Kathryn L. Kellar

Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance

Emerging evidence suggests critical roles for APCs in suppressing immune responses. Here, we show that zymosan, a stimulus for TLR2 and dectin-1, regulates cytokine secretion in DCs and macrophages to induce immunological tolerance. First, zymosan induces DCs to secrete abundant IL-10 but little IL-6 and IL-12(p70). Induction of IL-10 is dependent on TLR2- and dectin-1-mediated activation of ERK MAPK via a mechanism independent of the activation protein 1 (AP-1) transcription factor c-Fos. Such DCs stimulate antigen-specific CD4+ T cells poorly due to IL-10 and the lack of IL-6. Second, zymosan induces F4-80+ macrophages in the splenic red pulp to secrete TGF-beta. Consistent with these effects on APCs, injection of zymosan plus OVA into mice results in OVA-specific T cells that secrete little or no Th1 or Th2 cytokines, but secrete robust levels of IL-10, and are unresponsive to challenge with OVA plus adjuvant. Finally, coinjection of zymosan with OVA plus LPS suppresses the response to OVA via a mechanism dependent on IL-10, TGF-beta, and lack of IL-6. Together, our data demonstrate that zymosan stimulates IL-10+ IL-12(p70)- IL-6low regulatory DCs and TGF-beta+ macrophages to induce immunological tolerance. These data suggest several targets for pharmacological modulation of immune responses in various clinical settings.

Multiplexed microsphere-based flow cytometric assays.

Flow cytometry has become an indispensable tool for clinical diagnostics and basic research. Although primarily designed for cellular analysis, flow cytometers can detect any particles in the lower micron range, including inert microspheres of different sizes, dyed with various fluorochromes. Over the past 20 years, microspheres have been used as calibrators for flow cytometers and also as a solid support for numerous molecular reactions quantitated by flow cytometry. Proteins, oligonucleotides, polysaccharides, lipids, or small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule. More recently, assays for similar analytes have been multiplexed, or analyzed in the same assay volume, by performing each reaction on a set of microspheres that are dyed to different fluorescent intensities and, therefore, are spectrally distinct. Some recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interactions of nuclear receptors. The speed, sensitivity, and accuracy of flow cytometric detection of multiple binding events measured in the same small volume have the potential to replace many clinical diagnostic and research methods and deliver data on hundreds of analytes simultaneously.

Multiplexed microsphere-based flow cytometric immunoassays for human cytokines.

Cytokines play a pivotal role in the regulation of immunologic, hematologic and wound-healing processes. They function to stimulate as well as inhibit the proliferation, differentiation and maturation of a variety of cell types. Thus, their functions are pleiotropic as well as interdependent to the extent that any cytokine may have effects that are synergistic or antagonistic with other cytokines. Cytokines also display redundancy when one mimics the functions of others. These characteristics imply that measuring the levels of one cytokine in a biologic system provides only a fraction of the information that is relevant to the existing physiologic state. A more realistic indication of the complexity of cellular interactions would include measurements of multiple cytokines at any time point. One method of multiplexed analysis can be performed by capture of the cytokines on an array of fluorescent microspheres for quantitation by flow cytometry. This technology has been applied to a variety of biomolecules, but simultaneous quantitation of multiple cytokines in a small sample volume has become rapid, inexpensive, reliable and informative.

A Novel Recombinant Multisubunit Vaccine against Chlamydia

The administration of an efficacious vaccine is the most effective long-term measure to control the oculogenital infections caused by Chlamydia trachomatis in humans. Chlamydia genome sequencing has identified a number of potential vaccine candidates, and the current challenge is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. Vibrio cholerae ghosts (VCG) are nontoxic, effective delivery vehicles with potent adjuvant properties, and are capable of inducing both T cell and Ab responses in mucosal tissues. We investigated the hypothesis that rVCG could serve as effective delivery vehicles for single or multiple subunit chlamydial vaccines to induce a high level of protective immunity. rVCG-expressing chlamydial outer membrane proteins were produced by a two-step genetic process, involving cloning of Omp genes in V. cholerae, followed by gene E-mediated lysis of the cells. The immunogenicity and vaccine efficacy of rVCG-expressing single and multiple subunits were compared. Immunologic analysis indicated that i.m. immunization of mice with either vaccine construct induced a strong mucosal and systemic specific Th1 response against the whole chlamydial organism. However, there was an immunogenic advantage associated with the multiple subunit vaccine that induced a higher frequency of Th1 cells and a relatively greater ability to confer protective immunity, compared with the single subunit construct. These results support the operational theory that the ability of a vaccine to confer protective immunity against Chlamydia is a function of the level of Th1 response elicited.

