Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis.

The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.

Investigating mechanisms of alkalinization for reducing primary breast tumor invasion.

The extracellular pH (pHe) of many solid tumors is acidic as a result of glycolytic metabolism and poor perfusion. Acidity promotes invasion and enhances metastatic potential. Tumor acidity can be buffered by systemic administration of an alkaline agent such as sodium bicarbonate. Tumor-bearing mice maintained on sodium bicarbonate drinking water exhibit fewer metastases and survive longer than untreated controls. We predict this effect is due to inhibition of tumor invasion. Reducing tumor invasion should result in fewer circulating tumor cells (CTCs). We report that bicarbonate-treated MDA-MB-231 tumor-bearing mice exhibited significantly lower numbers of CTCs than untreated mice (P < 0.01). Tumor pHe buffering may reduce optimal conditions for enzymes involved in tumor invasion such as cathepsins and matrix metalloproteases (MMPs). To address this, we tested the effect of transient alkalinization on cathepsin and MMP activity using enzyme activatable fluorescence agents in mice bearing MDA-MB-231 mammary xenografts. Transient alkalinization significantly reduced the fluorescent signal of protease-specific activatable agents in vivo (P ≤ 0.003). Alkalinization, however, did not affect expression of carbonic anhydrase IX (CAIX). The findings suggest a possible mechanism in a live model system for breast cancer where systemic alkalinization slows the rate of invasion.

Immunological characterization of bronchoalveolar lavage fluid in patients with acute pulmonary coccidioidomycosis.

BACKGROUND: The specific cellular immunological characteristics of bronchoalveolar lavage (BAL) fluid in acute pulmonary coccidioidomycosis have not been defined. METHODS: BAL fluid from patients living in a coccidioidomycosis-endemic region of Arizona who were undergoing bronchoscopy because of pulmonary infiltrates was analyzed. Mononuclear cells from BAL fluid and peripheral blood mononuclear cells (PBMCs) were incubated with the coccidioidal antigen T27K in vitro, and cellular immunological assays were performed. RESULTS: Forty-six patients were studied. Twelve received a diagnosis of acute pulmonary coccidioidomycosis, 17 received other diagnoses, and 17 had no diagnosis established. There was an increased proportion of polyfunctional CD8(+) T cells after antigen stimulation from subjects with coccidioidomycosis as compared to those with another diagnosis (P = .025). In cells collected from BAL fluid and in PBMCs, the concentrations of interferon γ, tumor necrosis factor α, and interleukin 17 (IL-17) were all significantly increased in samples from those with acute pulmonary coccidioidomycosis, compared with the other 2 groups (for all, P<.05). CONCLUSIONS: When incubated in vitro with a coccidioidal antigen preparation, cells from both BAL fluid and peripheral blood obtained from patients with pulmonary coccidioidomycosis demonstrated specific cellular immune responses, including expression of IL-17.

Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system.

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.