Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.antigen discovery ͉ melioidosis ͉ diagnostic ͉ antigen prediction

Aspergillus fumigatus causes life-threatening invasive pulmonary aspergillosis in the immunocompromised patient. In this study we have used a murine model of intratracheal challenge with A. fumigatus to investigate the recruitment of inflammatory cells in the lung and the expression of proinflammatory cytokines and chemokines. Our results show that A. fumigatus causes an acute pulmonary inflammatory response which is dominated by neutrophils and to a lesser extent macrophages. During the peak of infection, proinflammatory cytokines (TNF-alpha, GM-CSF and IL-1beta) and chemokines (MIP-1alpha, MCP-1 and MIP-2), are induced within the lung. Furthermore, treatment of mice with neutralizing anti-TNF-alpha and anti-GM-CSF mAbs reduced the influx of neutrophils into the lung and delayed fungal clearance. Our observations show that Aspergillus conidia are effective inducers of host chemokine responses both in vitro and in vivo. Furthermore, TNF-alpha and GM-CSF play a central role in the recruitment of neutrophils into the lung in response to this clinically important pathogen.

The role of gut intraepithelial lymphocytes (IEL) in immunity to cryptosporidial infection was investigated with a murine infection model involving Cryptosporidium muris. Oocyst shedding was monitored in severe combined immunodeficiency (SCID) mice infected with C. muris following intravenous injection of mesenteric lymph node (MLN) cells or intestinal IEL from BALB/c donor mice which were naive or previously infected with C. muris. SCID mice receiving no lymphoid cells developed chronic infections and excreted large numbers of oocysts until the end of the experiment. SCID mice injected with IEL from immune animals, however, were able to overcome the infection, and furthermore, these animals produced fewer oocysts and recovered sooner than ones which received IEL or MLN cells from naive BALB/c donors. Similar levels of protection were obtained in SCID mice injected with either 2 ؋ 10 6 IEL or MLN cells from immune donor mice. Depletion of CD4 ؉ cells from immune IEL, however, abrogated the ability to transfer immunity to SCID mice, while depletion of CD8 ؉ cells only marginally reduced the protective capacity of immune IEL. Finally, control SCID mice which received no lymphocytes had <1% CD4 ؉ cells in the IEL from the small intestine, whereas the IEL from SCID mice recovered from infection, as a result of injection with immune IEL, contained 15% CD4 ؉ cells. Thus, the ability to control C. muris infection correlated with the presence of the protective CD4 ؉ cells in the gut epithelium.

This study was designed to investigate and characterize T-cell responses which lead to elimination of a primary infection of Cryptosporidium muris in BALB/c mice. The proliferative response of spleen cells to parasite antigen was measured by uptake of 3H-thymidine and, in parallel, supernatants were removed from cells to measure levels of IFN-gamma, TNF, IL-2 and IL-4 by ELISA. Oocyst excretion in faeces was first detected on day 10 post infection (p.i.); the level of shedding subsequently increased until day 14 and then declined until no oocysts were detected by day 25. The proliferative response of spleen cells from infected animals was similar to control levels up to day 14 p.i. but increased significantly on day 21 and was even greater on day 26. IFN-gamma and IL-2 were detected initially on day 14 p.i. and significantly higher concentrations were found on days 21 and 26. IL-4 secretion was also detected, but not until day 21 p.i., and production of TNF was not found at any time. Depletion of T-cells or CD4+ cells from spleen cells cultured with antigen resulted in a significant decrease in the levels of cytokine detected. These results indicated, therefore, that in BALB/c mice there was a correlation between the development of immunity to C. muris infection and both a parasite antigen-specific proliferative response and Th1 and Th2 cytokine production by spleen cells.