Immunohistologic identification of factor VIII related antigen in frozen sections of rat lung

Factor VIII related antigen (F VIII RAg) is localized in the cytoplasm of vascular endothelial cells. In this study, endothelial cells in frozen lung sections of Fischer-344 rat lungs were surveyed for F VIII RAg using a commercially available primary antibody against human F VIII RAg and an immunofluorescence assay. Rat pulmonary endothelial cells were positively labeled for F VIII RAg, whereas other lung cell types were not. Immunohistologic staining of F VIII RAg is useful in the rat model for identifying pulmonary endothelial cells in lung sections, and may be of use for detecting changes in lung endothelial cells due to environmental insults.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).     

Semi-automated method of quantifying vasculature of 1-methyl-1-nitrosourea-induced rat mammary carcinomas using immunohistochemical detection.

Studies of the vascularization of autochthonous rodent mammary tumors are limited in number, and the majority have used Factor VIII staining for blood vessel detection. Moreover, little effort has been directed at measuring the vascularization of tissue immediately adjacent to a tumor despite its central importance in the process of angiogenesis. Thirty-six chemically-induced mammary carcinomas and tissue immediately adjacent to these carcinomas were used to develop a census counting method for quantitative assessment of intra- and extra-tumor vascularization. Blood vessels were identified using antiserum directed against either CD31 or Factor VIII. Techniques used to create digitized images of all tumors and the semi-automated methods for circumscribing the extra-tumoral region are described. For Factor VIII, CD31 allowed greater discrimination of blood vessels with areas <25 microm(2) and demonstrated crisp staining of blood vessels, with minimal background and excellent preservation of tissue architecture. Census counting data support the use of CD31 for quantifying both intra- and extra-tumoral vascularization. This method provides a basis for standardizing the approach to evaluation of experimentally induced premalignant and malignant mammary lesions in rodent model systems used to investigate potential anti-angiogenic cancer preventive agents.     

卵巢上皮肿瘤淋巴转移与血管内皮生长因子C的表达

The aim of the present study was to explore the role of vascular endothelial growth factor-C (VEGF-C) in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign cystadenomas. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density (MVD) were assessed by image analysis. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than that in borderline tumors and benign cystadenomas (p < 0.05 or p < 0.01). In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage III-IV and with lymphatic metastasis than those of clinical stage I-II and without lymphatic metastasis respectively (p < 0.05 or p < 0.01), VEGFR-3 positive vessels and MVD was significantly higher in the VEGF-C protein positive tumors than negative tumors (p < 0.05), VEGFR-3 positive vessels was significantly correlated with MVD(p < 0.01). These data suggest that VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian carcinomas, and VEGF-C could be used as a biologic marker of metastasis in ovarian epithelial carcinomas.     

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth,Neovascularization, Angiogenesis and Macrophage Infiltration

CD73 (ecto-5''-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A₁, A_2A, and A₃ in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A₁AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A₃AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.     

Involvement of bone marrow-derived vascular progenitor cells in neovascularization during formation of the corpus luteum in mice

Neovascularization is necessary for formation of the corpus luteum (CL) and includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. Here we investigated whether vasculogenesis occurs in neovascularization during CL formation. Mice transplanted with bone marrow from transgenic mice expressing green fluorescent protein (GFP) were injected with equine chorionic gonadotropin and human chorionic gonadotropin (hCG) to induce ovulation and subsequent CL formation. Immunohistochemistry was performed on the ovaries obtained before hCG injection and at 6, 12, and 24 h after hCG injection using antibodies for CD34 or CD31 (an endothelial cell marker), platelet-derived growth factor receptor beta (PDGFR-beta, a pericyte marker), F4/80 (a macrophage marker), and GFP (a bone marrow-derived cell marker). Cells immunostained for CD34, PDGFR-beta, F4/80, and GFP were present in the theca cell layer of the preovulatory follicle before hCG injection. Each of these cell types invaded the granulosa cell layer after hCG injection, and a number of them were observed in the CL 24 h after hCG injection. Fluorescence-based immunohistochemistry or double immunohistochemical staining revealed that a few CD34/CD31-positive cells and PDGFR-beta-positive cells were also positive for GFP in the preovulatory follicle and CL, and that many of the GFP-positive cells recruited to the CL during CL formation were F4/80-positive macrophages. In conclusion, bone marrow-derived vascular progenitor cells and macrophages contribute to neovascularization during CL formation.     

