Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy

The acrosome reaction is an important marker for human sperm function. Since different laboratory techniques may be used for the detection of this exocytotic process, the purpose of the present study was to compare three common markers [Pisum sativum agglutinin (PSA), concanavalin A (ConA), double staining] and transmission electron microscopy for identification of acrosomal changes. Preliminary findings had demonstrated that similar results were achieved with Trypan Blue and Hoechst 33258 staining. Therefore, supravital stainings were omitted. In various experiments, human spermatozoa were treated with two concentrations (10 and 3.3 microM) of calcium ionophore A23187 for 15, 30 and 60 min after capacitation for 3 and 6 h at 37 degrees C. The percentages of spermatozoa with acrosomal loss detected by fluorescein isothiocyanate (FITC)-ConA were consistently lower than those obtained by double staining or FITC-PSA, which showed comparable results. Following 6 h of capacitation and incubation with 10 microM ionophore for 1 h at 37 degrees C, 25.9 +/- 15.7% of all spermatozoa showed almost complete loss of the acrosomal content. Binding of FITC- ConA to the acrosomal region was observed in 27.0 +/- 13.2% of spermatozoa obtained from the same sample. FITC-ConA and double staining or FITC-PSA detect different stages of the acrosome reaction and may be helpful for a differentiated evaluation of this sperm function.      

Value of CT in the preoperative assessment of lung cancer: a survey of thoracic surgeons

Practicing thoracic surgeons were randomly surveyed to evaluate how computed tomography (CT) has influenced the preoperative evaluation of bronchogenic carcinoma. Thirty-six percent of the 529 respondents routinely requested CT and 62% did so selectively. Approximately 40% indicated that CT provided useful information in most cases. Nearly all surgeons (98.7%) do not rely on the identification of enlarged lymph nodes with CT to spare the patient surgical staging; however, 77.5% are influenced by CT results in their staging procedures. Fifty-seven percent reported that a negative CT study eliminates surgical staging entirely unless the patient has a "coin lesion," in which case 75% are willing to proceed directly to thoracotomy. For surgeons who use CT selectively, an abnormal mediastinal contour on the radiograph was the most frequent radiologic abnormality to prompt CT (85%). Thirty-seven percent are influenced by tumor histology in their decision to request CT. There was little difference in the pattern of CT use between university and community hospital surgeons.     

Cancer vaccines.

It has been more than 100 years since the first reported attempts to activate a patient's immune system to eradicate developing cancers. Although a few of the subsequent vaccine studies demonstrated clinically significant treatment effects, active immunotherapy has not yet become an established cancer treatment modality. Two recent advances have allowed the design of more specific cancer vaccine approaches: improved molecular biology techniques and a greater understanding of the mechanisms involved in the activation of T cells. These advances have resulted in improved systemic antitumor immune responses in animal models. Because most tumor antigens recognized by T cells are still not known, the tumor cell itself is the best source of immunizing antigens. For this reason, most vaccine approaches currently being tested in the clinics use whole cancer cells that have been genetically modified to express genes that are now known to be critical mediators of immune system activation. In the future, the molecular definition of tumor-specific antigens that are recognized by activated T cells will allow the development of targeted antigen-specific vaccines for the treatment of patients with cancer.     

αE‐catenin is a candidate tumor suppressor for the development of E‐cadherin‐expressing lobular‐type breast cancer

