Lipopolysaccharide enhances apoptosis of corpus luteum in isolated perfused bovine ovaries in vitro.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL) in vivo. The objective was to investigate whether these effects were mediated directly by LPS or via LPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 µg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2α were measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2, -4) and LH (LHCGR); the cytokine TNFA; steroidogenic (STAR, HSD3B), angiogenic (VEGFA121, FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES, PGFS, PTGFR) and apoptosis (CASP3, -8, -9). Treatment with LPS abolished the hCG-induced increase in P4 (P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2α at 70 min after LPS challenge. Furthermore, mRNA abundance of TLR2, TNFA, CASP3, CASP8, PGES, PGFS, and VEGFA121 increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4 responsiveness to hCG in LPS-treated ovaries in vitro was not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2α could not be excluded.

Intramammary lipopolysaccharide infusion alters gene expression but does not induce lysis of the bovine corpus luteum.

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 µg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

[Round robin test for detection of genomic aberrations in non-Hodgkin lymphoma by in situ hybridization]. / Ringversuch zum Nachweis genomischer Veränderungen bei Non-Hodgkin-Lymphomen mittels In-situ-Hybridisierung.

BACKGROUND: The detection of characteristic genomic aberrations by fluorescence in situ hybridization (FISH) has a high diagnostic impact on lymphomas according to the World Health Organization (WHO). To investigate the reproducibility of non-isotopic ISH results a multicenter trial was carried out involving eight institutes for hematopathology. MATERIAL AND METHODS: Analyses were performed on two diffuse large B-cell lymphomas (DLBCL) without known aberrations, on one follicular lymphoma with a IGH/BCL2 translocation and BCL6 split and on two B-cell lymphomas intermediate between DLBCL and Burkitt's lymphoma with c-MYC and BCL2 rearrangements, one with an additional BCL6 split. Break-apart probes for BCL6 and c-MYC, as well as fusion probes for the c-MYC/IGH and the IGH/BCL2 translocations were used. RESULTS: All aberrations were correctly detected by all centres and no false positive or false negative results were obtained. The numbers of positive cells varied from 25% to 94%. Pearson's correlation coefficient between the centres was always > 0.8. CONCLUSIONS: The ISH analysis of recurrent genomic aberrations in formalin-fixed paraffin-embedded (FFPE) tissue is a highly reproducible technique which yields substantial additive help for lymphoma diagnostics.

[Intratumoral heterogeneity of HER2 status in breast carcinoma]. / Intratumorale HER2-Heterogenität des invasiven Mammakarzinoms.

Intratumoral heterogeneity of HER2 protein expression and HER2 gene amplification can negatively affect determination of HER2 status in a subset of invasive breast carcinomas. The frequency and clinical significance of HER2 genetic heterogeneity are unknown due to the lack of uniform criteria for diagnosis. Recent ASCO/CAP guidelines (2009) define HER2 genetic heterogeneity as the presence of between 5% and 50% of tumor cells with a HER2/CEP17 ratio >2.2. We describe a tool (a customized Excel spreadsheet) for easy and reproducible diagnosis of HER2 genetic heterogeneity according to ASCO/CAP criteria. Our tool may be useful for routine HER2 diagnostics and for studies to analyse the hitherto unknown predictive significance of HER2 genetic heterogeneity.

Is there an advantage to be gained from adding digital image cytometry of brush cytology to a standard biopsy protocol in patients with Barrett's esophagus?

BACKGROUND AND STUDY AIMS: The current gold standard in Barrett's esophagus monitoring consists of four-quadrant biopsies every 1-2 cm in accordance with the Seattle protocol. Adding brush cytology processed by digital image cytometry (DICM) may further increase the detection of patients with Barrett's esophagus who are at risk of neoplasia. The aim of the present study was to assess the additional diagnostic value and accuracy of DICM when added to the standard histological analysis in a cross-sectional multicenter study of patients with Barrett's esophagus in Switzerland. METHODS: One hundred sixty-four patients with Barrett's esophagus underwent 239 endoscopies with biopsy and brush cytology. DICM was carried out on 239 cytology specimens. Measures of the test accuracy of DICM (relative risk, sensitivity, specificity, likelihood ratios) were obtained by dichotomizing the histopathology results (high-grade dysplasia or adenocarcinoma vs. all others) and DICM results (aneuploidy/intermediate pattern vs. diploidy). RESULTS: DICM revealed diploidy in 83% of 239 endoscopies, an intermediate pattern in 8.8%, and aneuploidy in 8.4%. An intermediate DICM result carried a relative risk (RR) of 12 and aneuploidy a RR of 27 for high-grade dysplasia/adenocarcinoma. Adding DICM to the standard biopsy protocol, a pathological cytometry result (aneuploid or intermediate) was found in 25 of 239 endoscopies (11%; 18 patients) with low-risk histology (no high-grade dysplasia or adenocarcinoma). During follow-up of 14 of these 18 patients, histological deterioration was seen in 3 (21%). CONCLUSION: DICM from brush cytology may add important information to a standard biopsy protocol by identifying a subgroup of BE-patients with high-risk cellular abnormalities.

