method of evaluating the HER2 status of breast tumors is immunohistochemistry. Preliminary data suggest that the proportion of HER2-positive tumors in Hungary shows a wide range from laboratory to laboratory, and overall it just reaches the bottom levels of the incidence reported in the literature (12% 3+ and 11% 2+ cases). A 3-round quality-control test was implemented on a voluntary and anonymous basis. Participating laboratories had to immunostain and evaluate 4 tumor samples per circulation, according to their daily routine. The authors of the present article gave an expert opinion in all cases, and this was compared with the individual laboratories' evaluation and the real FISH-controlled HER2 status of the samples. On the basis of the participants' and experts' evaluation 22/218 and 21/218 HER2 3+ cases were underscored, corresponding to an underevaluation rate of 10%. As most samples were from HER2-positive tumors, overscoring was less common (1%), but reached 5% (individual laboratories' evaluation) or 7% (expert evaluation) when the 2+ scoring of negative cases was also considered. Each case was discussed interactively with the participants and technical advise was also given when deemed necessary. The evaluation of the HER2 status of breast cancers gives reliable results only if adequate quality control measures are implemented, and this study was an important step in this respect.

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

The four main aspects of applied medical information technology, which change the traditional systems of the entire health service are signal and data processing, digital modelling and interface optimisation. The information technology serving individual clinical specialties including clinical histopathology is changing at each of the four levels resulting in transformation of the communication paradigms. The object of investigation in histopathology is the digital slide, which is accessible throughout the world with no time or geographical limits. It permits the digital modelling of routine histological and/or cytological slide and it also allows measurements by using image analysis or stereology software packages. The electronic slide can be viewed, examined and diagnosed on a computer connected to a microscope, a new interface in diagnostic histopathology. This study describes the main theoretical and practical aspects, including challenges, of digital pathology and it also discusses the conditions required for successful information management. It reviews the experiences of the last one and half decades gained in the field of pathological information technology in Hungary including its main episodes and milestones of development. Introducing its present state, this paper describes the concept and the mode of investigation of the digital slide. It shows the development and use of a virtual microscope in Hungary. Based on know-how including the British experience this review describes the possible uses of digital slides, which by improving communication could have a positive effect on the entire health care system. It summarises the possible and necessary components of a digital pathology laboratory, which may include the new Slide Archive and Communication System (SACS). Using experimental data it mentions the possibility of generating primary digital pathological sample and producing the so called optical biopsy with no need for removing tissue from the patient.

Blood formation by pluripotent stem cells and their progeny is thought to be regulated by receptor-ligand interactions between cell-substrate, cell-cell and cell-matrix in the bone marrow. Primitive stem cells form progenitors and, in their turn, these give rise to haemopoietic progeny which are more specifically committed in that they can form progressively fewer types of blood cells. Recently we have established that direct cell-cell communication via gap junctions may be part of this regulatory system. Connexin43 gap junctions metabolically couple the three dimensional meshwork of bone marrow stromal cells to form a functional syncytium in which some blood-forming cells are also coupled. The expression of gap junctions in the bone marrow is markedly upregulated when there is an urgent and substantial demand for blood-formation; for example, following cytotoxic injury after 5-fluorouracil or irradiation; or during neonatal blood-formation and in the epiphysis of growing bones. Chemical blockade of gap junctions blocks blood-formation in long-term cultures but is reversible after the blockade has been relieved. This short review highlights briefly the known regulatory mechanisms of blood-formation with especial attention to gap junctional communication.

Deletion analysis of the dystrophin gene (Xp21) was carried out by examinations of the most frequently deleted 18 exons (3., 4., 6., 8., 12., 13., 17., 19., 43., 44., 45., 47., 48., 49., 50., 51., 52. and 60. exon) and the muscle specific promoter in 42 Duchenne and Becker muscular dystrophy (DMD/BMD) affected patients with multiple polymerase chain reaction (PCR). 22 (52%) of 42 patients were found to have one or more exon deletions. 9% BMD patients (milder allelic form) were found in the deletion group versus 35% in the non deletion group. This method seems to be useful for prenatal genetic diagnosis in the family of deletion patients.

The immunoreactivity of a CD1a monoclonal antibody (MAb), denoted 010, was investigated by means of the streptavidin-biotin-peroxidase method in formalin-fixed and paraffin-embedded tissues from 47 cases. The samples comprised reactive lymphoid proliferations of skin, tonsil, and lymph node including dermatopathic lymphadenopathy and Langerhans' cell histiocytosis, Hodgkin's and non-Hodgkin's lymphomas, and thymomas. Interdigitating and dermal dendritic cells, veiled cells, Langerhans' cells, and also cortical thymocytes and their neoplastic counterparts displayed immunostaining with MAb 010 in paraffin sections. These results are identical to previous ones reported for other CD1a MAbs in fresh or frozen specimens. The findings suggest that the binding site of 010 is a fixation-resistant epitope of CD1a antigen which has not been previously identified.

