The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen.

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.

Prognosis of metastatic carcinoma of the lung in the bevacizumab era: comparison between the major histologic types of lung cancer.

BACKGROUND: In October 2006, bevacizumab was approved for treatment for patients with metastatic non-small-cell lung cancer other than squamous carcinoma. Our hypothesis was that the change in survival after approval of bevacizumab for metastatic adenocarcinoma would show differences from that of small-cell carcinoma and squamous carcinoma. METHODS: Data was obtained from the National Cancer Institute Surveillance Epidemiology and End Results (SEER) registry for patients with lung cancer diagnosed between January 2004 and November 2007. In addition to known characteristics predicting survival differences (histotype, age, gender, and race) we compared 1-year survival experience in those diagnosed before (January 2004-September 2006) and after (October 2006-November 2007) introduction of bevacizumab. RESULTS: Of 24,575 patients meeting criteria, 16,081 (65.4%) died within 1 y. Adjusted for age, gender, and race, patients with squamous carcinoma showed a 13% decline (95% CI 7%-20%) in survival times. By contrast, the 1% increment for adenocarcinoma and the 1% decrement for small cell carcinoma might well have been due to chance (P > 0.05 for each analysis). CONCLUSIONS: Life expectancy for metastatic adenocarcinoma (for which bevacizumab is approved) and metastatic small-cell carcinoma (bevacizumab not approved) did not change statistically. On the other hand, life expectancy for patients with metastatic squamous carcinoma (bevacizumab not approved) of the lung has declined since the approval of bevacizumab. This likely reflects increased classification of tumors previously diagnosed as poorly differentiated non-small-cell carcinoma as poorly differentiated squamous carcinoma. Hence, life expectancy of metastatic adeno, squamous, and small-cell-lung cancer has not improved after introduction of bevacizumab.

CD4+ T lymphocytes are critical mediators of tumor immunity to simian virus 40 large tumor antigen induced by vaccination with plasmid DNA.

A mechanistic analysis of tumor immunity directed toward the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory for scenarios of recombinant Tag immunization in BALB/c mice. In the present study, we performed a preliminary characterization of the immune components necessary for systemic tumor immunity induced upon immunization with plasmid DNA encoding SV40 Tag as a transgene (pCMV-Tag). Antibody responses to SV40 Tag were observed via indirect enzyme-linked immunosorbent assay following three intramuscular (i.m.) injections of pCMV-Tag and were typified by a mixed Th1/Th2 response. Complete tumor immunity within a murine model of pulmonary metastasis was achieved upon two i.m. injections of pCMV-Tag, as assessed by examination of tumor foci in mouse lungs, without a detectable antibody response to SV40 Tag. Induction-phase and effector-phase depletions of T cell subsets were performed in vivo via administration of depleting rat monoclonal antibodies, and these experiments demonstrated that CD4(+) T lymphocytes are required in both phases of the adaptive immune response. Conversely, depletion of CD8(+) T lymphocytes did not impair tumor immunity in either immune phase and resulted in the premature production of antibodies to SV40 Tag. Our findings are unique in that a dominant role could be ascribed to CD4(+) T lymphocytes within a model of DNA vaccine-induced tumor immunity to Tag-expressing tumor cells. Additionally, our findings provide insight into the general mechanisms of vaccine-induced tumor immunity directed toward tumors bearing distinct tumor-associated antigens.

Vaccines and immunotherapeutics for the treatment of malignant disease.

The employment of the immune system to treat malignant disease represents an active area of biomedical research. The specificity of the immune response and potential for establishing long-term tumor immunity compels researchers to continue investigations into immunotherapeutic approaches for cancer. A number of immunotherapeutic strategies have arisen for the treatment of malignant disease, including various vaccination schemes, cytokine therapy, adoptive cellular therapy, and monoclonal antibody therapy. This paper describes each of these strategies and discusses some of the associated successes and limitations. Emphasis is placed on the integration of techniques to promote optimal scenarios for eliminating cancer.

