Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States.

OBJECTIVE: To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. DESIGN: Prospective study. ANIMALS: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. PROCEDURE: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: Of the 82 serum samples tested, 37 (45.1%) and 13 (15.9%) had detectable antibodies to B burgdorferi or E equi, respectively. Test results indicated that 12 horses had been exposed to both agents, 11 of these horses had granulocytic ehrlichiosis. The ELISA regularly detected antibodies to the following recombinant protein (p) antigens of B burgdorferi: p29, p37, p39, and p41-G. The use of immunoblot analysis confirmed ELISA results by indicating antibody reactivities to antigens of whole-cell B burgdorferi having molecular masses of predominantly 31, 34, 37, 39, 41, 58, and 93 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Horses living in areas where ticks (Ixodes scapularis) abound are sometimes exposed to multiple pathogens. Analyses for specific recombinant borrelial antibodies using an ELISA can help separate horses with borreliosis from those with granulocytic ehrlichiosis, even when antibodies to both etiologic agents are detected in serum samples. Analysis using immunoblots is sensitive, and along with ELISA or IFA procedures, is suitable for confirming a clinical diagnosis of each disease.

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens.

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.

The nuclear receptor PPAR gamma and immunoregulation: PPAR gamma mediates inhibition of helper T cell responses.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR gamma has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR gamma ligand, 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR gamma 1. The PPAR gamma expressed is of functional significance in that two ligands for PPAR gamma, 15d-PGJ2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC. Finally, we demonstrate that the two ligands for PPAR gamma mediate inhibition of IL-2 secretion by the T cell clones while not inhibiting IL-2-induced proliferation of such clones. The demonstration of the expression and function of PPAR gamma in T cells reveals a new level of immunoregulatory control for PPARs and significantly increases the role and importance of PPAR gamma in immunoregulation.

Human exposure to a granulocytic Ehrlichia and other tick-borne agents in Connecticut.

Indirect fluorescent-antibody (IFA) staining methods with Ehrlichia equi (MRK or BDS strains) and Western blot analyses containing a human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain) were used to confirm probable human cases of infection in Connecticut during 1995 and 1996. Also included were other tests for Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), Babesia microti, and Borrelia burgdorferi. Thirty-three (8.8%) of 375 patients who had fever accompanied by marked leukopenia or thrombocytopenia were serologically confirmed as having HGE. Western blot analyses of a subset of positive sera confirmed the results of the IFA staining methods for 15 (78.9%) of 19 seropositive specimens obtained from different persons. There was frequent detection of antibodies to a 44-kDa protein of the HGE agent. Serologic testing also revealed possible cases of Lyme borreliosis (n = 142), babesiosis (n = 41), and HME (n = 21). Forty-seven (26.1%) of 180 patients had antibodies to two or more tick-borne agents. Therefore, when one of these diseases is clinically suspected or diagnosed, clinicians should consider the possibility of other current or past tick-borne infections.

Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis.

Use of recombinant antigens of Borrelia burgdorferi in serologic tests for diagnosis of lyme borreliosis.

Recombinant antigens of outer surface proteins (Osps) OspA, OspB, OspC, OspE, and OspF of Borrelia burgdorferi sensu stricto and of p41-G, an antigenic region of flagellin of this spirochete, were tested with human sera in class-specific and polyvalent enzyme-linked immunosorbent assays (ELISAs). In analyses for immunoglobulin M (IgM) antibodies, 18 (85.7%) of 21 serum samples from persons who had been diagnosed as having Lyme borreliosis on the basis of the presence of erythema migrans reacted positively in ELISAs with one or more Osp antigens or the p41-G antigen. Eleven serum samples contained antibodies to OspC antigen, and of these, six also reacted to the p41-G antigen and to one or more of the other recombinant antigens. The remaining five serum samples reacted solely to OspC (n = 4) or to OspC plus OspA and OspE without reactivity to p41-G (n = 1). In analyses for IgG antibodies, seropositivity was comparable to that of IgM analyses and was marked by predominant reactivity to p41-G, OspC, and OspF. Similarly, all 21 serum samples were positive in polyvalent and class-specific ELISAs with whole-cell B. burgdorferi. Minor cross-reactivity was noted when sera from persons who had syphilis, periodontitis or other oral infections, or rheumatoid arthritis were tested with OspC, OspE, OspF, and p41-G. With relatively high degrees of specificity, ELISAs with recombinant antigens, particularly OspC and p41-G, can help to confirm B. burgdorferi infections.

Presentation of synthetic peptide antigen encoded by the MAGE-1 gene by granulocyte/macrophage-colony-stimulating-factor-cultured macrophages from HLA-A1 melanoma patients.