Immunohistochemical studies prove the most of the neurons of the central nervous system express one or more protein kinase C isoenzymes. However, there are only a few descriptions of the glial localization. In the present study, we detected protein kinase C immunohistochemically in guinea pig sensory neocortex, hippocampus and hippocampal slices following in vitro maintenance. Monoclonal antibodies against types II and III of protein kinase C were used. Consecutive sections were stained with polyclonal anti-glial-fibrillary-acidic protein serum. Scattered astrocytes contained protein kinase C in layers I and VI of the cerebral cortex, in the subcortical white matter, the subiculum, alveus and molecular layer of Ammon's horn, in the hilus and molecular layer of the dentate fascia. Ultrastructurally, immunostaining of glial processes in the neuropil and around blood vessels was observed. Strong staining of the endoplasmic reticulum and nuclear envelope was noted in glial cell bodies. The ultrastructural localization of protein kinase C suggests its participation in receptor-mediated processes, which may influence the shape and function of astrocytes.

In a 81-years old man synchronous triple cancers (prostate, kidney and lung) were found at autopsy. The poorly differentiated prostatic adenocarcinoma metastasized to the clear cell carcinoma of the right kidney and to its solitary lung metastasis. The "neoplasm to neoplasm" metastasis and the "metastasis in metastasis" was confirmed by immunohistochemistry.

The cellular infiltration in 42 needle and wedge biopsies of transplanted kidneys was investigated immunohistochemically. The percentages of helper/inducer (CD 4+) cells, suppressor/cytotoxic cells (CD 8+), B lymphocytes, macrophages, plasma cells (Pc) and granulocytes were determined. The proportions of the various inflammatory cell populations were established in acute interstitial rejection (AIR), acute vascular rejection (AVR), chronic rejection (CR) and cyclosporin A nephrotoxicity (CsAN). The most prominent differences were detected as regards the Pc, whose number was much higher in CR than in AIR, AVR or CsAN. The striking difference between CR and CsAN in the number of Pc may be of differential diagnostic importance: the presence of many Pc in the biopsies can be regarded as a sign of CR. Over 80% of the Pc in CR contained IgG, whereas in chronic interstitial nephritis (CIN) the IgA-positive Pc predominated. In AIR, AVR and CsAN, too, the majority of the Pc contained IgG, but the numbers of IgM and IgA-positive cells were also relatively high. The great number of IgG-positive Pc indicates an important role of a secondary type humoral immune response in CR.

A case of ceruminous adenocarcinoma is reported. The tumor destroyed the right pyramid, widely invaded the base of the skull and caused death shortly after the diagnosis. Distant metastases were not found by autopsy. The tumour cells reacted with epithelial markers and with the antibody against S-100 protein. Heparan sulphate proteoglycan seemed to be a good marker for detecting basement membrane ruptures and concomitant tumour invasion. Among the lectins BS-I and PNA gave the strongest reactions in the stroma. This is the first immunohistochemical and lectin histochemical report on ceruminous adenocarcinoma.

Arteries were investigated ultrastructurally in material from 40 needle and wedge biopsies of renal allografts, and immunohistochemically in another 10 cases with signs of chronic obliterative transplantation arteriopathy. In the early biopsies, but even in the control kidneys, thin extensions of the smooth muscle cells of the media were observed, which were in direct contact with the endothelial cells through the lamina elastica interna. These extensions may contain receptors mediating endothelial noxae to the smooth muscle cells thus initiating their proliferation, migration to the intima presumably begins in the early post-transplant period and continues until the lumen is occluded. Concomitantly, inflammatory cells (mainly macrophages, with a smaller number of CD4 and CD8-positive T lymphocytes) invade the intima. The proliferating myointimal cells, possibly having become HLA-DR-positive, may behave as antigen-presenting cells, enhancing the anti-graft immune response further, and aggravating the arterial injury.