Confocal Laser Endomicroscopy for the Morphometric Evaluation of Microvessels in Human Colorectal Cancer Using Targeted Anti-CD31 Antibodies

Introduction

Numerous anti-angiogenic agents are currently developed to limit tumor growth and metastasis. While these drugs offer hope for cancer patients, their transient effect on tumor vasculature is difficult to assess in clinical settings. Confocal laser endomicroscopy (CLE) is a novel endoscopic imaging technology that enables histological examination of the gastrointestinal mucosa. The aim of the present study was to evaluate the feasibility of using CLE to image the vascular network in fresh biopsies of human colorectal tissue. For this purpose we have imaged normal and malignant biopsy tissue samples and compared the vascular network parameters obtained with CLE with established histopathology techniques.

Materials and Methods

Fresh non-fixed biopsy samples of both normal and malignant colorectal mucosa were stained with fluorescently labeled anti-CD31 antibodies and imaged by CLE using a dedicated endomicroscopy system. Corresponding biopsy samples underwent immunohistochemical staining for CD31, assessing the microvessel density (MVD) and vascular areas for comparison with CLE data, which were measured offline using specific software.

Results

The vessels were imaged by CLE in both normal and tumor samples. The average diameter of normal vessels was 8.5±0.9 µm whereas in tumor samples it was 13.5±0.7 µm (p = 0.0049). Vascular density was 188.7±24.9 vessels/mm² in the normal tissue vs. 242.4±16.1 vessels/mm² in the colorectal cancer samples (p = 0.1201). In the immunohistochemistry samples, the MVD was 211.2±42.9/mm² and 351.3±39.6/mm² for normal and malignant mucosa, respectively. The vascular area was 2.9±0.5% of total tissue area for the normal mucosa and 8.5±2.1% for primary colorectal cancer tissue.

Conclusion

Selective imaging of blood vessels with CLE is feasible in normal and tumor colorectal tissue by using fluorescently labeled antibodies targeted against an endothelial marker. The method could be translated into the clinical setting for monitoring of anti-angiogenic therapy.     

Rapid and potent induction of cell death and loss of NK cell cytotoxicity against oral tumors by F(ab′)2 fragment of anti-CD16 antibody

Freshly isolated untreated NK cells undergo rapid apoptosis and lose their cytotoxic function upon the addition of F(ab′)₂ fragment of anti-CD16 antibodies. Loss of NK cell cytotoxic function after treatment with F(ab′)₂ fragment of anti-CD16 antibody can be seen against K562 and UCLA-2 oral tumor cells when either added immediately in the co-cultures of NK cells with the tumor cells or after pre-treatment of NK cells with the antibody before their addition to the tumor cells. Addition of Interleukin-2 (IL-2) in combination with anti-CD16 antibody to NK cells delayed the induction of DNA fragmentation in NK cells, and even though decreased cytotoxicity could still be observed against K562 and UCLA-2 oral tumors when compared to IL-2 alone treated NK cells, the cytotoxicity levels remained relatively higher and approached those obtained by untreated NK cells in the absence of antibody treatment. No increases in IFN-γ, Granzymes A and B, Perforin and TRAIL genes could be seen in NK cells treated with anti-CD16 antibody. Neither secretion of IFN-γ nor increased expression of CD69 activation antigen could be observed after the treatment of NK cells with anti-CD16 antibody. Furthermore, IL-2 mediated increase in CD69 surface antigens was down-modulated by anti-CD16 antibody. Finally, the addition of anti-CD16 antibody to co-cultures of NK cells with tumor target cells was not inhibitory for the secretion of VEGF by oral tumor cells, unlike those co-cultured with untreated or IL-2 treated NK cells. Thus, binding and triggering of CD16 receptor on NK cells may enhance oral tumor survival and growth by decreased ability of NK cells to suppress VEGF secretion or induce tumor cell death during the interaction of NK cells with oral tumor cells. This work was supported by RO1-DE18830 from NIH.     

