Rational approaches to immune regulation

Our studies are mainly focused on developing strategies of immune regulation. In the case of infectious and neoplastic disease, our approach is to upregulate cell-mediated immunity to viral of tumor antigens using an intracellular bacterium as a vector for targeting these antigens to the major histocompatibility complex (MHC) class I and class II pathways of antigen processing, in addition to exploiting the adjuvant properties of the vector to stimulate innate immunity. In the area of autoimmunity, we are attempting to downregulate the immune response by specific immune intervention directed against autoreactive T cells. In these studies we use murine models for multiple sclerosis. Our approach is to use both rationally designed T cell receptor (TCR) peptide analogs and recombinant viral vectors that express TCR components to regulate the disease.     

Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.     

Rational approaches to immune regulation

Our laboratory is interested in the properties of proteins that render them immunogenic, and how such immunogenicity may be modulated in vivo. We are attempting to enhance the immune response in the design of more effective vaccines against viral diseases, such as HIV, and against tumor antigens expressed on breast, ovarian, and cervical cancer and B cell lymphomas. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic cytotoxic T lymphocytes (CTL) epitopes. In the field of tumor immunotherapy, we are also developing nonliving vaccine vectors for tumor antigens.     

Short-circuiting autoimmune disease by target-tissue-derived nitric oxide

A previous report from this laboratory suggested that expression of skeletal-muscle-derived, inducible nitric oxide synthase (iNOS), is associated with resistance to the autoimmune model of myasthenia gravis (MG) demonstrated by Wistar Furth rats following the passive transfer of antibody reactive with the nicotinic acetylcholine receptor (AChR). The study reported below demonstrates an association between increased expression of iNOS/NO in Wistar Furth rats and the induction of programmed cell death (apoptosis) in both macrophages and CD4+ T cells that attempt to traffic through targeted muscles. It is concluded that production of muscle-derived NO is protective in experimental MG, and in part, dictates the severity of eventual immunopathology.     

Adhesion of carcinoma cells to rat hepatocytes and rat fibronectin is inhibited by the OPAR monoclonal antibody,which is directed against a rat liver-specific carbohydrate epitope

The OPAR mouse monoclonal antibody (mAb) directed against rat hepatocytes was previously shown to inhibit adhesion of TA3/Ha mammary carcinoma cells to hepatocytes. The antigen is abundantly present at the surface of hepatocytes beneath the endothelium of liver capillaries where we have observed invasion of carcinoma cells to occur. The OPAR mAb reacted with three major bands on a Western blot of liver plasma membrane proteins. The same proteins were also seen upon immunoprecipitation from iodinated liver plasma membrane proteins. We have isolated OPAR antigens by lectin wheat germ agglutinin (WGA) and OPAR affinity chromatography. Amino acid sequence analysis revealed that two of the bands were 1-macroglobulin and C4-binding protein, which are serum components produced by hepatocytes. The presence of the epitope on distinct proteins and our previous observation that it can be detected in the Golgi apparatus but not in the endoplasmic reticulum, suggested that OPAR reacts with a liver-specific glycoconjugate. Loss of OPAR reactivity after neuraminidase and N-glycosidase F treatment showed that the epitope contains sialic acid residues on N-linked sugar moieties. OPAR also reacted with rat fibronectin, and inhibited adhesion of TA3/St cells to fibronectin. This explains the inhibition by the OPAR mAb of TA3/St cell adhesion to hepatocytes, which we have shown to be due mainly to interaction with hepatocyte surface-associated fibronectin. However, adhesion of the related TA3/Ha cells to hepatocytes, which is mediated by the ₆P4 integrin, and does not involve binding to fibronectin, is also inhibited. This suggests that ₆₄ on liver-metastasizing carcinoma cells binds to an OPAR epitope-carrying glycoprotein produced by hepatocytes.     

The gene for prolactin-inducible protein (PIP), uniquely expressed in exocrine organs,maps to chromosome 7

The hormonally responsive prolactin-inducible protein (PIP), gene is expressed in benign and malignant breast tumor tissues and in such normal exocrine organs as sweat, salivary, and lacrimal glands. In this communication we report the regional chromosome localization of the PIPgene locus to chromosome 7 by Southern hybridization to DNA from human-hamster somatic cell hybrids, and to 7q32–36 by in situ hybridization.     

Genotyping of clinical methicillin-susceptible Staphylococcus aureus isolates in a Dutch teaching hospital

Methicillin-susceptible Staphylococcus aureus isolates, recovered from 204 patients in our hospital in a 22-month period, were characterized by pulsed-field gel electrophoresis. Among the multiple S. aureus types six clonal lineages dominated, comprising isolates from 158 patients. Despite the limited genetic variation, cross-transmission was made plausible only sporadically.     

Chemotherapie mit Cisplatin-Bleomycin und nachfolgende Strahlentherapie bei fortgeschrittenen Kopf-Hals-Tumoren

Summary 42 patients with advanced stage III and IV squamous cell carcinoma of the head and neck were treated with initial cisplatin and bleomycin chemotherapy and subsequent radiotherapy. 39 were evaluable for results, and 3 for toxicity only. 8 patients suffered from stage III and 31 from stage IV tumors, of these 10 with distant metastases. 5 patients underwent later a rescue operation. 27 were previously untreated (= group A) including 2 cases with localized relapses beyond the margins of surgical and/or radiotherapeutic treatment fields. 12 patients had a recurrence within pretreated areas (= group B). The induction chemotherapy alone showed the following results: In group A 4 (15%) CR, 10 (37%) PR, 7 (26%) MR; in group B 3 (25%) PR, 2 (17%) MR. The subsequent radiotherapy mostly consisted of a 65 Gy tumor dose given in 61/2 weeks. The results after completion of the combined modality therapy were: In group A 44% CR, 28% PR; in group B 10% CR and 30% PR. No patient resistant to the initial chemotherapy responded to the radiotherapy. The median survival time of stage III patients was 20 months but only 7 months in stage IV patients. 4 of all live with NED now between 30+ and 41+ months; 1 patient is alive with relapse. All the others are dead after a survival time of max. 32 months, included the 5 with rescue operation. In general, hematologic and renal toxicities were not severe, but nausea and vomiting were the worst tolerated side effects. 1 patient died from septic myocarditis having a WBC nadir of 2000/µl, probably due to additional immunologic deficiency because of inactive liver cirrhosis. The poor results concerning NED survival are discussed. We conclude, that an initial chemotherapy with CDDP and BLM gives good remission effects but its combination with radiotherapy at present fails to produce a NED survival benefit. Further efforts are necessary to improve the CR and the long time survivor rates.

Die Autoren danken folgenden Kollegen für ihre Mitarbeit: Drs. Garbea (HNO), B. Bienko, M. Danker, N. Birke, M. Klessing, K. Lottner, W. Palm     

The self antigen heme evades immune recognition by sequestration in some hemoproteins

Heme is a non-protein autoantigen which is ubiquitous in vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4⁺, class II-restricted T cells, freshly explanted from unprimed mice, to proliferate in vitro. In this study, we show that heme incorporated into various species of mammalian cytochrome c (cyt c), including murine cyt c, represents a facultative cryptic determinant, able to be recalled only at high doses of native cyt c. By contrast, avian cyt c is of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cyt c exhibited greatly increased antigenicity, comparable to that of heme and avian cyt c, indicating that the crypticity of heme in native mammalian cyt c is due to the resistance of the native conformation of this molecule to antigen processing within murine antigen-presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of hemereactive T cells. Given the high frequency of heme-reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory response.