Major histocompatibility complex class II-restricted presentation of an internally synthesized antigen displays cell-type variability and segregates from the exogenous class II and endogenous class I presentation pathways.

Although reported examples of endogenous antigen (Ag) presentation by major histocompatibility complex (MHC) class II molecules have increased, the mechanisms governing this process remain poorly defined. In this communication, we describe an experimental system designed to examine the mechanisms governing class II presentation of internal Ag. Our target peptide is processed from a transmembrane protein constitutively expressed by a variety of nucleated cells (MHC class I, H-2Ld), is naturally displayed by MHC class II molecules in vivo, and is recognized by a class II-restricted, CD4+ T cell hybridoma. Our results indicate that presentation of the Ld target Ag is independent of its plasma membrane expression, may not involve endosomal proteolysis, and thus may be distinct from the classically defined class II presentation pathway. In addition, the observations that Ld presentation does not require a functional TAP-1 complex, is not blocked by invariant chain, and cannot utilize cytoplasmic forms of H-2Ld, suggest that a classical class I pathway is not involved in this presentation event. Finally, our data suggest that different cofactors participate in MHC class II presentation of exogenous and endogenous Ag, and that disparate Ag presenting cells, such as B, T, and pancreatic islet cells, may differentially express these two class II pathways of Ag presentation.

Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions.

Lymphocytotropic mouse parvoviruses can perturb immune responses. For example the recently identified mouse parvovirus designated MPV-1 persistently infects lymphoid tissues and interferes with the ability of cloned T cells to proliferate. As a consequence of these findings the present studies were undertaken to characterize further the immunomodulatory effects of MPV-1 on T cell-mediated immune responses in vivo and in vitro. To evaluate the effect of MPV-1 on CD8+ T cell-mediated responses sarcoma I (SaI) cells, devoid of class II major histocompatibility (MHC) antigens, were administered to MPV-1-infected adult BALB/c mice. MPV-1 infection accelerated tumor allograft rejection. Immunofluorescence staining and in situ hybridization studies of tumors suggested that direct infection of the tumor cells was not responsible for accelerated rejection. Furthermore, compared with uninfected mice, T cells from infected mice that had rejected SaI tumors had a diminished cytolytic capacity. Taken together these results suggest that MPV-1 may induce "bystander help." To examine the in vivo effect of MPV-1 on CD4+ T cell mediated responses adult mice were primed with ovalbumin (OVA) and infected with MPV-1. Spleen and popliteal lymph node cells from OVA-primed mice 3 or 7 days after MPV-1 inoculation had reduced proliferation responses, whereas the proliferative capacity of mesenteric lymph node cells from these mice was increased. Similarly, MPV-1 reduced cytokine-induced proliferation of allospecific CD8+ cloned L3 T cells and OVA-reactive CD4+ T cells without effecting cell viability. Since parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infections in mice used for transplantation studies and in cultures of mouse T lymphocytes.

Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection.

BACKGROUND: The recently identified autonomous mouse parvovirus designated mouse parvovirus-1 (MPV-1) persists in adult BALB/c mice for at least 9 weeks, infects lymphoid tissues, interferes with the ability of cloned T cells to proliferate, and exhibits immunomodulatory properties. As a consequence of these findings, the present studies were undertaken to characterize further the inmunomodulatory effects of MPV-1 on T cell-mediated immune responses in vivo and in vitro. METHODS: To evaluate the effect of MPV-1 infection on CD8+ T cell-mediated responses, BALB/c-H2dm2 mice were infected after transplantation of allogeneic BALB/c skin. RESULTS: MPV-1 potentiated the rejection of allogeneic skin grafts. This potentiation was not a result of virus infecting the cellular or vascular component of the graft as determined by in situ hybridization, but was mediated by T cells. However, the proliferative capacity of alloantigen-reactive lymphocytes from graft-sensitized infected mice was diminished. MPV-1 also induced the rejection of syngeneic skin grafts, and T cells from these infected graft-sensitized mice lysed syngeneic P815 target cells. CONCLUSIONS: These results suggest that MPV-1 infection of skin-grafted mice may disrupt normal mechanisms of peripheral tolerance and provide a unique model to study virus-induced autoimmunity.

Evaluation of diagnostic methods for Helicobacter bilis infection in laboratory mice.

Disease-susceptible (C3H) and -resistant (B6) immunocompetent and immunodeficient (C3H-scid and B6-rag1) mice were examined up to 10 weeks after inoculation with Helicobacter bilis (a prototype species of proven virulence). Infection was monitored weekly by use of fecal culture, polymerase chain reaction (PCR) nucleic acid amplification, membrane extract enzyme-linked immunosorbent assay (ELISA), and histologic examination. All mice became infected by three to five weeks after inoculation, on the basis of results of culture and PCR analysis of feces. The PCR analysis was more sensitive than culture at determining infection status, particularly during early infection. None of the mice had evidence of disease by week 10. Immunoglobulin G seroconversion was detectable in C3H mice by week eight and in B6 mice by week nine. Results indicated that culture and PCR analysis are more sensitive than is membrane extract ELISA serologic testing for detecting early infection in individual mice, regardless of genotype or immune status. Results underscore the need for improved seroassays for this important group of murine pathogens.