Role of the innate immune response and tumor immunity associated with simian virus 40 large tumor antigen.

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.

Tumor immunity against a simian virus 40 oncoprotein requires CD8+ T lymphocytes in the effector immune phase.

The required activities of CD4(+) T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8(+) T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8(+) T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-gamma), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8(+) T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8(+) T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8(+) T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.

Detection of macrocyclic trichothecene mycotoxin in a caprine (goat) tracheal instillation model.

This study demonstrates the detection and dynamics of macrocyclic trichothecene mycotoxin (MTM) tissue loading using a commercially available assay in a goat model. The detection of MTMs has been difficult and complex due to the uncertainty of what tissues to examine and when to sample. Twelve goats (two groups of each) were instilled with Stachybotrys chartarum conidial suspension via the trachea. The first group was challenged repeatedly with fungal conidia containing 1 mg/kg of MTM per instillation whereas the second group was exposed once, to spores with a calculated concentration of 5 microg/kg of mycotoxin. These toxin estimates were generated by the QuantiTox(TM) Kit assay; a conidium of S. chartarum possessed 8.5 pg of MTM. After repeated exposure of 3 days, MTM was detected in one of six animals. This animal and two others from the same group had mycotoxin detected in their serum 24 hours after challenge at a comparable level (1.69 ng/mL) to the six animals challenged with a single dose (2.02 ng/mL) at the same time post-instillation. Results showed that MTMs are detectable in experimental animals soon after challenge and contribute to the understanding of the role of these mycotoxins in the disease process following mold exposure.

Evidence of simian virus 40 exposure in a colony of captive baboons.

Simian virus 40 (SV40) is a polyomavirus for which non-human primates are the permissive host. The baboon (Papio spp.) is an old world monkey that is used in a variety of research investigations; however, natural infection of SV40 among baboons has not been thoroughly examined or reported. Initially, we were interested in determining the prevalence of SV40 infection among a captive colony of baboons based on the presence of antibodies to SV40 large T-antigen (Tag). An overall seroprevalence rate of >50% was found after screening sera from 142 baboons in the colony based on ELISA. Endpoint titer values for serum antibody binding to SV40 Tag reached as high as 1280 for 5 out of 142 baboons. Peptide binding assays revealed that a range of SV40 Tag epitopes are immunogenic in the baboon, and that individual animals differ in their humoral immune responses to SV40 Tag based on epitope recognition. Specificity to SV40 Tag and not some other primate polyomavirus encoded large Tag was further examined by serologic reactivity to peptide epitopes unique to SV40 Tag. Additional serology was performed to assess SV40 Tag reactivity by Western blot and whether antibodies were capable of neutralizing SV40 infectivity in vitro. Although antibodies with high levels of SV40 neutralization were observed in a number of the baboons, there was a lack of correlation between viral neutralization and antibodies to SV40 Tag. Further examination using molecular-based diagnosis and SV40 Tag specific real-time quantitative PCR determined that some of the baboons appeared to be exposed to SV40. DNA sequence analysis of the PCR products confirmed that SV40 Tag specific sequences were detected in baboons.

Growth and mycotoxin production by Chaetomium globosum is favored in a neutral pH.

Chaetomium globosum is frequently isolated in water-damaged buildings and produces two mycotoxins called chaetoglobosins A and C when cultured on building material. In this study, the influence of ambient pH on the growth of C. globosum was examined on an artificial medium. This fungus was capable of growth on potato dextrose agar ranging in pH from 4.3 to 9.4 with optimal growth and chaetoglobosin C production occurring at a neutral pH. In addition, our results show that sporulation is favored in an acidic environment.

Growth and mycotoxin production by Chaetomium globosum.

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.

Mold contamination and air handling units.

