Dermatology
PO Box 208030
New Haven, CT 06520-8030
Over the past decade, a sizable cluster of YSDRCC investigators with complementary areas of expertise have been involved in productive collaborative associations involving the pathogenesis and treatment of Lyme disease. Many of these studies of the mechanisms of spirochete persistence and pathogenesis in a mouse model of Lyme borreliosis were initiated by Dr. Stephen Barthold (Comparative Medicine), who was a YSDRCC investigator from 1993 until his very recent departure in July, 1998 for U.C. Davis. Dr. Barthold developed strikingly productive collaborative associations with a sizable number of investigators whose interests and expertise complemented and extended his own; YSDRCC investigators involved in these collaborations include Erol Fikrig, Ruth Montgomery and Stephen Malawista (Rheumatology), and Richard Flavell (Immunobiology). Mice, like humans and other mammalian species, develop multisystemic infections following intradermal syringe or tick-borne inoculation with B. burgdorferi. The ID(50) for intradermal inoculation is fewer than 10 spirochetes, whereas it is >100-fold higher via other routes, such as intraperitoneal inoculation. Following dissemination, disease evolves in joints and heart, but not other infected tissue (including skin), then undergoes immune-mediated resolution and periodic bouts of exacerbation over the course of persistent infection. At 1 year of infection, the skin is the most frequent site of spirochete persistence. Despite the discernible presence of extracellular spirochetes in the skin at 1 year, there is no inflammatory response to their presence. Nevertheless, transfer of skin biopsies from persistently infected mice to naive mice, or inoculation of naive mice with spirochetes cultured from persistently infected mice results in disseminated infection and disease. Their efforts are directed at clarifying the kinetics of spirochete populations in different tissues during different stages of infection and disease; identification of spirochete antigens that elicit host immunity; and manipulation of infection by immunization or tolerization against specific antigens. Their recent finding that spirochetes may express antigens differentially within the context of specific tissues, such as joints, may explain why inflammation occurs selectively in joints, but not other tissues, such as skin. Inoculation of skin with cultured spirochetes or their lipoproteins elicit strong local dermal inflammation, yet infection of skin by dissemination of host-adapted spirochetes does not.
The goal of research in the laboratory of Dr. Erol Fikrig is to use experimental models of Lyme borreliosis to characterize B. burgdorferi genes that are selectively induced in the infected host as well as the functional properties of these synthesized antigens, and to identify the antigens responsible for eliciting biologically active antibody responses in the infected host. These studies have involved active and very productive collaboration with Drs. Barthold and Flavell. Screening a B. burgdorferi genomic expression library with immune serum from low-dose infected mice, and from patients with Lyme disease, has resulted in identification of a number of clones containing genes of potential significance. Mouse bioassays for detecting protective and arthritis modulating activity in serum, and polyclonal antibodies against candidate proteins have been developed. These experiments are likely to define the antigens responsible for protective immunity generated by active infection and those that induce arthritis resolution. These antigens have the most potential for providing a therapeutic vaccine for actively infected individuals that circumvents the problem of OspA, which is not expressed in the infected host and therefore is not vulnerable to OspA vaccine- induced immunity.
