Differential effects of intranasal vaccination with recombinant NcPDI in different mouse models of Neospora caninum infection

In this study, mice were vaccinated intranasally with recombinant N. caninum protein disulphide isomerase (NcPDI) emulsified in cholera toxin (CT) or cholera toxin subunit B (CTB) from Vibrio cholerae. The effects of vaccination were assessed in the murine nonpregnant model and the foetal infection model, respectively. In the nonpregnant mice, previous results were confirmed, in that intranasal vaccination with recNcPDI in CT was highly protective, and low cerebral parasite loads were noted upon real-time PCR analysis. Protection was accompanied by an IgG1-biased anti-NcPDI response upon infection and significantly increased expression of Th2 (IL-4/IL-10) and IL-17 transcripts in spleen compared with corresponding values in mice treated with CT only. However, vaccination with recNcPDI in CT did not induce significant protection in dams and their offspring. In the dams, increased splenic Th1 (IFN-γ/IL-12) and Th17 mRNA expressions was detected. No protection was noted in the groups vaccinated with recNcPDI emulsified in CTB. Thus, vaccination with recNcPDI in CT in nonpregnant mice followed by challenge infection induced a protective Th2-biased immune response, while in the pregnant mouse model, the same vaccine formulation resulted in a Th1-biased inflammatory response and failed to protect dams and their progeny.

AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^

Mechanisms of UV induced systemic immunosuppression: An essential role for interleukin-10

The recognition of the skin as an immunocompetent organ has focused attention on the complex interaction between ultraviolet radiation and the immune system. How UV-radiation, which hardly penetrates past the epidermis, induces systemic immune suppression is not entirely clear. We propose that suppressive cytokines, released by UV-irradiated keratinocytes, play a role in the induction of immune suppression. Injecting supernatants from UV-exposed murine keratinocytes into mice impairs their ability to mount a delayed-type hypersensitivity response against allogeneic histocompatibility antigens. We tested the hypothesis that the down regulation of the immune response by UV is precipitated by the release of IL-10 after keratinocytes are UV-irradiated. After UV exposure IL-10 mRNA was upregulated. Western analysis indicated immunoreactive IL-10 was secreted by UV-exposed keratinocytes. The addition of supernatants from UV-irradiated keratinocytes to Th1 clones diminished their IFN production, whereas the addition of supernatants from normal keratinocytes had no suppressive effect on IFN production. Furthermore, treating supernatants from UV-irradiated keratinocytes with anti-IL-10 antibodies blocked the induction of immune suppression. To determine if IL-10 was responsible for the immunosuppression seen after total-body UV irradiation, UV-exposed mice were treated with anti-IL-10 antibodies. Treating UV-irradiated mice with anti-IL-10 reversed the induction of immune suppression. These findings suggest that keratinocyte-derived IL-10 was mediating UV-induced suppression in vivo. We also tested the hypothesis that UV-induced suppressor cells are Th2 cells. Mice were injected with spleen cells from either normal or UV-exposed donor mice immunized with alloantigen. At the time of spleen cell infusion, the recipient mice were then resensitized. Spleen cells from UV-exposed mice suppressed DTH. Mice treated identically and injected with anti-IL-10 antibodies were able to generate a DTH response. Taken together these data suggest that the suppressor cells that are induced by UV radiation are Th2 cells which mediate their suppressive effect by release of IL-10. ^

Radiolabeled human monoclonal IgM for intracompartmental cancer therapy

Radioimmunotherapy (RIT) with i.v. administered radiolabeled IgG can selectively irradiate tumor cells in vivo. However, it only provides effective therapy for lymphomas. Intracompartmental RIT with radiolabeled human monoclonal IgM may allow curative treatment of solid tumors by increasing tumor deposition of radioactivity, reducing systemic toxicity and allowing repeated administration. This hypothesis was tested in nude mouse models with IgM radiolabeled with indium-111 $\rm(\sp{111}In)$ or yttrium-90 $\rm(\sp{90}Y).$ The use of two radioisotopes, $\rm\sp{111}In$ for imaging and $\rm\sp{90}Y$ for therapy, allow for more quantitative and cautious development of RIT.^ Radiolabled 2B12, an IgM reactive with human ovarian carcinomas was tested by i.v. and intraperitoneal (i.p.) administration in nude mice bearing i.p. nodules of a human ovarian carcinoma cell line (SKOV3 NMP2). Radiolabeled CR4E8, an IgM reactive with human squamous cell carcinomas was tested by i.v. and intralesional (i.l.) administration in nude mice bearing subcutaneous tumors of a human head and neck squamous cell carcinoma cell line (886). These two models were selected to test proof of concept. Radiolabeled irrelevant IgM (CH-1B9), and $\rm\sp{90}Y$-aggregate served as specificity controls. Biodistribution was performed by excising, weighing and then measuring the radioactivity of tumor and normal organs. Therapy was conducted with i.p. $\rm\sp{90}Y$-labeled 2B12 using both single and fractionated administration and with i.l. $\rm\sp{90}Y$-labeled CR4E8 using single administration. Mice were monitored for tumor response, survival and systemic toxicity.^ Intracompartmental administration of radiolabeled IgM produced immediate high and prolonged tumor deposition of radioactivity with low normal tissue uptake. In contrast, i.v. administration resulted in low tumor, but high liver and spleen uptake. Similar biodistributions were demonstrated for $\rm\sp{111}In$- and $\rm\sp{90}Y$-labeled IgM. Intraperitoneal therapy with $\rm\sp{90}Y$-labeled 2B12 increased survival by approximately 12 days for every 100 $\rm\mu Ci$ of activity without significant toxicity for single (0-300 $\rm\mu Ci)$ and fractionated (150-510 $\rm\mu Ci)$ administration. Intralesional therapy with $\rm\sp{90}Y$-labeled CR4E8 (150-400 $\rm\mu Ci)$ induced prolonged complete regressions. Significant local or systemic toxicity was not observed.^ Intracompartmental RIT with radiolabeled tumor-reactive human monoclonal IgM can selectively irradiate tumor cells. Intracompartmental radiolabled IgM can significantly extend the survival of treated mice with minimal toxicity. It deserves further development as a new cancer therapy. ^