An investigation was conducted on selected locations in air handling units (AHUs) to (a) identify common mold species found on these locations, (b) determine whether some locations (and subsets) featured mold growth sites more frequently than others, (c) ascertain whether the operating condition of AHUs is related to mold contamination, and (d) provide a basis for a microbial sampling protocol for AHUs. A total of 566 tape lifts and 570 swab samples were collected from the blower wheel fan blades, insulation, cooling coil fins, and ductwork from 25 AHUs. All AHU conditions were numerically rated using a heating, ventilation and air-conditioning (HVAC) survey. Results showed that Cladosporium sp. fungi were commonly recovered in terms of growth sites and deposited spores, and they were found mainly in the blower wheel fan blades, the ductwork, and the cooling coil fins. Subsections of the fan blades, insulation, and cooling coil fins showed no preferred area for mold growth sites. Other organisms such as Penicillium sp., Aspergillus sp., and Paecilomyces sp. were recovered from the cooling coil fins and insulation. Because of the widespread prevalence of Cladosporium sp., there was no relationship between mold growth and operating condition. However, the presence of different species of molds in locations other than the blower wheel blades may indicate that the AHU condition is not optimal. A suggested microbial sampling protocol including interpretations of sample results is presented.

Fc gamma receptors play a dominant role in protective tumor immunity against a virus-encoded tumor-specific antigen in a murine model of experimental pulmonary metastases.

Simian virus 40 (SV40) large tumor antigen (Tag) represents a virus-encoded tumor-specific antigen expressed in many types of human cancers and a potential immunologic target for antitumor responses. Fc receptors are important mediators in the regulation and execution of host effector mechanisms against conditions including infectious diseases, autoimmunity, and cancer. By examining tumor protection in SV40 Tag-immunized wild-type BALB/c mice using an experimental pulmonary metastasis model, we attempted to address whether engagement of the immunoglobulin G Fc receptors (FcgammaRs) on effector cells is necessary to mediate antitumor responses. All immunized BALB/c FcgammaR-/- knockout mice developed anti-SV40 Tag antibody responses prior to experimental challenge with a tumorigenic cell line expressing SV40 Tag. However, all mice deficient in the activating FcgammaRI (CD64) and FcgammaRIII (CD16) were unable to mount protective immunologic responses against tumor challenge and developed tumor lung foci. In contrast, mice lacking the inhibitory receptor FcgammaRII (CD32) demonstrated resistance to tumorigenesis. These results underscore the importance of effector cell populations expressing FcgammaRI/III within this murine tumor model system, and along with the production of a specific humoral immune response, antibody-dependent cell-mediated cytotoxicity (ADCC) may be a functioning mechanism of tumor clearance. Additionally, these data demonstrate the potential utility of ADCC as a viable approach for targeting vaccination strategies that promote FcgammaRI/III scavenging pathways against cancer.

In vitro simian virus 40 large tumor antigen expression correlates with differential immune responses following DNA immunization.

Simian virus 40 (SV40) contains an essential protein, large tumor antigen (Tag), which assists in viral replication and causes cell transformation and immortalization. Our laboratory has examined plasmid DNA, expressing SV40 Tag under two different promoters, for use in potential cancer vaccination strategies. One plasmid, pSV3-neo, failed to induce SV40 Tag antibody, produced a weak cell-mediated response, and only partial protection in murine experimental tumor challenge systems. The second plasmid, pCMV-Tag, induced antibodies to SV40 Tag, produced a robust cell-mediated response, and invoked complete tumor immunity in vivo. The induction of CD4+ and CD8+ T cell responses following plasmid DNA immunization and tumor cell challenge reflected a type 1 cytokine secretion profile. Our hypothesis for this differential immune response is that pCMV-Tag exhibits a higher level of transgene expression due to a more efficient promoter. We determined that pCMV-Tag levels of SV40 Tag mRNA and protein expression were higher when compared to pSV3-neo. A threshold amount of SV40 Tag may be required to stimulate antibody production and provide complete systemic tumor immunity.