兔VX2肺外周肿瘤超声造影特征与CD31和CD34微血管密度的相关性研究

摘要 目的：探讨兔VX2肺外周肿瘤超声造影特征与CD31和CD34微血管密度的相关性研究。方法：15只雌性新西兰白兔进行VX2 肺肿瘤模型，总共有30个VX2 癌植入15只兔子的肺中。通过高倍镜分析不同期VX2肿瘤的MVD。通过双重免疫荧光化学染色分析不同生长期VX2肿瘤Ki67和CD31阳性表达。分析肿瘤形态学组织病理学结果与微血管分布和CEUS结果之间的相关性。结果：随着生长周期的进展，VX2肿瘤的MVD增大（P<0.05）。随着生长周期的进展，VX2肿瘤内Ki67和CD31的阳性表达率增加（P<0.05）。CEUS显示VX2 肿瘤在早期动脉期有明显的外周增强和短暂的内部增强。肺外周VX2 肿瘤中 PI和CD31 MVD值之间呈显著正相关性（r=0.734，P<0.05）。CD31 MVD和CD34 MVD之间呈负相关（r=-0.691，P<0.05）。PI和CD34 MVD值之间不存在显著相关性（r=-0.412，P>0.05）。结论：与CD34 MVD 相比，CD31 MVD 可以更有效地评估肿瘤血管生成。CEUS作为一种无创成像方法，可以有效评估兔周围型肺癌的肿瘤血管生成情况。     

C-myc expression in the microvessels of medulloblastoma

The increased expression of c-myc is related to neoplastic transformation and angiogenesis. Therefore, the assessment of expression of c-myc in endothelial cells and neovascularization could help to determine the biological behavior of the tumor. We analyzed neovascularization and c-myc expression in 36 medulloblastoma specimens. The results were shown by determining immunohistochemical staining index (ISI), the sum of staining intensity (SI) and the percentage of positive cells (PPC) in the blood vessels endothelium of the tumor. We also performed the microvessel count (MVC) in 10 high-power fields (400X) with the most prominent vascularization and expressed it as microvessel density per mm2 (MVD). C-myc immunostaining intensity index in blood vessel endothelium is grouped into four groups, 0--no reaction, I-weak reaction (ISI = 1 or 2), II--moderate reaction (ISI = 3 or 4), III--strong reaction (ISI = 5 or 6). Statistically significant differences (p = 0.0214) have been found between groups 0 and 1 compared to groups 2 and 3. A higher percentage of positive cells has been found in male patients than in female ones (p = 0.0483). C-myc PPC 0 or 1 has on the average smaller density of blood vessels per mm2 than c-myc PPC 2 or 3, but the difference is not statistically significant. C-myc ISI 0 or 1 has, on the average, smaller density of blood vessels per mm2 than c-myc ISI 2 or 3, but the difference is not statistically significant. We concluded that c-myc staining intensity was associated with higher microvessels density.     

CD40 is overexpressed by HPV16/18-E6 positive cervical carcinoma and correlated with clinical parameters and vascular density

CD40 is expressed in many tumor cells, however, its role in tumor biology is yet to be demonstrated. In the present study, we investigated the role of CD40 in cervical carcinoma. In vivo, we evaluated CD40 expression in 56 cervical carcinoma tissues, 43 cervicitis and 38 normal cervix, and investigated the relationship between CD40 and HPV antigen, histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in cervical carcinoma than in the cervicitis and normal cervix. The expression of CD40 was significantly correlated with HPV and VEGF expressions and microvessel density (MVD). These observations provide evidence that CD40 may be involved in neovascularization of cervical carcinoma, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing cervical carcinoma, and may also be used as a target for therapy.     

