Prognostic importance of tumor angiogenesis in breast carcinoma with adjuvant chemotherapy

Tumor angiogenesis is believed to be related to prognostic factors involved in tumor development and metastasis. Using immunohistochemical methods, we evaluated tumor angiogenesis in 42 early invasive breast cancer patients (T1-2, NO-1-2, M0). Four patients received tamoxifen, 25 patients received CAF or CA, and 15 patients received CMF as adjuvant therapy. The median follow-up was 47 (range 24-119) months. Ten patients (43.5%) in the node-positive group and 2 patients (10.5%) in the node-negative group relapsed (p = 0.019). The mean microvessel count (MVC) was 60.3 3.05 per 200x field (range: 16-95). MVCs of postmenopausal and premenopausal patients were 50.13 +/- 5.74 and 68.64 +/- 4.11, respectively, in the axillary lymph node (ALN)-negative patient group (p = 0.04). Staining was moderate to strong in 13 (68%) ALN-negative and in 17 (74%) ALN-positive patients (p > 0.05), and was also moderate to strong in 82% of premenopausal patients and in 50% of postmenopausal patients (p = 0.037). There was no significant relationship between angiogenesis and p53, nor was angiogenesis significantly associated with the patient ER status and tumor size. No significant correlations were found between OS/DFS and Factor VIII staining or p53 (log rank test, p > 0.05). Of all ALN-negative patients with increased angiogenesis, one patient of the CMF group relapsed, but no recurrence occurred in patients undergoing anthracycline-based chemotherapy (p > 0.05). On the other hand, of all ALN-positive patients with increased angiogenesis, 5/14 patients treated with anthracylcine and 2/2 CMF-treated patients relapsed (p = 0.175). Despite the statistical insignificance, anthracycline-based adjuvant chemotherapy appears to be more effective than CMF as regards relapse prevention particularly in early ALN-positive breast cancer patients with increased angiogenesis. Additional studies are necessary to demonstrate the clinical importance of angiogenesis.     

Soluble mediators and cytokines produced by human CD3- leucocyte clones from decidualized endometrium.

CD3- granulated leucocyte clones have been generated from human first-trimester decidualized endometrial tissue following culture in interleukin-2 (IL-2). Supernatants from both CD3- decidual granulated leucocyte (dGL) and CD3- peripheral blood natural killer (PBNK) cell clones inhibited the proliferation of choriocarcinoma cell lines. A panel of CD3- dGL clones, with or without phytohaemagglutinin stimulation, was assayed for cytokine secretion compared with CD3- PBNK clones and fresh tissue extracts. Levels of interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and IL-10 produced by stimulated CD3-CD8- dGL clones were greater than those produced by stimulated CD3-CD8+ dGL clones. In contrast, CD8+ dGL clones were more effective in production of IL-6 than CD8- dGL clones. Immunoreactive transforming growth factor-beta 2 (TGF-beta 2) was undetectable in supernatants from CD3- dGL and PBNK clones. CD3- dGL clones generally produced higher levels of all cytokines than PBNK clones. Some unstimulated CD3- dGL and PBNK clones spontaneously produced these cytokines, but usually at a reduced level. Fresh extracts of first-trimester decidual tissue contained detectable GM-CSF, TNF-alpha, IL-10,IL-6 and TGF-beta 2. Cytokine production by fresh CD3- dGL and CD3- dGL clones indicates that these cells could play an important role in the regulation of placental growth.     

Human lymphocyte antigens (HLA) and Graves' disease in Turkey

To evaluate the association of HLA types with Turkish patients with Graves' disease, HLA typing, clinical findings, and thyroid antibodies were correlated. The HLA types, clinical findings (ophthalmopathy and age at onset), and thyroid stimulating hormone (TSH) receptor (TRAb) and antithyroid microsomal antibodies (MAb) were analyzed. Seventy Turkish patients with Graves' disease and 306 control subjects were assessed. Serological HLA typing was performed in HLA A, B, C, DR, and DQ loci. There was a significantly increased prevalence of HLA B8, B49, DR3, DR4, and DR10 in Graves' disease. The association of Graves' disease with HLA DR3 was found to be less strong than previously described. The HLA DR4 antigen may contribute to the predisposition of Graves' disease in Turkey. The results suggest that HLA B7, B13, DR7, DQw2, and DQw3 may confer a protective effect for Graves' disease in Turkey. Patients carrying HLA B12, B18, and B44 haplotypes had a tendency to develop the disease at a later age. The difference from the other studies may be the result of the selection of the controls; in part, of the variability in serological typing reagents; and, also, of the rather weak HLA associations with the disease.This study was presented in part at the Annual Meeting of the National Endocrinology and Diabetes Association, Bursa, Turkey, May 25–28, 1992.     