Immunological treatment of liver tumors.

Although multiple options for the treatment of liver tumors have often been described in the past, including liver resection, radiofrequency ablation with or without hepatic pump insertion, laparoscopic liver resection and the use of chemotherapy, the potential of immunotherapy and gene manipulation is still largely unexplored. Immunological therapy by gene manipulation is based on the interaction between virus-based gene delivery systems and dendritic cells. Using viruses as vectors, it is possible to transduce dendritic cells with genes encoding tumor-associated antigens, thus inducing strong humoral and cellular immunity against the antigens themselves. Both chemotherapy and radiation therapy have the disadvantage of destroying healthy cells, thus causing severe side-effects. We need more precisely targeted therapies capable of killing cancer cells while sparing healthy cells. Our goal is to establish a new treatment for solid liver tumors based on the concept of cytoreduction, and propose an innovative algorithm.

Acneiform eruption induced by Iressa (gefitinib) tablets used to treat non-small cell lung cancer.

Many medications have been reported to induce acneiform eruptions. A relatively new chemotherapy drug, gefitinib (Iressa), approved by the Food and Drug Administration in 2003 for the treatment of advanced-stage non-small cell lung cancer (NSCLC), has been reported to cause acne or an acne-like eruption. We report an Asian female who presented with hundreds of erythematous papules and pustules on her face, chest, and back, all of which appeared after starting gefitinib to treat non-small cell lung cancer.

Anti-idiotype responses abrogate anti-CD4-induced tolerance to a tumor-specific antigen and promote systemic tumor immunity.

PURPOSE: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. METHODS: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. RESULTS: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. CONCLUSIONS: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4+ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.

Characterization of exposure to low levels of viable Penicillium chrysogenum conidia and allergic sensitization induced by a protease allergen extract from viable P. Chrysogenum conidia in mice.

BACKGROUND: Previous evidence by our laboratory has shown that mice inoculated with viable Penicillium Chrysogenum conidia or spores at levels comparable to those found in contaminated buildings induced spore antigen-specific allergic responses. We proposed that mice exposed to low levels of viable P. Chrysogenum conidia would not develop allergic symptoms. We also hypothesized that the symptoms induced by high numbers of conidia were the result of sensitization to allergens released by the conidia. METHODS: C57BL/6 and BALB/c mice were exposed to 1 x 10(2) viable P. Chrysogenum conidia by intranasal instillation weekly for a period of 11 weeks. C57BL/6 mice were also sensitized to a viable P. Chrysogenum conidia protease extract by intraperitoneal injections for a period of 6 weeks followed by intranasal challenge with protease extract, viable, or nonviable P. Chrysogenum conidia for 2 weeks. RESULTS: C57BL/6 mice inoculated with low numbers of conidia developed no significant lung inflammation or increased serum immunoglobulins. Mice sensitized to the protease extract and challenged with both protease extract and viable conidia produced significant increases in serum IgE and IgG1. Mice sensitized to and challenged with the protease extract developed significant eosinophilia and mucus hyperproduction as determined by bronchoalveolar lavage and histopathological examination of lung tissue. CONCLUSIONS: Mice did not develop allergic symptoms in response to challenge with low levels of P. Chrysogenum conidia. Protease allergens from viable conidia induced specific allergic responses in mice, indicating the importance of P. Chrysogenum conidia in allergic sensitization to the organism.

Studies on the role of fungi in Sick Building Syndrome.

Sick Building Syndrome is a term used to describe symptoms in humans which result from problems with indoor air quality. Common complaints include dyspnea, flu-like symptoms, watering eyes, and allergic rhinitis. Although there is likely no single cause for Sick Building Syndrome, fungal contamination in buildings has increasingly been associated with this spectrum of symptoms. The authors describe 2 case studies, and other experimentation, that have investigated the role of fungi in the occurrence of Sick Building Syndrome.