Authors examined cells participating in intimaproliferation in transplantation arteriopathy ultrastructurally in needle and wedge biopsy material from 40 transplanted kidneys, and immunohistochemically in 10 cases. In early biopsies--even in two control kidneys--it could be observed that the smooth muscle cells of media are in direct contact with endothel cells by their small processes. Processes can fulfil a receptor function and can transmit endothel noxa to smooth muscle cells. Smooth muscle cells of media react to endothel damage caused by rejection with migration to intima and during this period they are transformed to myofibroblasts (myointimal cells). In the mean time inflammatory cells (mainly macrophages, helper and cytotoxic cells in lower number) from the lumen infiltrate the intima, and mediators, enzymes released from them can inspire smooth muscle cells to further proliferation, migration to intima and transformation to myofibroblast. To effect of mediators (gamma interferon) released from inflammatory cells, the myointimal cells during rejection will press out 2nd class transplantation antigens (HLA-DR), and as vicious circle it further aggravates immune reply to graft, causing vascular damage, intimaproliferation.

This study was undertaken to evaluate the number of the immunoglobulin producing cells in the lamina propria of the small intestine by immunocytochemical techniques, using peroxidase-antiperoxidase (PAP) complex in normacid patients with CSBS. 25 patients were studied including 13 patients with bacterial overgrowth, where the bacterial concentration was higher than 10(4) colony forming units/ml, and 12 subjects with normal bacterial concentration, served as control. The patients with CSBS were treated with antibiotics according to the antibiotic resistance. After treatment the luminal bacterial concentrations was lower than 10(4) cfu/ml in 7 of 13 patients (CSBS I. group). In 6 patients the bacterial concentration remained high (CSBS II. group). The immunoglobulin producing cells were determined in biopsy specimens taken from the lower part of duodenum. The number of the IgA and IgM producing immunocytes was significantly decreased only in the CSBS II. group. Our results show that temporary immunological alterations may play an important role in the mechanism of the recurrent CSBS.

Case of ceruminal adenocarcinoma is reported. Tumor was diagnosed in advance stage, and within a month following diagnosis it caused death. Tumor started from right auditory canal, undermined the pyramidal bone and infiltrated great part of right side of base of skull, narrowing great foramen to a certain extent. Metastasizes were only the right of dura. Besides electron microscopic studies, authors performed the immune-histochemical and lectin-histochemical examination of tumor the first time.

7 gastrinomes and 1 gastrin-producer complex carcinoma-carcinoid tumor were examined by light and electron microscopical-method and by immunohistochemical method. In six cases, the tumor was in the pancreas or in the wall of duodenum; in two cases its localisation was of extra-gastroenteropancreatic (liver, lymph node). All patients developed Zollinger-Ellison syndrome, three patients bled and one had diarrhea. One patient had other tumors, besides gastrinome, which were characteristic of MEN-I syndrome. By immunohistochemical methods all tumors proved to be gastrin and neuron-specific-enolase positive. In four cases somatostatin positivity, in some cases glucagon, pancreatic polypeptide, S-100 protein, keratin and carcinoembryonal antigen positivity were detected. Relation could not be detected between other polypeptide hormones, produced besides gastrin, and biological behaviour of tumor and clinical symptoms.

An immunocytochemical investigation of sarcoplasmic reticulum (SR) Ca2(+)-ATPase (SR-Ca-ATPase) was performed on formalin-fixed paraffin-embedded specimens of different types of rhabdomyosarcomas such as variants of embryonal and pleomorphic forms. Immunostaining frequency of tumours using SR-Ca-ATPase was compared with that of traditionally used muscle specific markers myoglobin, and desmin. Utilizing the possible cleaving of ester bounds sodium methoxide pretreatment was found to be very effective in enhancement of SR-Ca-ATPase immunostaining reaction. In 11 of 15 tissue specimens of 5 cases round shaped and elongated rhabdomyoblasts with definite cytoplasm exhibited positive immunoreactions with all of the polyclonal antibodies tested, using the streptavidin-biotinylated peroxidase complex (S-ABC-method). In formalin-fixed and paraffin-embedded material of 2 cases of undifferentiated rhabdomyosarcomas composed of small round tumour cells with scanty cytoplasm pretreatment with sodium methoxide induced the immunostaining of SR-Ca-ATPase. After that pretreatment a staining of the paranuclear cytoplasm occurred in many of these undifferentiated tumour cells. In these 2 cases, neither myoglobin nor desmin antibodies could react. However, when frozen sections of one of the poorly differentiated tumours were used monoclonal and polyclonal desmin antibodies reacted immunocytochemically in all of the small cells. Sodium methoxide induced or enhanced SR-Ca-ATPase immunocytochemical reaction can be a further addition to the diagnosis of rhabdomyosarcomas in formalin-fixed paraffin-embedded sections, even when desmin antibody fails to react.