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

The immunology of the porcine skin and its value as a model for human skin.

The porcine skin has striking similarities to the human skin in terms of general structure, thickness, hair follicle content, pigmentation, collagen and lipid composition. This has been the basis for numerous studies using the pig as a model for wound healing, transdermal delivery, dermal toxicology, radiation and UVB effects. Considering that the skin also represents an immune organ of utmost importance for health, immune cells present in the skin of the pig will be reviewed. The focus of this review is on dendritic cells, which play a central role in the skin immune system as they serve as sentinels in the skin, which offers a large surface area exposed to the environment. Based on a literature review and original data we propose a classification of porcine dendritic cell subsets in the skin corresponding to the subsets described in the human skin. The equivalent of the human CD141(+) DC subset is CD1a(-)CD4(-)CD172a(-)CADM1(high), that of the CD1c(+) subset is CD1a(+)CD4(-)CD172a(+)CADM1(+/low), and porcine plasmacytoid dendritic cells are CD1a(-)CD4(+)CD172a(+)CADM1(-). CD209 and CD14 could represent markers of inflammatory monocyte-derived cells, either dendritic cells or macrophages. Future studies for example using transriptomic analysis of sorted populations are required to confirm the identity of these cells.

The teleostean liver as an immunological organ: Intrahepatic immune cells (IHICs) in healthy and benzo[a]pyrene challenged rainbow trout (Oncorhynchus mykiss).

The existence of a resident population of intrahepatic immune cells (IHICs) is well documented for mammalian vertebrates, however, it is uncertain whether IHICs are present in the liver of teleostean fish. In the present study we investigated whether trout liver contains an IHIC population, and if so, what the relative cellular composition of this population is. The results provide clear evidence for the existence of an IHIC population in trout liver, which constitutes 15-29% of the non-hepatocytes in the liver, and with a cellular composition different to that of the blood leukocyte population. We also analyzed the response of IHICs to a non-infectious liver challenge with the hepatotoxic and immunotoxic chemical, benzo[a]pyrene (BaP). Juvenile trout were treated with BaP (25 or 100mg/kgbw) at levels sufficient to induce the molecular pathway of BaP metabolism while not causing pathological and inflammatory liver changes. The IHIC population responded to the BaP treatments in a way that differed from the responses of the leukocyte populations in trout blood and spleen, suggesting that IHICs are an independently regulated immune cell population.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and β (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

Imaging of the spleen in malaria

Splenomegaly, albeit variably, is a hallmark of malaria; yet, the role of the spleen in Plasmodium infections remains vastly unknown. The implementation of imaging to study the spleen is rapidly advancing our knowledge of this so-called "blackbox" of the abdominal cavity. Not only has ex vivo imaging revealed the complex functional compartmentalization of the organ and immune effector cells, but it has also allowed the observation of major structural remodeling during infections. In vivo imaging, on the other hand, has allowed quantitative measurements of the dynamic passage of the parasite at spatial and temporal resolution. Here, we review imaging techniques used for studying the malarious spleen, from optical microscopy to in vivo imaging, and discuss the bright perspectives of evolving technologies in our present understanding of the role of this organ in infections caused by Plasmodium.

IN VITRO INDUCTION OF XENOIMMUNE RESPONSES TO LIPOSOMES CONTAINING HUMAN COLON TUMOR ANTIGENS

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl(,2) to a dried lipid film. Cytoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposomes also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposomes were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens.^ The adjuvant effect of liposomes was shown to be mediated primarily by tumor antigens exposed on the outer surface of liposomes. Trypsinization of the liposomes which eliminated 96% of the surface protein reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes of increasing protein concentration showed that while 10 (mu)g protein was threshold, 100 (mu)g protein induced maximal responses. In addition, membrane fluidity studies revealed that rigid liposomes were significantly (P < 0.05) more efficacious than fluid liposomes in inducing cytotoxicity.^ The induction of the primary response required the presence of nonadherent splenocytes bearing the Thy-1, Lyt-1, and Lyt-2 surface markers. The role of a Lyt-123 subpopulation was suggested by the inability of both the Lyt-1 and Lyt-2 depleted populations to completely restore the cytolytic levels to normal. In addition, the interaction of I-A('+) spleen adherent cells with liposomes for at least 8 hours was required to generate maximal cytotoxic activity. The phenotype of the cytotoxic effector was Thy-1('+), Lyt-2('+), and I-A('d-).^ Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select specific immune responses, as well as to identify colon tumor-specific determinants.^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^