Previous studies on borrelial antigens have been focused on those that are expressed in spirochetes found in culture; this in vitro environment is, in a sense, artifactual for it does not mimic the natural life cycle of Borrelia. Borrelia is normally found either in its vertebrate host or in the tick vector, and it is likely that changes in borrelial gene expression and surface protein synthesis accompany the adaptations that must occur as this organism migrates within the tick vector and adapts to life in the mammalian host. Dr. Fikrig and his collaborators have, therefore, recently followed an approach to identify and characterize those proteins and their genes which are selectively expressed in these different borrelial environments in order to understand the mechanisms of pathogenesis and to develop novel vaccine and diagnostic targets. In addition, they have devised a novel genetic system to identify Borrelia genes expressed in vivo. Their approach is to use differential screening with antibodies in which a Borrelial expression library is screened with two antisera, one generated against Borrelia in a given state, for example a tick, and a second generated against Borrelia after infection. Those colonies selectively detected by one or the other antisera provide a means of identifying those gene products which are expressed in only one or other of these two differential states. Using this approach, they identified the first two differentially expressed genes, p21, and the unpublished V1/V3 pair of gene products. They have gone on to screen borrelial libraries for genes which are specifically expressed in infected mice early after infection, and have identified a total of 5 gene clusters which are expressed in that time period. Two of these genes have been partially characterized (p35 and p37), and studies with these are ongoing. Their long term goals are: (a) To characterize genes expressed differentially in the tick and mammalian cell environment by DNA sequence, timing of expression, location of the genetic information (plasmid or chromosome), etc. (b) To test whether such molecules are protective against borrelial infection by either bactericidal action in the tick vector, or within the mammalian host; and (c) To test whether such agents are useful in diagnostic tests for Lyme disease. The possibility that borrelial proteins expressed specifically after infection of the mammalian host might be valuable diagnostics is intriguing, since they by definition provide the opportunity to diagnose infection rather than merely exposure to the organism. The output from these experiments should be new borrelial genes which should substantially advance their understanding of borrelial pathogenesis and provide new targets for commercial development.
Research in the laboratories of Dr. Stephen Malawista and Dr. Ruth Montgomery (Rheumatology) focuses on the interaction of phagocytes with the spirochetal agent of Lyme disease, Borrelia burgdorferi. Initial studies of spirochetes and macrophages in vitro showed that the spirochetes were ingested and killed easily and quickly. Spirochetes persist in skin at the site of introduction in mice and in human Lyme disease. Indeed, skin is believed to be the preferred reservoir of the spirochetes. Spirochetes in human skin (from 48 hours after inoculation for >10 days) are surrounded by an immune cell infiltrate, but they are sighted in the periphery of the rash giving the appearance that they are escaping from the lesion.
Drs. Montgomery and Malawista collaborated with Dr. Stephen Barthold (who has just left Yale for UC Davis) in a P/F study funded during the YSDRCC's 03-04 years which investigated the interaction of macrophages and spirochetes in the skin, the portal of entry for Lyme infection; these studies, which involved immunofluorescent light microscopy, confocal microscopy and immunoEM, as well as quantitative biochemical and molecular methods (including RTPCR), made productive use of the Tissue Acquisition and Analysis Core, as well as the (now disbanded) Molecular Analysis Core. The data obtained during the course of this P/F study enabled them to obtain alternative independent funding from the Arthritis Foundation for studies which are focusing on the antigenic substitution of the spirochete in the skin and phagocyte function there.
Their in vitro data show that macrophages readily ingest and kill unopsonized spirochetes in large numbers; thus, it is unclear how spirochetes avoid the initial immune response in vivo and survive to disseminate. Several projects are ongoing which study the failure of phagocytes to clear the spirochetes. The in vivo persistence of spirochetes leads to the question of whether phagocytes are entirely functional or can be compromised by the infectious agent, its products, or, in the initiation of infection in the skin, by the delivery vehicle, tick saliva, which has been shown to downregulate phagocyte function.
Dr. Montgomery has developed new double-label immunofluorescent techniques in skin that establish our ability to stain both spirochetes and macrophages in situ. She has succeeded in staining spirochetes in mouse skin at the site of inoculation. Her double label confocal analysis of infected skin demonstrate the feasibility of staining in this tissue and preliminary calculations of the abundance of spirochetes and macrophages responding to them. With such methodology firmly in hand, Dr. Montgomery is positioned to examine thoroughly the interaction of spirochetes and phagocytes in the skin.
