Quantitative Immunohistochemical Analyses of the Expression of E-Cadherin, Thrombomodulin, CD44H and CD44v6 in Primary Tumours of pharynx/larynx Squamous Cell Carcinoma and their Lymph Node Metastases

The quantitative expression of E‐cadherin, thrombomodulin, CD44H and CD44v6 in 32 specimens of primary tumours of pharynx/larynx squamous cell carcinoma and their lymph node metastases was studied by immunohistochemistry. With the aim of obtaining comparative and objective data, image acquisition conditions were kept unaltered for all the measurements and the immunostaining intensity was quantified by applying an image processing system. On the one hand, correlations were only observed between CD44H and CD44v6, both in primary tumours and metastases, and between E‐cadherin and TM in metastases. On the other hand, statistical analyses of paired data did not show significant differences in the expression of these markers between the two tumour sites. In agreement with previous reports, E‐cadherin expression was rather low or negative in primary tumours and metastases of the three poorly differentiated specimens we studied, as well as that of TM, but otherwise some of these samples showed intermediate immunostaining levels of CD44H/CD44v6. It may be concluded from the present study that the quantitative expression of these adhesion molecules in well established lymph node metastases of pharynx/larynx squamous cell carcinoma is essentially unaltered in relation to their primary sites

Performance of a Pneumolysin Enzyme-Linked Immunosorbent Assay for Diagnosis of Pneumococcal Infections▿

A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children

Overexpression of proCOL11A1 as a stromal marker of breast cancer

Background: Our previous studies demonstrated the expression of procollagen11A1 in fibroblasts of pancreatic cancer desmoplasia and the lack of expression in fibroblasts of pancreatitis by means of the polyclonal antibody (anti-proCOL11A1 pAb) we generated. In a similar way, we decided to compare the expression of procollagen11A1 in fibroblasts of infiltrating ductal carcinoma of the breast and fibroblasts of benign sclerosing lesions of the breast, in order to validate the anti-proCOL11A1 pAb in this setting and to study how proCOL11A1 expression relates to other prognostic and predictive factors, as well as to survival. Methods: 45 core biopsies of sclerosing adenosis and 50 core biopsies of infiltrating ductal carcinoma of the breast were stained with anti-proCOL11A1 pAb, a polyclonal antibody highly specific to the less homologous fraction of proCOL11A1 (in comparison with proCOL5A1 and proCOL11A2). In addition, the expression of the proCOL11A1 gene was measured by RT-qPCR. On the other hand, the expression of proCOL11A1 was compared to the expression of estrogenic receptors, progestagen receptors, the state of the epidermal growth factor receptor 2 (HER2), the histologic grade and the stage of the disease. We also compared the immunohistochemical expression of proCol11A1 to the disease-free interval, and to overall survival. Results: The immunohistochemical analysis showed that proCOL11A1 was expressed in 100% of infiltrating ductal carcinomas, but only focally expressed in 2.2% (1 case) of sclerosing adenosis, in agreement with RT-qPCR results. ProCOL11A1 expression did not prove to have a prognostic value in relation to the disease-free interval or to overall survival in infiltrating ductal carcinoma. Conclusion: The anti-proCOL11A1 pAb is a stromal marker for breast cancer and the expression of proCOL11A1 does not seem to have a prognostic value in infiltrating ductal carcinoma of the breast

Protection against Pneumococcal Pneumonia in Mice by Monoclonal Antibodies to Pneumolysin

Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups

A new aggressive xenograft model of human colon cancer using cancer-associated fibroblasts.

Background Colorectal cancer is the second leading cause of cancer death. Almost half of the patients present recurrence within 5 years after the treatment of the primary tumor, the majority, with metastasis. On the other hand, in the search for new animal models that simulate metastatic cancer, it has been suggested that fibroblasts immersed in the peritumoral stroma (cancer-associated fibroblasts (CAFs)), play a relevant role in the development of cancer. The objective of this study was to identify an adequate animal model to study metastatic colon cancer and the application of new treatments. Methods Human CAFs and normal fibroblasts (NF) for transplant and culture were obtained from surgical fresh samples of patients with adenocarcinoma of sigmoid colon. Stromal cell purity was evaluated by morphology and immunostaining with vimentin (VIM) as a fibroblast marker and anti-proColXIα1 as a specific human CAF marker. Phenotypic characterization of cultured stromal cells was performed by co-staining with mesenchymal and epithelial cell markers. For identification in mice, human CAFs were labeled with the PKH26 red fluorescence dye. Cell line HT-29 was used as tumor cells. Transplant in the head of the pancreas of 34 SCID mice was performed in four different groups, as follows: I. 150,000 CAFS (n = 12), IIa. 1.5 million HT29 cells (n = 7), IIb. 150,000 NF+1.5 million HT29 cells (n = 5), III. 150,000 CAFS+1.5 million HT29 cells (n = 10). After euthanasia performed one month later, histological analysis was made using hematoxylin-eosin and anti-proColXIα1. A histopathological score system based on three features (tumor volume, desmoplasia and number of metastasized organs) was established to compare the tumor severity. Results The CAFs and NF cultured were proColXIα1+/VIM+, proColXIα1/alphaSMA+ and proColXIα1+/CK19+ in different proportions without differences among them, but the CAFs growth curve was significantly larger than that of the NF (p < 0.05). No tumor developed in those animals that only received CAFs. When comparing group II (a + b) vs. group III, both groups showed 100% hepatic metastases. Median hepatic nodules, tumor burden, lung metastases and severity score were bigger in group III vs group II (a + b), although without being significant, except in the case of the median tumor volume, that was significantly higher in group III (154.8 (76.9-563.2) mm3) vs group II (46.7 (3.7-239.6) mm3), p = 0.04. A correlation was observed between the size of the tumor developed in the pancreas and the metastatic tumor burden in the liver and with the severity score. Conclusion Our experiments demonstrate that cultured CAFs have a higher growth than NF and that when human CAFs are associated to human tumor cells, larger tumors with liver and lung metastases are generated than if only colon cancer cells with/without NF are transplanted. This emphasizes the importance of the tumor stroma, and especially the CAFs, in the development of cancer

Plot of all data of immunostaining score with anti-proCOL11A1 mAb in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC).

<p>Bars: ± 1 SD.</p

Confocal microscopy of pancreatic cultured CAFs.

<p>Double fluorescence stain illustrates the presence of cells proCOL11A1+/CK7+, proCOL11A1+/αSMA+ and proCOL11A1+/VIM+. <i>Red</i>: proCOL11A1; <i>green</i>: CK7, αSMA and VIM, respectively; <i>blue</i>: nuclei. Insets: randomly taken high power fields of culture. Scale bar 100 μm (X200) and 20 μm (X630). </p

Co-staining of anti-proCOL11A1 mAb with different fibroblastic markers and CK 7, in Chronic Pancreatitis.

<p><b>A</b>, anti-proCOL11A1 (brown) <i>vs</i>. desmin (magenta); <b>B</b>, Anti-proCOL11A1 (brown) <i>vs</i>. αSMA (magenta); <b>C</b>, anti-proCOL11A1 (brown) <i>vs</i>. VIM (magenta); <b>D</b>, anti-proCOL11A1 (brown) <i>vs</i>. GFAP (not staining); <b>E</b>, anti-proCOL11A1 (brown) vs. CK7 (magenta); <b>F</b>, CK7 (epithelial tumor cells: <i>brown</i>) vs. VIM (magenta).(all photomicrographs at ×200, Scale bar 200 μm). .</p