Multiple Cytokines Are Released When Blood from Patients with Tuberculosis Is Stimulated with Mycobacterium tuberculosis Antigens

Background Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection. Methods Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens. Results Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines. Conclusions Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.

Multiplex analysis of circulating cytokines in the sera of patients with different clinical forms of visceral leishmaniasis

The clinical spectrum of visceral leishmaniasis (VL), a chronic intracellular parasitic disease, ranges from a subclinical, asymptomatic infection to severe clinical disease (kala‐azar). In experimental leishmaniasis, mice that have a Th1 response to infection tend to have limited disease while a Th2 response is associated with disease progression. Humans with VL most often have mixed rather than polarized responses. However, most clinical studies have used methods that require a relatively large sample volume, thus limiting their scope. Measuring multiple cytokine levels in blood samples using a multiplexed microsphere assay (MMA) may be useful to further evaluate the Th1/Th2 paradigm in humans.

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a persons lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test‐negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage‐specific CD68+ epithelioid macrophages and small round CD3+ lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid‐fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead‐based assay using the Luminex 100 and were found to contain interleukin (IL)‐6, IL‐8, interferon‐γ and tumor necrosis factor‐α, cytokines known to be involved in human granuloma formation, in quantities from two‐fold to 7000‐fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.

Rapid Microsphere Assay for Identification of Cryptosporidium hominis and Cryptosporidium parvum in Stool and Environmental Samples

ABSTRACT Cryptosporidium hominis and Cryptosporidium parvum are associated with massive disease outbreaks worldwide. Because these two species have different transmission cycles, identification of these parasites to the species level in clinical samples may provide laboratory data of crucial importance in epidemiologic investigations. To date, the most reliable way to differentiate C. hominis and C. parvum is based on DNA sequencing analysis of PCR amplicons. Although this approach is very effective for differentiation of Cryptosporidium species, it is labor-intensive and time-consuming compared with methods that do not require DNA sequencing analysis as an additional step and that have been successfully used for specific identification of a number of pathogens. In this study, we describe a novel Luminex-based assay that can differentiate C. hominis from C. parvum in a rapid and cost-effective manner. The assay was validated by testing a total of 143 DNA samples extracted from clinical specimens, environmental samples, or samples artificially spiked with Cryptosporidium oocysts. As few as 10 oocysts per 300 μl of stools could be detected with this assay. The assay format includes species-specific probes linked to carboxylated Luminex microspheres that hybridize to a Cryptosporidium microsatellite-2 region (ML-2) where C. hominis and C. parvum differ by one nucleotide substitution. The assay proved to be 100% specific when samples that had been characterized by direct fluorescent antibody test (DFA) and DNA sequencing analysis were tested. In addition, the assay was more sensitive than DFA and provided species identification, which is an advantage for epidemiologic studies.

Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

Background The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. Methods and Principal Findings In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. Conclusion and Significance Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

Multiplexed Microsphere‐Based Flow Cytometric Immunoassays

Multiplexed microsphere‐based immunoassays can be developed to simultaneously measure multiple analytes in a biologic system by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. A multiplexed sandwich immunoassay is based on an ELISA format that is transferred directly to microspheres to quantitate multiple antigens. These assays require smaller sample volumes, are less expensive, and are as reproducible, reliable, and sensitive as ELISAs. However, potential cross‐reactivities between multiplexed antibodies, antigens, and specimens need to be systematically eliminated during the validation process. Sandwich and competitive immunoassays, which require only one antigen‐specific antibody, can be combined in the same multiplexed array. Antibody‐capture immunoassays are used to detect multiple antibodies from a specimen for diagnostic or surveillance purposes. The protocols for these three multiplexed immunologic assays are accompanied by methods for coupling analytes to microspheres and biotinylation of antibodies with a water‐soluble derivative.