The abilities of cancer-associated fibroblasts (CAFs) to regulate immune responses in the context of radiotherapy remain largely unknown. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on macrophages. CAFs were isolated from freshly-resected non-small cell lung cancer tumors, while monocyte-derived macrophages were prepared from peripheral blood of healthy donors. Experimental settings included both (CAF-macrophage) co-cultures and incubations of M0 and M1-macrophages in the presence of CAF-conditioned medium (CAF-CM). Functional assays to study macrophage polarization/activation included the expression of cell surface markers, production of nitric oxide, secretion of inflammatory cytokines and migratory capacity. We show that CAFs promote changes in M0-macrophages that harmonize with both M1-and M2-phenotypes. Additionally, CAFs inhibit pro-inflammatory features of M1-macrophages by reducing nitric oxide production, pro-inflammatory cytokines, migration, and M1-surface markers expression. Radiation delivered as single-high dose or in fractioned regimens did not modify the immunoregulatory features exerted by CAFs over macrophages in vitro. Protein expression analyses of CAF supernatants showed that irradiated and non-irradiated CAFs produce approximately the same protein levels of immunoregulators. Thus, CAF-derived soluble factors mediate measurable changes on uncommitted macrophages and down-regulate pro-inflammatory features of M1-polarized macrophages. Notably, ionizing radiation does not curtail the CAF-mediated immunosuppressive effects.

A simplified method to produce Paracoccidioides brasiliensis exoantigens for immunodiffusion testing is proposed. It uses technology accessible for small laboratories with few resources in Latin America, where paracoccidioidomycosis is endemic. This procedure may replace the more complex procedure, originally proposed by Camargo et al. in 1988, that is currently commonly used. It is based on the production of exoantigen by P. brasiliensis isolate B339, a good secretor of the characteristic 43000-Da glycoprotein gp43.

Recent studies have demonstrated that radiotherapy is able to induce anti-tumor immune responses in addition to mediating direct cytotoxic effects. Cancer-associated fibroblasts (CAFs) are central constituents of the tumor stroma and participate actively in tumor immunoregulation. However, the capacity of CAFs to influence immune responses in the context of radiotherapy is still poorly understood. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on natural killer (NK) cells. CAFs were isolated from freshly resected non-small cell lung cancer tissues, while NK cells were prepared from peripheral blood of healthy donors. Functional assays to study NK cell immune activation included proliferation rates, expression of cell surface markers, secretion of immunomodulators, cytotoxic assays, as well as production of intracellular activation markers such as perforin and granzyme B. Our data show that CAFs inhibit NK cell activation by reducing their proliferation rates, the cytotoxic capacity, the extent of degranulation, and the surface expression of stimulatory receptors, while concomitantly enhancing surface expression of inhibitory receptors. Radiation delivered as single high-dose or in fractioned regimens did not reverse the immunosuppressive features exerted by CAFs over NK cells in vitro, despite triggering enhanced surface expression of several checkpoint ligands on irradiated CAFs. In summary, CAFs mediate noticeable immune inhibitory effects on cytokine-activated NK cells during co-culture in a donor-independent manner. However, ionizing radiation does not interfere with the CAF-mediated immunosuppressive effects.

Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally occurs in patients with degenerative and neoplastic diseases and is considered the fourth most frequent cause of bloodstream infections. Diagnosis of candidemia or hematogenous candidiasis has been problematic because clinical signs and symptoms are nonspecific, leading to delays in diagnosis and, consequently, delays in appropriate antifungal therapy. We developed an inhibition enzyme-linked immunosorbent assay (ELISA) for detection of a 65-kDa antigen in an experimental model of candidemia and for diagnosis of patients in intensive care units (ICUs) with suspected candidemia. An anti-65-kDa monoclonal antibody was tested for detection of the 65-kDa antigen produced by Candida albicans, Candida tropicalis, and Candida parapsilosis in murine candidemia models. The 65-kDa antigen was detected in sera at concentrations ranging from 0.012 to 3.25 g/ml. A total of 20 human patients with candidemia were then evaluated with the inhibition ELISA using sequential sera. Sixteen (80%) patients had the 65-kDa antigen in concentrations ranging from 0.07 to 5.0 g/ml. Sequential sera from patients with candidemia presented three different patterns of antigenemia of the 65-kDa molecule: (i) total clearance of antigenemia, (ii) initial clearance and relapse of antigenemia, and (iii) partial clearance of antigenemia. Our results indicate detection of the 65-kDa protein may be a valuable tool for the diagnosis of candidemia by C. albicans, C. tropicalis, and C. parapsilosis.

The main objectives of this study were to obtain clones of Paracoccidioides brasiliensis by two methods (micromanipulation and plating assay) and to determine if the secretion of the 43-kDa glycoprotein (gp43) is dependent on the clonal culture. The results show that the secretion of gp43 is not dependent on clonal cultures. Clones that originally were secretors of this molecule, after subculturing, lost this characteristic; on the other hand, clones that originally did not secrete gp43 began to secrete gp43 after subculturing.Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis. P. brasiliensis secretes and excretes various proteins and glycoproteins, and the 43-kDa molecule (gp43) is the immunodominant antigen and the main antigenic component, useful for diagnosis of paracoccidioidomycosis.The occurrence of instability in the synthesis of antigenic components by the same P. brasiliensis isolate under controlled incubation conditions was first observed by Franco et al. (5). When a specific isolate is used to produce antigens for immunodiffusion tests or Western blot assays, the gp43 molecule must be present in these preparations. However, this condition is not reproducible, and sometimes the final preparation does not contain gp43. Our hypothesis was that a culture is composed of different clones and for some unknown reason only some of them express gp43. A more reasonable approach to obtain a homogenous antigen preparation is to work with clonal cultures in which all cells contain the same genetic information.In the present study, we compared two ways to isolate single cells (clones) of P. brasiliensis, i.e., by a micromanipulation technique and by plating of single cells, and determined which medium is better in order to obtain clonal cultures. In addition, these clonal cultures were tested for their potential to secrete gp43 in order to confirm or reject our hypothesis about the relationship between gp43 secretion and clonal cultures. Also, clonal cultures were analyzed by rapidly amplified polymorphic DNA to verify possible polymorphism at the DNA level.