中国人前列腺癌VEGF—C mRNA、VEGFR-3和CD31表达与肿瘤转移的关系

The expressions of VEGF-C mRNA, VEGFR-3 and CD31 were studied in order to investigate the correlation between them and neoangiogenesis, hyperplasia of micro-lymphatics and tumor metastases. 34 cases of prostate cancer tissue and 12 cases of adjacent nontumorous tissue specimens were tested. They were marked by VEGFR-3 and CD31 with immunohistochemistic staining and analyzed with image, the micro-lymphatics count (MLC) and microvessel density (MVD) were counted using Weidner's highest vessel density count method; the expression of VEGF-C mRNA was inspected in situ hybridization. The expression of VEGF-C mRNA was 44.12% positive in 34 cases of prostate cancer, the MLC (8.26 +/- 2.73)mm2 and MVD (74.82 +/- 11.76)mm2 in prostate cancer were significantly higher than those in adjacent nontumorous tissue (MLC, 4.82 +/- 3.48/mm2; MVD, 32.86 +/- 5.41/mm2). In addition, there was a correlation between the expression of VEGF-C mRNA and micro-lymphatics metastases and there was a positive correlation between the expression of VEGFR-3 and CD31. The expressions of VEGF-C mRNA , MLC, MVD in stage III and IV and those who have lymph metastasis were higher than those in stage I and II and those who have no lymph metastasis; the expressions of VEGFR-3 and CD31 in VEGF-C mRNA positive groups were significantly higher than those in negative groups. The difference of histopathologic grading in prostate cancer had no statistical significance. VEGF-C could accelerate the hyperplasia of micro-lymphatics and neoangiogenesis induced by tumor and play an important role in tumor lymph metastases. There was a close correlation between the expressions of VEGFR-3, CD31 and tumor metastases. The increase of MLC and MVD on prostate cancer indicated the hyperplasia of new micro-lymphatics and neoangiogenesis in the tumor tissue, which could also be a signal to determine the tumor metastases in clinic.     

食管鳞癌VEGF—C mRNA和CD31表达及其意义

To investigate the expression of vascular endothelial growth factor-C (VEGF-C) mRNA and CD 31 in esophageal squamous cell carcinoma (ESCC) and its promotion of lymphatic metastasis. The expression of VEGF-C mRNA was examined in 43 ESCC by in situ hybridization. Intratumoral microvessel density (MVD) was assessed by immunostaining endothelial cells, using anti-CD31 antibody. The positive rate of VEGF-C mRNA expression was 41.86%. The average rank of MVD was 76.36 +/- 20.30/mm2. VEGF-C mRNA expression correlated significantly with lymph node metastasis, TNM stage and depth of invasion (p < 0.05 or p < 0.01) in statistic, but not with histological grade (differentiation) (p > 0.05). MVD correlated significantly with lymph node metastasis and TNM stage (p < 0.05) in statistic, but not with depth of invasion and histological grade (differentiation) (p > 0.05). MVD was significant higher in the VEGF-C positive tumors than negative tumors (p < 0.05). The present study indicated that VEGF-C might play a role in lympatic metastasis via lymphangiogenesis and angiogenesis in ESCC.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Measuring microvascular density in tumors by digital dissection

OBJECTIVE: To develop and validate a digital dissection techniquefor measuring the cross-sectional area of blood vessels in histologic sections of tumors routinely stained with hematoxylin and eosin. STUDY DESIGN: The procedure was first validated in four experimental tumors in rats by comparing the results of the digital dissection technique to functional estimates of the blood volume in the tumors as measured by dynamic, contrast-enhanced magnetic resonance imaging. The method was then tested on a variety of experimental and human tumors. RESULTS: The digital dissection technique yielded results that exactly matched the functional measurements of blood volume infour experimental tumors. Digital dissection of 40 additional tumors in rats showed that 21 infiltrating ductal carcinomas had significantly greater microvascular density (MVD) than 19 benign fibroadenomas (12% vs. 7.9%, P=.028 by two-tailed t test). In 10 human breast carcinomas the MVD was consistently greater than the measurement of blood vessel density as identified by immunohistochemical staining for factor VIII. The between-run coefficients of variation for the MVD assay were 12% (n = 5) for a human breast cancer and 18% (n = 5)for an experimental rat tumor. CONCLUSION: The digital dissection technique is a reproducible, objective and accurate method of measuring MVD in sections of tumors that are routinely stained with hematoxylin and eosin.     