Lugol chromoendoscopy combined with brush cytology in patients at risk for esophageal squamous cell carcinoma.

BACKGROUND AND STUDY AIMS: Patients with achalasia or malignancies of the head and neck are at increased risk for esophageal squamous cell carcinoma. The discussion of a screening and surveillance program is controversial. The aim of the present study was to determine the diagnostic potential of Lugol chromoendoscopy combined with brush cytology to diagnose esophageal squamous cell carcinoma and high-grade dysplasia. Secondly, the benefit of additional biomarkers was investigated. PATIENTS AND METHODS: A total of 61 patients (21 patients with achalasia and 40 patients with malignancies of the head and neck) were included. Chromoendoscopy with 1.2% Lugol iodine solution with targeted biopsies and brush cytology processed by digital image cytometry (DICM) and fluorescence in situ hybridization (FISH) from unstained lesions (USLs) and stained mucosa were performed. RESULTS: Six of the 61 patients had USLs ≥2 cm. Four patients had high-grade dysplasia (HGD) or carcinoma in situ (CIS). One patient with HGD and one patient with CIS were detected only after Lugol chromoendoscopy. The sensitivity and specificity for detected HGD or CIS in USLs ≥2 cm were 100% and 96.5%. No dysplasia was found in USLs <2 cm. DNA ploidy by DNA cytometry and p53 loss of heterozygosity (LOH) by fluorescence in situ hybridization showed no additional impact on diagnostic accuracy. CONCLUSIONS: Lugol chromoendoscopy enhances the detection rate of high-risk lesions with dysplasia or carcinoma in situ in large unstained lesions. Biomarkers such as aneuploidy and p53 LOH from brush cytology were not of additional benefit in this setting.

Immunocytochemical characterization of lymphocytes in benign and malignant lymphocyte-rich serous effusions.

The cytological diagnosis of malignant Lymphoma in serous effusions can be difficult because reactive lymphocytes may be morphologically indistinguishable from malignant cells in lymphocytic and other low grade Non-Hodgkin's lymphomas. As a result of the present study, diagnostic accuracy can be improved by means of B- and T-cell enumeration using an immunoalkaline-phosphatase method (IAP). 30 cytological specimens, including 28 pleural, 1 pericardial and 1 ascitic fluids, were studied with a panel of monoclonal anti B- and anti T-cell antibodies (PAN B, kappa, lambda, T1, T2, OKT4, T8). Reactive lymphocytic effusions were characterized by a predominance of T cells constituting greater than or equal to 80% of all lymphocytes with an excess of helper/inducer cells (mean helper to suppressor ratio 3.0) and by a surface kappa to surface lambda ratio of 1.6 on B-cells. Tuberculous effusions showed a similar distribution of lymphocyte-subpopulations whilst most of the carcinomatous fluids showed a lower percentage of T cells (lowest value 67%) and lower Th: Ts ratio (mean 2.0). Lymphoid cells in samples of five B-cell lymphomas were characterized by T-cell depression (less than 70%). B-cells in three cases expressed clear cut light chain monoclonality which was at least suggested in the other two cases. Lymphoid cells from two cases of Hodgkin's disease expressed an indistinct immunological pattern. Labelling of cytoplasmic immunoglobulins (heavy and light chains) using the peroxidase antiperoxidase method (PAP) may be important to characterize neoplasms of the plasma cell series. It is concluded that the chosen panel of antibodies in combination with IAP labelling method may be of great value in identifying B-cell lymphomas. The technique can be used in the routine laboratory and storage of unlabelled and labelled slides over long periods is possible.

Bone-marrow plasmocytosis--an immunohistological study.

Bone-marrow biopsies and smears from 59 patients with reactive plasmocytosis (22), multiple myeloma (24), solitary plasmocytoma (3) and monoclonal gammopathy of undetermined significance (MGUS) (10) were examined. To demonstrate cytoplasmic immunoglobulin the immunoperoxidase method was applied and evaluated quantitatively. Immunohistology yielded different ranges in kappa/lambda ratio for reactive plasmocytosis (0.4-3.5), multiple myeloma (less than or equal to 0.1 and greater than or equal to 11.2) and MGUS (0.2-3.0). As a result this method seems to be helpful in characterizing a plasmocytosis and distinguishing overt myeloma from monoclonal gammopathy of undetermined significance and reactive plasmocytosis. A differentiation of monoclonal gammopathy of undetermined significance from reactive plasmocytosis is not possible histologically and immunohistologically.