The clinical, microscopic, immunohistochemical and ultrastructural features of 7 gastrinomas and 1 combined carcinoma-carcinoid tumor were evaluated. The tumors were located in the pancreas or duodenal wall in 6 cases, and on extragastro-enteropancreatic sites in 2 (liver or peripancreatic lymph node). All patients had the Zollinger-Ellison syndrome, 3 of them with additional bleeding and 1 with diarrhea. One patient with gastrinoma had additional tumors characteristic of the MEN-I syndrome. Immunohistochemistry showed gastrin and neuron-specific enolase-positivity in all of the tumors. Somatostatin was found in 4 cases, and single cell glucagon, pancreatic polypeptide. S-100 protein, keratin as well as carcino-embryonic antigen positivity in another few. Additional hormone production did not appear to be connected with biological behaviour of the tumors or with the clinical symptoms.

By the immunohistochemical demonstration of SR calcium ATPase and myoglobin a fibre classification method was developed. Fast fibres showed intense, while slow fibres weak SR calcium ATPase reactivity. Immunohistochemical reaction of myoglobin characterized the oxidative metabolic state of fibres similar to the succinate dehydrogenase (SDH) reaction. By means of SR calcium ATPase and myoglobin immunohistochemistry fibres were classified as slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (Fg) groups. The SR calcium ATPase activity of the different fibres varied in the FG greater than FOG greater than SO order, while myoglobin immunoreactivity in the FOG greater than SO greater than FG order. Both proteins studied preserved their antigenicities in Bouin's fixative or in formol-acetate and paraffin embedding. The light microscopic immunogold-silver method was found suitable also for electron microscopy. The silver intensification of small particle-size (5 nm) gold conjugate results in a reaction with the joint advantages of high sensitivity and optimal visibility. The described immunohistochemical method proved to be suitable for the retrospective differentiation of human biopsy materials.

Authors have studied in apostematous pyelonephritis induced by the ligation of the ureter and the intravenous injection of E coli bacteria the localization and elimination with time of the pathogen. The pathogen was demonstrated by light and electron microscopy, its parietel antigen was localized with the light microscopic peroxidase antiperoxidase and post-embedding electron microscopic immunogold techniques. Two days after inoculation the suppurative inflammation of tubulo-interstitial foci was observed; in the capillaries, interstitium, and tubuli, free and phagocyted bacteria were encountered. In the interstitium, in the proximal tubuli and in the capillary space of some glomeruli bacterial groups were observed. Intracapillary bacteria were attached by their outer wall to the surface of endothelial cells. In the tubuli this adherence occurred with pili or with the outer layer of bacterial wall. From the seventh day after inoculation macrophages containing PAS-positive globuli appeared in the interstitium. Under the electron microscope these globuli proved to be features composed of myelin figures of phagolysosomal origin. Globuli and the myelin figures possessed an E. coli antigenicity. Thirteen weeks after inoculation E. coli antigen positivity was found in the cytoplasm of inflammatory cells in the tubular walls and in the suppurative cylinders, The organism was apparently unable to eliminate the materials derived from the pathogenic microorganisms.

Acute pyelonephritis was induced in rats by temporary unilateral ureteric obstruction and the intravenous injection of Escherichia coli. Animals were sacrificed 48 h after infection and changes in renal cortical tubules due to the presence of bacteria were studied. Bacteria appeared and multiplied in the tubular lumina and proximal tubular epithelial cells endocytosed the microorganisms in large numbers. Coalescence of phagosomes with lysosomes resulted in the surrounding of engulfed bacteria with acid phosphatase. However, the lysosomal apparatus of the cells did not eliminate Escherichia coli since the bacteria multiplied within phagosomes and destroyed the normal cell architecture. The peritubular interstitial inflammatory infiltrate caused ischemia of tubules, enhancing bacterial damage to the proximal tubules. The cytoplasm of the injured tubular cells was sometimes detached from the basement membrane. Cells of the distal tubules and collecting ducts did not show significant endocytosis or bacterial tubular damage.

S-100 protein-containing cells were demonstrated by immunogold silver staining in human islets of Langerhans from patients with chronic pancreatitis (CP) with (n = 6) (Group I) or without (n = 6) (Group II) diabetes mellitus, (DM) and from nondiabetic, non-pancreatic controls (n = 6) (Group III). In all three groups S-100 protein containing cells were observed in all islets of Langerhans throughout the pancreas. Quantitative analysis of cell composition of islets did not reveal significant differences in S-100 protein cell content between the three groups. When double immunohistochemical staining was used to demonstrate different endocrine cell types (insulin, glucagon and pancreatic polypeptide) and S-100 protein immunoreactive cells, the latter proved to be a distinct cell type. Somatostatin-producing cells and S-100 protein-containing cells were usually also present as two distinct cell populations, but positive staining for both S-100 protein and somatostatin was occasionally observed within the same cells.