A major focus of Dr. Debra Bessen's research is to understand the basis for tissue tropism by group A streptococci. Impetigo and pharyngitis are the most common diseases caused by this bacterium, and the throat and skin serve as the two principal reservoirs. In collaboration with Dr. S. Hollingshead (University of Alabama at Birmingham), her studies have identified genetic markers which distinguish "skin" and "throat" strains of group A streptococci. The genetic markers encode for emm genes, which give rise to surface fibrils that play a major role in virulence. The identification of genetic markers for principal tissue reservoir has lead to numerous ongoing studies on the molecular epidemiology and evolution of this bacterial species. In collaboration with investigators at the Menzies School of Health Research (Northern Territory, Australia), the differences between streptococcal disease in tropical and non-tropical regions are being studied at a molecular level.
In a P/F study funded during the 04 year of this Center and involving the collaboration of Dr. Jennifer (Madison) McNiff and the services of the Tissue Acquisition and Analysis Core, Dr. Bessen investigated several animal models for impetigo and pharyngeal colonization with the goal of defining the pathogenic mechanisms that define each disease. These pilot studies facilitated Dr. Bessen receiving support from the American Heart Association to continue this line of investigation, one major focus of which involves use of immunodeficient scid mice grafted with human skin. These studies involve utilization of the YSDRCC's SCID Mouse: Skin Xenograft Core Superficial damage to engrafted human skin, followed by application of certain strains of streptococci, leads to an inflammatory response on a microscopic level which closely parallels that observed for human impetigo. In collaboration with investigators at University of Lund (Sweden), the role of high-affinity plasminogen binding by streptococcal M proteins in impetigo is being explored.
In other studies, Dr. Bessen has been evaluating the role of group A streptococcal virulence factors in the initiation of post-streptococcal autoimmune sequelae; some of these efforts have been directed towards guttate psoriasis.
Dr. Janet Brandsma's laboratory (Comparative Medicine) is devoted to the study of papillomavirus pathogenesis and immunity and is utilizing several animal models to investigate the genetic determinants of papilloma formation and malignant progression. In a P/F study funded as a part of this Center's original competitive application in 1992, Dr. Brandsma collaborated with Dr. Daniel DiMaio (Genetics) on a study of the use of particle bombardment ("gene gun") inoculation of infectious bovine papillomavirus DNA in calves; this project enabled Dr. Brandsma to test the infectivity of a molecular clone of BPV in vivo.
High risk human papillomaviruses (HPVs) induce benign epithelial proliferative growths with the potential to progress to cervical, penile and anal cancer, other types of HPV are also associated with skin cancer in epidermodysplasia verruciformis, and studies carried out in collaboration with Jack Longley prior to his departure to Columbia identified a novel association between type 73 human papillomavirus and esophageal squamous carcinoma.. She also has been involved in a productive collaboration with Dr. Jordan Pober (Scid Mouse:Skin Xenograft Core) to generate suitable animal hosts by engrafting human skin, including foreskin, onto these immunodeficient mice; the grafts are inoculated with recombinant HPV genomes and monitored for clinical, histologic and molecular effects. Future experiments will determine the effects of individual viral gene functions, by infecting grafts with HPV genomes engineered to contain mutations in viral genes whose functions have been well characterized in vitro.
Dr. Brandsma is also developing strategies for human vaccination to HPV using the well characterized Shope or cottontail rabbit papillomavirus (CRPV)-rabbit model. This system is being used to characterize malignant progression, with the goal of better understanding its molecular basis. Genetically tagged papilloma stemlines will be followed prospectively and analyzed retrospectively to determine viral properties associated with malignant progression. The natural history of disease will be further characterized to determine the effects of CRPV on genetic stability and on cellular responses to ultraviolet B-irradiation, a potential carcinogenic co-factor. Other studies, which involve the active collaboration of Dr. Robert Tigelaar, are using mammalian expression vectors encoding individual CRPV genes inoculated intracutaneously using an Agracetus "gene gun", and rabbits are monitored for humoral immunity, cell-mediated immunity, and protection against challenge with CRPV virus. Future studies will determine if vaccines that induce prophylactic immunity can have immunotherapeutic effects and if cell-mediated immunity can be enhanced by co-inoculation of cytokines genes, e.g. encoding IL-12 or GM-CSF.