The recent identification of the sequences of the peptides derived from a number of human melanoma-associated antigens has presented opportunities for developing a specific-peptide-based vaccine in this form of cancer. Since antigen-presenting cells (APC) play a crucial role in the induction of the T-cell-mediated immune response, we examined whether or not ex vivo cultured APC, bearing the appropriate MHC restricting elements, when pulsed with a relevant melanoma-specific cytotoxic-T-lymphocyte (CTL)-determined peptide, can present the peptide to the CTL. Here we show that a population of cells, derived from the monocyte/macrophage lineage from peripheral blood and grown in granulocyte/macrophage-colony-stimulating factor, exhibit many essential characteristics of "professional" APC (dendritic-type morphology with a proportion of the population, the B7 molecule, and high levels of MHC class I and class II molecules, CD11b and CD54 molecules) and are capable of efficiently presenting the nonapeptide, EADPTGHSY, encoded by the melanoma antigen MAGE-1 gene, to the MAGE-1-specific CTL clone, 82/30. These results suggest that this type of autologous ex vivo cultured population of professional APC, when pulsed with the relevant-CTL-determined peptide, can serve as a novel type of candidate vaccine for active specific immunization against HLA-A1-positive patients with melanoma expressing the MAGE-1 antigen.

Recombinant outer surface protein C ELISA for the diagnosis of early Lyme disease.

To compare the sensitivity of a new ELISA for IgM antibodies to Borrelia burgdorferi that uses a recombinant outer surface protein C (rOspC) with those of a whole cell (WC) ELISA and an immunoblot assay for the diagnosis of early Lyme disease, serum specimens from 82 consecutive patients with physician-documented erythema migrans were analyzed. To compare the specificities of the three assays, serum specimens from 50 patients without a history of Lyme disease and from an area in which B. burgdorferi is not endemic were analyzed. The sensitivities of the WC ELISA, immunoblot assay, and IgM rOspC ELISA were 28%, 29%, and 46%, respectively, while the specificities were 100%, 100%, and 98%, respectively. The IgM rOspC ELISA is a convenient, readily automated, easily standardized serologic test that is significantly more sensitive for the diagnosis of early Lyme disease than either WC ELISA or immunoblot assay.

Increased collagen synthesis in skin fibroblasts from patients with primary hypertrophic osteoarthropathy. Evidence for trans-activational regulation of collagen transcription.

OBJECTIVE: To investigate collagen synthesis in skin fibroblasts from patients with primary hypertrophic osteoarthropathy (HOA), a disorder characterized clinically by skin thickening. METHODS: Collagenase-digestible protein, messenger RNA (mRNA) levels, and transcriptional activity of the alpha 1(I) procollagen gene were assessed in skin-derived fibroblast lines. RESULTS: Compared with fibroblasts from uninvolved skin, fibroblasts from involved skin had elevated levels of collagen synthesis and alpha 1(I) procollagen mRNA, and increased transcriptional activity of the alpha 1(I) procollagen promoter. CONCLUSION: Abnormalities of collagen synthesis in fibroblasts from patients with primary HOA can be accounted for, at least in part, by a trans-activated up-regulation of collagen transcription.

Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease.

Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC). We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S. J. Padula, A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan, Infect. Immun. 61:5097-5105, 1993). Now we have compared the detection of B. burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA. Seventy-six negative controls were also studied. With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result. With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74%. Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B. burgdorferi which expresses abundant levels of OspC. We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.

Molecular characterization and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi.

We have found that sera from patients with early stages of Lyme disease contain predominant immunoglobulin M reactivity to a major 23-kDa protein (p23) from Borrelia burgdorferi 2591 isolated in Connecticut. To characterize this immunodominant antigen, we cloned and sequenced p23 and found it to be 83% identical by nucleotide sequence and 75% identical by amino acid sequenced to pC (recently renamed OspC), an abundantly expressed protein on the outer surface of PKo, a European strain of B. burgdorferi (B. Wilske, V. Preac-Mursic, S. Jauris, A. Hofmann, I. Pradel, E. Soutschek, E.Schwab, G. Will, and G. Wanner, Infect. Immun. 61:2182-2191, 1993). In addition, immunoelectron microscopy localized p23 to the outer membrane, confirming that p23 is the strain 2591 homolog of OspC. The North American strain B31, commonly used in serologic assays for Lyme disease, does not express OspC. Northern (RNA) blot analysis detected low levels of ospC mRNA in B31, and DNA sequencing of the ospC gene from B31 revealed a 54-bp deletion in the upstream regulatory region, possibly accounting for the low transcriptional activity of ospC. The ospC coding region from B31 was cloned and antibody-reactive OspC was expressed in Escherichia coli. An immunoglobulin M enzyme-linked immunosorbent assay using recombinant OspC as the target antigen shows promise for the serodiagnosis of early stages of Lyme disease.

Inhibition of interleukin-2 synthesis and interleukin-2 receptor alpha expression on T cells by a cell-free factor derived from a CD4+ regulatory T cell clone.