ACE Genotype May Have an Effect on Single versus Multiple Set Preferences in Strength Training

A polymorphic variant of the human angiotensin converting enzyme (ACE) gene was identified. The 'D' (rather than 'I') variant was associated with improvements in strength related to physical training. We set out to determine whether the response to different patterns of strength training might also differ. Ninty-nine Caucasian male non-elite athletes were randomly allocated into one of three groups: 31 non-training/control (CG: 31), single-set (SSG: 35) and multiple-set (MSG: 33). SSG and MSG trained three times a week for 6 weeks. Both training groups were underwent a strength-training program with two mesocycles (12-15 repetition maximum (RM) and 8-12 RM mesocycles). One RM loads in half squat and bench press were assessed before training and after the first and second mesocycles. ACE polymorphisms analysed by polymerase chain reaction (PCR) methods. Subjects with ACE II genotype in the MST group had improved strength development in 12-15 RM, while SST and MST groups had similar gains in 8-12 RM. Subjects with ACE DD genotype in both the SSG and the MSG had similar benefits from both 12-15 RM and 8-12 RM. Strength gains for subjects with ACE ID genotype in the SSG were similar to MSG gains in response to 8-12 RM loads but not with 12-15 RM loads. Additionally, subjects with DD genotype had superior strength gains in both strength training groups. Tailoring strength training programmes (single-set vs. multiple set) according to the athlete's ACE genotype may be advantageous.     

Human NK1 and NK2 subsets determined by purification of IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells

The in vivo existence of human NK cell subsets similar to Th1 and Th2 cells was demonstrated in freshly isolated IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells. The IFN-gamma-secreting NK subset showed a typical cytokine pattern with predominant expression of IFN-gamma, but almost no IL-4, IL-5 and IL-13. In contrast, the IFN-gamma-nonsecreting NK subset was composed of IL-4, IL-5 and IL-13-producing NK cells. Short-time stimulation or 2 weeks of in vitro differentiation of NK cells led to distinct patterns of cytokine production similar to freshly-purified IFN-gamma (+) or IFN-gamma (-) NK cell subsets. NK cells stimulated with IL-12 produced increased levels of IFN-gamma and decreased levels of IL-4. In contrast, stimulation of NK cells with IL-4 inhibited IFN-gamma, but increased IL-13 production. Freshly-purified IFN-gamma (+) and IFN-gamma (-) or in vitro differentiated NK1 and NK2 subsets showed similar cytotoxicity to K562 cells. These results demonstrate that circulating NK cells retain effector subsets in humans with distinct cytokine profiles and may display different inflammatory properties.     

Infective agents in fixed human cadavers: a brief review and suggested guidelines

Cadavers remain a principal teaching tool for anatomists and medical educators teaching gross anatomy. Infectious pathogens in cadavers that present particular risks include Mycobacterium tuberculosis, hepatitis B and C, the AIDS virus HIV, and prions that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). It is often claimed that fixatives are effective in inactivation of these agents. Unfortunately cadavers, even though they are fixed, may still pose infection hazards to those who handle them. Specific safety precautions are necessary to avoid accidental disease transmission from cadavers before and during dissection and to decontaminate the local environment afterward. In this brief review, we describe the infectious pathogens that can be detected in cadavers and suggest safety guidelines for the protection of all who handle cadavers against infectious hazards.     

Differential sensitivity to Arg side chain modification of IL-1β binding to type I and type II receptors

The central role of interleukin-1 (IL-1) in several disease processes, including fever and inflammation, makes the characterization of ligand-receptor interaction of prime importance.The role of arginine (Arg) side chains of hr-IL-1 in receptor recognition was studied by the modification of Arg residues with the specific reagent 1,2-cyclohexanedione. It was found that chemical modification of Arg residues decreased the binding potential of IL-1 to type I receptor dramatically (by 230-fold) while the affinity to type II receptor was reduced only moderately (by 10-fold), with an insignificant reduction of the dissociation rate.These studies suggest that intact Arg side chains of IL-1 may be necessary for high affinity binding to type I IL-1 receptor, but have less importance for the interaction of IL-1 with type II IL-1 receptor.This observation may be useful in the study of type II IL-1 receptor-mediated biological responses and design of receptor-subtype specific ligands as well.     

Automatic analysis of immunocytochemically stained tissue samples

An automatic colour image segmentation and cell counting software system has been developed for immunocytochemical analysis of stained tissue samples. The system was designed to count the total number of positive and negative cells in tissue samples treated with cytokine DNA probes from pigs naturally parasitised with Taenia solium metacestodes, using in situ hybridisation. A reaction index was calculated as the ratio of the number of cells with a positive reaction to the total number of cells (positives plus negatives) for each of five different probes. The objectives of automatic counting were to improve the reproducibility of the analysis and reduce the processing time of large image batches. A fast KNN classifier was used for colour segmentation. Watershed segmentation combined with edge detection was used to isolate individual cells that were then automatically labelled, using the results of the corresponding colour segmented image. Validation was performed on 122 non-training digital images with a total of 1069 positive cells and 1459 negative cells, with the following results: a mean true positive rate of 90.2% for positive cells and a mean true positive rate of 85.4% for negative cells. The corresponding mean false positive rates were 9.6% and 6.6%. The mean reaction index error of the automatic analysis was 5.35%. The processing of each digital image took 10 s on a Pentium IV PC.     

Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1α are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin–biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK⁺ cells was 236 (range, 20–847) per 5 × 10⁴ enriched BM cells. The presence of CK⁺ cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK⁺ per 5 × 10⁴ enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.