Heat shock proteins with molecular masses of ∼60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungus Histoplasma capsulatum displays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) to H. capsulatum Hsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected with H. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modify H. capsulatum pathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Histoplasma capsulatum (Hc), is a facultative intracellular fungus that binds to CD11/CD18 receptors on macrophages (Mφ). To identify the ligand(s) on Hc yeasts that is recognized by Mφ, purified human complement receptor type 3 (CR3, CD11b/CD18) was used to probe a Far Western blot of a detergent extract of Hc cell wall and cell membrane. CR3 recognized a single 60-kDa protein, which was identified as heat shock protein 60 (hsp60). Biotinylation of viable yeasts, followed by precipitation with streptavidin-coated beads, and Western blotting with anti-hsp60 demonstrated that hsp60 was on the surface of Hc yeasts. Electron and confocal microscopy revealed that hsp60 resided on the yeast cell wall in discrete clusters. Recombinant hsp60 (rhsp60) inhibited attachment of Hc yeasts to Mφ. Recombinant hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese hamster ovary cells transfected with CR3 (CHO3). Polystyrene beads coated with rhsp60 bound to Mφ, and attachment was inhibited by Abs to CD11 and CD18. Freeze/thaw extract (F/TE), a preparation of Hc yeast surface proteins that contained hsp60, inhibited the attachment of Hc yeasts to Mφ. Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mφ. Interestingly, rhsp60 did not inhibit binding of Hc yeasts to dendritic cells (DC), which recognize Hc via very late Ag 5. Moreover, F/TE inhibited attachment of Hc to DC even when depleted of hsp60. Thus, Hc hsp60 appears to be a major ligand that mediates attachment of Hc to Mφ CD11/CD18, whereas DC recognize Hc via a different ligand(s).

The function of macrophage Fc gamma receptors is impaired in patients with alcoholic cirrhosis, and this impairment probably contributes to the high incidence of bacterial infections among such patients.

Prompt diagnostic work-up of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of three immunoassays detecting anti-PF4/heparin-antibodies, derived a diagnostic algorithm with a short analytical turnaround-time (TAT) and prospectively validated it. Plasma samples were analysed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG and ID-H/PF4-PaGIA in retrospective (n=221) and prospective (n=305) derivation cohorts. We calculated likelihood ratios (LR) of result intervals and cut-off values with 100% negative (NPV) and positive (PPV) predictive value for a positive gold-standard functional assay (HIPA). We established a diagnostic algorithm based on the Bayesian combination of pre-test probability and LR of first- and second-line immunoassays. Cut-offs with 100% PPV for positive HIPA were >3.0 U/ml (HemosIL-AcuStar-HIT-IgG) and titre {greater than or equal to}16 (ID-H/PF4-PaGIA); cut-offs with 100% NPV were <0.13 U/ml and {less than or equal to}1, respectively. During the prospective validation of the derived algorithm (n=687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566/687 cases (82.4%) (analytical TAT 30 min). In 121/687 unresolved cases (17.6%), ID-H/PF4-PaGIA was used as second-line testing (additional TAT 30 min). The algorithm accurately predicted HIT in 51/687 (7.4%) and excluded it in 604/687 (87.9%) patients, leaving only 20/687 (2.9%) cases unresolved. Additionally, we identified 12/687 (1.7%) positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and two possible false positive algorithm predictions. The combination of pre-test probability with first- and second-line immunoassays for anti-PF4/heparin-antibodies is accurate for ruling in or out HIT in {greater than or equal to}95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH 2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated ϳ70 and ϳ50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice. MATERIALS AND METHODS Animals. BALB/c (H-2 d) mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Female New Zealand White rabbits were purchased from Myrtle Rabbitry, Thompson's Station, Tenn. Antigen. HIS-62 was isolated from CW/M as described previously (10). Antibodies. To prepare anti-HIS-62 antibody, a rabbit was initially phlebotomized to obtain preimmune control serum. The rabbit was then given an intradermal injection of 50 g of HIS-62 emulsified in Titermax (Cyt-Rx Corp., Norcross, Ga.). Four weeks later, the immunization was repeated in the absence of adjuvant. Serum was collected and tested for reactivity against HIS-62. Monospecific antibody was prepared as described by Smith and Fisher (25); 50 g of HIS-62 was electrophoresed in a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) gel and electroblotted onto a nitrocellulose membrane. The membrane was stained with Ponceau S, and the band corresponding to HIS-62 was excised. Subsequently, the nitrocellulose strip was blocked with 5% dried milk in Tris-buffered saline (pH 7.4), air dried, and then incubated with the rabbit polyclonal serum for 24 h at 4ЊC. The strip was washed three times with 0.1% Tween 20 in Tris-buffered saline, and the bound antibody was eluted with 2 ml of a 200 mM glycine buffer, pH 3.0. Eluted antibody was neutralized with 0.1 volume of 1 M Tris, pH 7.4. Anti-GroEL antibody was a kind gift of Roger Hendrix, University of Pittsburgh. ...

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Infection is a frequent complication in patients undergoing hemodialysis for end-stage renal disease and is the primary cause of mortality among such patients. Macrophages are important in host defense against infection largely because their Fc gamma receptors recognize antibody-coated bacteria. We therefore studied macrophage Fc gamma-receptor function in vivo and in vitro in 56 patients with end-stage renal disease who were on hemodialysis and in 20 healthy volunteers. The clearance of IgG-coated (sensitized) autologous red cells was decreased in 53 patients. The inhibition of clearance in the 56 patients was 52 +/- 3 percent at 1 hour, 41 +/- 5 percent at 1 1/2 hours, and 29 +/- 5 percent at 2 hours (P less than 0.001). The clearance of unsensitized erythrocytes and heat-altered autologous erythrocytes was normal. The impairment of clearance was not correlated with age, sex, nutritional status, HLA haplotype, or the presence of circulating immune complexes. The recognition of these IgG-sensitized red cells in vitro by Fc gamma RI (an Fc gamma-receptor protein that binds monomeric IgG) on blood monocytes from the patients was also significantly decreased (P less than 0.001) but was partially improved by hemodialysis. Nine patients had severe infections during a two-year follow-up period. The clearance of IgG-coated cells in these patients (half-time, 12.9 +/- 1.7 hours) was significantly impaired, as compared with that in the 47 patients without severe infections (half-time, 4.4 +/- 1.8 hours; P less than 0.001). We conclude that macrophage Fc gamma-receptor function is impaired in patients with end-stage renal disease who are undergoing hemodialysis, and that this impairment probably contributes to the observed immunodepression and high prevalence of infection among such patients.