Angiogenesis in gliomas

Brain gliomas are characterized by invasive growth and neovascularisation potential. Angiogenesis plays a major role in the progression of gliomas and its determination has a great prognostic value. The aim of the study was to assess the vascularisation of chosen brain gliomas and to estimate how it is correlated with tumour histological type, malignancy grade, location and size, and with age and sex of patients. Tumour vascularisation analysis was based on the determination of microvascular proliferation (MVP) and microvessel density (MVD). Microvascular proliferation was measured with immunohistochemical methods using mouse monoclonal antibodies to detect cell proliferation antigens. The following antibodies were used Ki-67 and PCNA (DAKO). Identification of vessels was performed by CD31 antibody and anti-human von Willebrand factor (DAKO). The highest microvascular proliferation and microvascular density were observed in multiform glioblastomas and the lowest in oligodendrogliomas. Significant correlation was observed between the vascularisation and malignancy grade.     

Essential Contribution of CD4+ T Cells to Antigen-Induced Nasal Hyperresponsiveness in Experimental Allergic Rhinitis

Nasal hyperresponsiveness (NHR) is a characteristic feature of allergic rhinitis (AR); however, the pathogenesis of NHR is not fully understood. In this study, during the establishment of an experimental AR model using ovalbumin-immunized and -challenged mice, augmentation of the sneezing reaction in response to nonspecific proteins as well as a chemical stimulant was detected. Whether NHR is independent of mast cells and eosinophils was determined by using mast cell- and eosinophil-deficient mice. NHR was suppressed by treatment with anti-CD4 antibody, suggesting the pivotal contribution of CD4⁺ T cells. Furthermore, antigen challenge to mice to which in vitro-differentiated Th1, Th2, and Th17 cells but not naïve CD4⁺ T cells had been adoptively transferred led to the development of equivalent NHR. Since antigen-specific IgE and IgG were not produced in these mice and since antigen-specific IgE-transgenic mice did not develop NHR even upon antigen challenge, humoral immunity would be dispensable for NHR. CD4⁺ T cells play a crucial role in the pathogenesis of AR via induction of NHR, independent of IgE-, mast cell-, and eosinophil-mediated responses.     

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.     

Angiogenesis in male breast cancer

BACKGROUND: Male breast cancer is a rare but aggressive and devastating disease. This disease presents at a later stage and in a more advanced fashion than its female counterpart. The immunophenotype also appears to be distinct when compared to female breast cancer. Angiogenesis plays a permissive role in the development of a solid tumor and provides an avenue for nutrient exchange and waste removal. Recent scrutiny of angiogenesis in female breast cancer has shown it to be of significant prognostic value. It was hypothesized that this holds true in invasive ductal carcinoma of the male breast. In the context of male breast cancer, we investigated the relationship of survival and other clinico-pathological variables to the microvascular density of the tumor tissue. METHODS: Seventy-five cases of primary male breast cancer were identified using the records of the Saskatchewan Cancer Agency over a period of 26 years. Forty-seven cases of invasive ductal carcinoma of the male breast had formalin-fixed paraffin-embedded tissue blocks that were suitable for this study. All cases were reviewed. Immunohistochemical staining was performed for the angiogenic markers (cluster designations 31 (CD31), 34 (CD34) and 105 (CD105), von Willebrand factor (VWF), and vascular endothelial growth factor (VEGF)). Microvascular density (MVD) was determined using average, centre, and highest microvessel counts (AMC, CMC, and HMC, respectively). Statistical analyses compared differences in the distribution of survival times and times to relapse between levels of MVD, tumor size, node status and age at diagnosis. In addition, MVD values were compared within each marker, between each marker, and were also compared to clinico-pathological data. RESULTS: Advanced age and tumor size were related to shorter survival times. There were no statistically significant differences in distributions of survival times and times to relapse between levels of MVD variables. There was no significant difference in MVD between levels of the different clinico-pathological variables. MVD was strongly and significantly correlated between AMC, CMC and HMC for CD31, CD34, and CD105 (p < 0.01) and remained moderate to weak for VWF and VEGF. CONCLUSION: Microvascular density does not appear to be an independent prognostic factor in male breast cancer. However, the likelihood of death for men with breast cancer is increased in the presence of increased age at diagnosis and advanced tumor size. This is perhaps linked to inherent tumor vasculature, which is strongly related throughout a tumor section.