The major area of investigation of the laboratory of Dr. Daniel DiMaio is elucidating the mechanisms of altered growth control in cells infected by the papillomaviruses, where gene transfer techniques are being used to assay the activity of viral mutants and individual viral genes in cultured mesenchymal, hematopoietic, and epithelial cells. As noted above, Dr. DiMaio was a co-investigator with Dr. Brandsma on one of the original P/F studies funded by the YSDRCC. His research is summarized more completely in the Keratinocyte Biology cluster (see above).
Dr. Peter Hotez (Pediatrics) and Dr. Michael Cappello (Pediatrics) have major research interests in the molecular pathobiology of hookworm infections, a major cause of anemia and malnutrition in the developing countries of the tropics. The major goals of their research are to understand the molecular mechanisms of invasion and development by infective hookworm larvae and to use the information to genetically engineer vaccine candidates for hookworm infection. A productive collaboration involving Dr. Hotez and Drs. Len Milstone and John Haggerty (Keratinocyte Biology cluster) demonstrated the presence of substantial amounts of hyaluronidase activity in hookworm larvae of the genus Ancylostoma, and particularly in A. braziliense, the major etiologic agent of cutaneous larva migrans, strongly suggesting this enzyme's function as a virulence factor in tissue invasion and in cutaneous larva migrans. Five years ago, Dr. Cappello was a postdoctoral fellow supported by the Dermatology Department's Research Fellowship Training Grant and working under the mentorship of Drs. Milstone and Hotez; a subsequent publication showed that hyaluronidases were also virulence factors for the gastrointestinal invasive stages of two other nematodes. Subsequently Drs. Cappello and Hotez identified the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss, an Ancylostoma canium anticoagulant peptide (AcAP) which acts as a specific inhibitor of factor Xa, which they then cloned and expressed. In other studies being carried out at the same time, Dr. Michael Bromberg (Hematology, Division, see Melanocyte/Melanoma cluster) in collaboration with Drs. Jennifer Madison (Dermatology) and Alan Garen (MB&B), showed that tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation. Drs. Cappello and Bromberg recently initiated a collaborative study which merged their independent observations into a preliminary study which showed that recombinant AcAP completely protected against pulmonary metastases following tail vein injection of a human melanoma cell line into scid mice; these observations formed the basis for a P/F study funded in this Center's 05 year; see Melanocyte/Melanoma cluster for additional details.
In addition, Dr. Hotez and Dr. Cappello are pursuing recombinant vaccine development against human hookworm infection, using a new class of hookworm secreted proteins known as the ASP family. Both ASP-1 and ASP-2 have predicted amino acid homologies to the major venom allergens from Hymenopteran venoms, suggesting that they have a role in the pathobiology and immunobiology of hookworm infection. ASP-1 is the major hookworm protein released by the parasite upon host entry. Recent studies in mice suggest that rASP-1 offers promise as a protective antigen. Of interest is the finding that ASP-1 localizes to the parasite amphidial glands associated with the major chemosensory organ of the parasite. This suggests a novel mechanism by which anti-ASP-1 antibodies directly interfere with parasite migration after host entry.