Although mutational inactivation of E‐cadherin (CDH1) is the main driver of invasive lobular breast cancer (ILC), approximately 10–15% of all ILCs retain membrane‐localized E‐cadherin despite the presence of an apparent non‐cohesive and invasive lobular growth pattern. Given that ILC is dependent on constitutive actomyosin contraction for tumor development and progression, we used a combination of cell systems and in vivo experiments to investigate the consequences of α‐catenin (CTNNA1) loss in the regulation of anchorage independence of non‐invasive breast carcinoma. We found that inactivating somatic CTNNA1 mutations in human breast cancer correlated with lobular and mixed ducto‐lobular phenotypes. Further, inducible loss of α‐catenin in mouse and human E‐cadherin‐expressing breast cancer cells led to atypical localization of E‐cadherin, a rounded cell morphology, and anoikis resistance. Pharmacological inhibition experiments subsequently revealed that, similar to E‐cadherin‐mutant ILC, anoikis resistance induced by α‐catenin loss was dependent on Rho/Rock‐dependent actomyosin contractility. Finally, using a transplantation‐based conditional mouse model, we demonstrate that inducible inactivation of α‐catenin instigates acquisition of lobular features and invasive behavior. We therefore suggest that α‐catenin represents a bona fide tumor suppressor for the development of lobular‐type breast cancer and as such provides an alternative event to E‐cadherin inactivation, adherens junction (AJ) dysfunction, and subsequent constitutive actomyosin contraction. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Tumor-specific CD4+ T cells from a patient with renal cell carcinoma recognize diverse shared antigens

Murine models for immune-mediated tumor regression have defined an essential role for CD4+ T helper (Th) cells, but the contribution of these cells to antitumoral immune responses in humans remains poorly defined. Here, we investigated the Th cell response against the autologous tumor in a patient with metastasized renal cell carcinoma (RCC) exhibiting objective clinical response to immunotherapy. Peripheral blood T cells of the patient were repeatedly stimulated in vitro using either autologous IFNgamma-treated whole tumor cells or Epstein-Barr virus-immortalized B cells (EBV-B) pulsed with tumor cell lysate. CD4+ T-cell clones recognizing autologous tumor cells but not EBV-B cells were efficiently reactivated and expanded with both types of stimulator cells, establishing the latter as potentially useful for isolating CD4+ T cells reactive against MHC class II-negative tumors. Two T-cell clones from both stimulation protocol were further characterized. The restricting MHC class II molecules were defined by using allogeneic EBV-B cells pulsed with tumor lysate, and the expression pattern of the antigens was examined by analyzing lysates from normal kidney cells, allogeneic RCCs as well as tumors of different histologic origin. Furthermore, the subcellular localization of the antigens recognized by the T-cell clones was examined by fractionating the tumor lysate, and the Th phenotype was determined by assessing the cytokines released after T cell activation. These experiments show that a dual Th1/Th2, MHC class II-restricted T-helper-cell response against diverse shared tumor antigens has been elicited in this patient.     

Diverse CD8+ T-cell responses to renal cell carcinoma antigens in patients treated with an autologous granulocyte-macrophage colony-stimulating factor gene-transduced renal tumor cell vaccine

A phase I clinical trial with granulocyte-macrophage colony-stimulating factor tumor cell vaccines in patients with metastatic renal cell carcinoma (RCC) showed immune cell infiltration at vaccine sites and delayed-type hypersensitivity (DTH) responses to autologous tumor cells indicative of T-cell immunity. To further characterize RCC T-cell responses and identify relevant RCC-associated antigens, we did a detailed analysis of CD8+ T-cell responses in two vaccinated RCC patients who generated the greatest magnitude of DTH response and also displayed a strong clinical response to vaccination (>90% reduction in metastatic tumor volume). Three separate CD8+ T-cell lines (and subsequent derived clones) derived from patient 24 recognized distinct RCC-associated antigens. One recognized a shared HLA-A*0201-restricted antigen expressed by both renal cancer cells and normal kidney cells. This recognition pattern correlated with a positive DTH test to normal kidney cells despite no evidence of impairment of renal function by the patient's remaining kidney after vaccination. A second line recognized a shared HLA-C7-restricted antigen that was IFN-gamma inducible. A third line recognized a unique HLA-A*0101-restricted RCC antigen derived from a mutated KIAA1440 gene specific to the tumor. In addition, two independent CTL lines and three clones were also generated from patient 26 and they recognized autologous tumor cells restricted through HLA-A*0205, HLA-A/B/C, and HLA-B/C. These results show that paracrine granulocyte-macrophage colony-stimulating factor tumor vaccines may generate a diverse repertoire of tumor-reactive CD8+ T-cell responses and emphasize the importance of polyvalency in the design of cancer immunotherapies.