We have shown that certain CD4+ T cell lines can function as suppressor cells in a cell culture system. In this context, the CD4+ T cells (AS-9) cloned from the peripheral blood lymphocytes (PBL) of a melanoma patient are capable of suppressing the induction of cytolytic response in autologous PBL in coculture. Here we show that a trypsin-sensitive cell-free culture supernatant factor from the AS-9 cells, AS-9 SF, interferes with IL-2 synthesis by T cells when they are stimulated. AS-9 SF also selectively blocks the expression of interleukin-2 receptor alpha (IL-2R alpha) on T cells during activation. Expression of transferrin receptors and the CD3 molecules is not down-regulated by this factor. The AS-9 SF consequently blocks proliferation of T cells when they are stimulated by lectin or activated through the T cell receptors. AS-9 SF suppresses the IL-2R alpha induction and the T cell proliferation at the induction phase only because it has no suppressive effect on preactivated T cells. Interleukin-2, IL-2R alpha, and beta messages are not down-regulated by the AS-9 SF and the suppressive effect of the AS-9 SF on IL-2R alpha expression and on T cell proliferation is not neutralized by the addition of exogenous recombinant IL-2. The factor does not appear to be IL-4, IL-10, or TGF-beta, three known cytokines possessing regulatory properties on T cell activation.

Analysis of cytolytic effector cell response in vitro against autologous human tumor cells genetically altered to synthesize interleukin-2.

Recently, there has been a surge of interest in gene therapy in cancer particularly with cytokine transduced tumor cells as a novel form of tumor vaccine. In two autologous human tumor systems, using the tumor cells engineered to produce interleukin-2 by gene transduction techniques, we have examined whether or not such genetically altered cells are capable of inducing a tumor specific cytolytic T cell (CTL) response, in vitro, in co-culture with the respective autologous peripheral blood lymphocytes (PBL). We found that in neither system did co-cultures of the IL-2 producing tumor cells and the autologous PBL generate much cytolytic effector cell activity directed against the respective tumor cells, although these co-cultures did lead to the generation of substantial levels of natural killer (NK) cell activity when measured against the prototype NK sensitive target K562 line. More surprisingly, the levels of lymphokine activated killer cell responses against the respective autologous targets that could be generated in the PBL with exogenous IL-2 alone were compromised by the presence of the autologous tumor cells in the co-culture.

Adoptive transfer of activated human autologous macrophages results in regression of transplanted human melanoma cells in SCID mice.

The potential anti-tumor activity of human macrophages, grown in macrophage colony stimulating factor (M-CSF), was examined in mice homozygous for the mutation severe combined immune deficiency (scid) bearing xenografts of autologous human melanoma. Injection of scid mice, bearing subcutaneous melanoma xenografts, with the cultured macrophages or with the macrophage culture supernatant, once or repeatedly, resulted in partial to complete regression of tumors. Since a large number of such macrophages (greater than 1 x 10(9)) could be grown in vitro for repeated injection, the scid-human chimera can serve as an in vivo model to examine the role of human macrophages in tumor immunity and to explore the potential of the in vitro cultured macrophages in the therapy of cancer.

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.

An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.

Effects of H-7 are not exclusively mediated through protein kinase C or the cyclic nucleotide-dependent kinases.

Culturing murine T cell tumor lines in the presence of the protein kinase inhibitor H-7 for 4 days led to their dependence on H-7 for maximal constitutive proliferation. Withdrawal of H-7 from H-7-conditioned cells led to inhibition of proliferation and cell death. The mechanism underlying this H-7 dependence does not appear to be related to clonal selection or to effects on protein kinase C or the cyclic nucleotide-dependent kinases. This suggests that all the effects of the widely used H-7 may not be completely understood, and that H-7 may be useful in the dissection of the complex patterns of growth regulation in T cell malignancies.

Subtypes of helper cells. Non-inflammatory type 1 helper T cells.

Class II MHC-restricted T cells recently have been characterized as being either type 1 (Th1) or type 2 (Th2) based on their ability to both secrete different lymphokines and perform different functions. Characterization of these subtypes to date have indicated that Th1 cells secrete IL-2, IFN-gamma, lymphotoxin, and IL-3, whereas Th2 cells secrete IL-4, IL-5, and IL-3. Functionally, Th1 cells mediated cytotoxicity and delayed-type hypersensitivity, and have been termed "inflammatory cells," whereas Th2 cells mediate helper function for Ig secretion and have been termed, "regulatory cells." We now present evidence that not all Th1 clones are inflammatory and capable of mediating cutaneous delayed-type hypersensitivity. We have generated a number of myelin basic protein-specific Th1 clones that do not mediate swelling when injected together with myelin basic protein directly into the footpads of syngeneic mice. These results suggest that Th1 cells can be further subdivided based on their ability to mediate delayed-type hypersensitivity, and that the Th1/Th2 characterization of Th cells may be insufficient to adequately characterize all functional subtypes of class II MHC-restricted T cells.

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.