The focus of the research in Dr. Diane McMahon-Pratt's laboratory is the genus of parasitic protozoan, Leishmania, specifically in the New World species L. amazonensis and L. braziliensis, the causative agents of cutaneous, diffuse cutaneous and mucocutaneous leishmaniasis. Using biochemical and molecular genetic approaches, they have been investigating molecules that are developmentally regulated by the parasite during its life cycle [vectormammalian host]; these molecules should provide clues as to how the parasite survives and/or manipulates its environment within either the insect vector or mammalian host. They are also interested in understanding and elucidating the immune effector mechanisms involved in the control of infection by the mammalian host. Leishmania are particularly intriguing organisms, in that within the mammalian host, the parasites reside and multiply within the phagolysosome of the macrophage, a key immunoregulatory cell. Immunologic approaches have involved both the use of immunohistopathology, cellular reconstitution, and genetically modified ("knock-out") mice as well as vaccine studies employing molecules specifically expressed either by the promastigote [insect] or amastigote [mammalian host] life cycle stage. These studies, which have involved productive collaboration with YSDRCC investigators Richard Flavell, Charles Janeway, and Jack Longley (now at Columbia) indicate the critical importance of CD40L-CD40 interactions, MHC class II molecules, as well as both CD4+ and CD8+ T cells in the control of cutaneous infection. These studies indicate that Th1-like CD4+ T cells are responsible for the maintenance of L. amazonensis; these results differ from those found for L. major and suggest that these two cutaneous organisms have evolved different strategies for survival in the mammalian host. Studies of endogenous antigen presentation, indicate that leishmanial parasites residing within the phagolysosome specifically downregulate MHC class II presentation and access the MHC class I pathway; they are currently investigating the cell biological mechanisms involved and continuing to explore the mechanisms of CD8+ T cell control of cutaneous infection in vaccinated/immunized mice. In addition, recent work has developed a murine model for visceral leishmaniasis that is initiated through intradermal injection. This model mimics the situation found in nature, where the disease is transmitted by the bite of the phlebotomine sandfly. Interestingly, the parasite is eliminated at the site of cutaneous infection and liver, yet persists (increases) in both the draining lymph node and spleen. Comparative studies are now in progress to elucidate the immunological mechanisms responsible for this site-specific immunity. In collaborative studies with colleagues in Brazil, they are investigating the human immune response to leishmanial antigens found to be protective in the murine model for cutaneous leishmaniasis.
Dr. Eric Pamer (Infectious Diseases) has become widely recognized for his pioneering studies of the antigens recognized by cytotoxic T cells generated by the infection of mouse macrophages with the intracellular bacterial pathogen, Listeria monocytogenes. His laboratory is investigating the processing and presentation of Listeria antigens to cytolytic T lymphocytes, and have focused their efforts on understanding the degradation of proteins into peptides in the cytosol of cells and the subsequent transport of these peptides into the endoplasmic reticulum for association with newly synthesized MHC class I molecules. His previous work, which has involved isolating and sequencing Listeria peptides eluted from MHC class I molecules from infected macrophages and which utilizes a series of CD8+ T cell lines against these peptides to monitor their production, has indicated that the cytolytic T lymphocyte response to this pathogen involves the recognition of at least several different bacterial peptides. However, the immune response is dominated by T cells with specificity for one particular epitope. The basis for the immunodominance of this particular epitope was poorly understood and Dr. Pamer's P/F project in this Center's 05 year (in collaboration with Dr. Charles Janeway) tested the hypothesis that immunodominant epitopes are recognized by a large number of different T cells, each with a distinctive different TCR, while subdominant epitopes are detected by a smaller number of T cells with a more restricted range of TCRs. The strategy ultimately chosen involved use of MHC class I tetramers complexed with Listeria-derived peptides. The data obtained was consistent with this hypothesis and was instrumental in Dr. Pamer's very recent receipt of a new R01 grant to further test this hypothesis. The realization that many of the methods and strategies Dr. Pamer has used to so successfully investigate T cell responses to Listeria are readily applicable to analyses of the T cell responses to as yet uncharacterized antigens CTCL, led to his continuing collaborative association with several investigators in that interest group, particularly Drs. Berger, Edelson, and Imaeda in their studies of T cell responses to MHC I-associated T cell receptor peptides in both human CTCL and in murine models of T cell lymphoma.
©
Yale School of Medicine
Last updated
4/21/08
Privacy policy Site editor