Neuromodulation for movement disorders.

Surgical neuromodulation has emerged as the primary method to treat the medically refractory symptoms of essential tremor and Parkinson disease. With reversible manipulation of CNS neurons, neuromodulation can be used to intraoperatively localize and verify a stereotactic target, and to chronically treat movement disorders. This article discusses the historical advances in stereotactic surgery using various modalities of neuromodulation leading to contemporary treatment. Electrical neuromodulation, or deep brain stimulation, is emphasized as the major surgical intervention with a discussion of the technique, surgical targets, and clinical outcomes. A comparison of neuromodulation techniques is presented.

Vascular smooth muscle cells in cerebral aneurysm pathogenesis.

Vascular smooth muscle cells (SMC) maintain significant plasticity. Following environmental stimulation, SMC can alter their phenotype from one primarily concerned with contraction to a pro-inflammatory and matrix remodeling phenotype. This is a critical process behind peripheral vascular disease and atherosclerosis, a key element of cerebral aneurysm pathology. Evolving evidence demonstrates that SMCs and phenotypic modulation play a significant role in cerebral aneurysm formation and rupture. Pharmacological alteration of smooth muscle cell function and phenotypic modulation could provide a promising medical therapy to inhibit cerebral aneurysm progression. This study reviews vascular SMC function and its contribution to cerebral aneurysm pathophysiology.

Sonothrombolysis: an emerging modality for the management of stroke.

OBJECTIVE: Ischemic stroke and intracranial hemorrhage remain a persistent scourge in Western civilization. Therefore, novel therapeutic modalities are desperately needed to expand the current limitations of treatment. Sonothrombolysis possesses the potential to fill this void because it has experienced a dramatic evolution from the time of early conceptualization in the 1960s. This process began in the realm of peripheral and cardiovascular disease and has since progressed to encompass intracranial pathologies. Our purpose is to provide a comprehensive review of the historical progression and existing state of knowledge, including underlying mechanisms as well as evidence for clinical application of ultrasound thrombolysis. METHODS: Using MEDLINE, in addition to cross-referencing existing publications, a meticulous appraisal of the literature was conducted. Additionally, personal communications were used as appropriate. RESULTS: This appraisal revealed several different technologies close to broad clinical use. However, fundamental questions remain, especially in regard to transcranial high-intensity focused ultrasound. Currently, the evidence supporting low intensity ultrasound's potential in isolation, without tissue plasminogen, remains uncertain; however, possibilities exist in the form of microbubbles to allow for focal augmentation with minimal systemic consequences. Alternatively, the literature clearly demonstrates, the efficacy of high-intensity focused ultrasound for independent thrombolysis. CONCLUSION: Sonothrombolysis exists as a promising modality for the noninvasive or minimally invasive management of stroke, both ischemic and hemorrhagic. Further research facilitating clinical application is warranted.

T-cell-independent responses to Borrelia burgdorferi are critical for protective immunity and resolution of lyme disease.

The humoral immune response to Borrelia burgdorferi during persistent infection is critical to both protective and disease-resolving immunity. This study examined the role of B cells in the absence of T cells during these events, using mice with selected immune dysfunctions. At 6 weeks postinfection, an interval at which arthritis resolves in immunocompetent mice, arthritis severity was equivalent among immunocompetent mice, alphabeta(+)-T-cell-deficient mice, and mice lacking both alphabeta(+) and gammadelta(+) T cells. Arthritis severity was worse in SCID mice, which lack T and B lymphocytes. Carditis regressed in immunocompetent mice and those lacking both alphabeta(+) and gammadelta(+) T cells but remained active in mice lacking only alphabeta(+) T cells and in SCID mice. Mice lacking only alphabeta(+) T cells and those lacking both alphabeta(+) and gammadelta(+) T cells generated immunoglobulin M (IgM) and IgG3 B. burgdorferi-reactive antibodies. Sera from infected immunocompetent mice, mice lacking only alphabeta(+) T cells, and mice lacking both alphabeta(+) and gammadelta(+) T cells passively protected naive mice against challenge inoculation with B. burgdorferi. However, only sera from infected immunocompetent mice, but not sera from infected T-cell-deficient mice, were able to resolve arthritis when passively transferred to actively infected SCID mice. These data demonstrate that B-cell activation during a T-cell-independent response may be critical for resolution of arthritis and carditis and that protective antibodies are generated during this response.

Cytolytic activity of murine CD4+ T cell clones correlates with IFN-gamma production in mouse strains having a BALB/c background.

CD4+ murine T cell clones were derived from various strains of mice, and their pattern of lymphokine secretion and cytolytic activity was compared. Limiting dilution cultures were established with lymph node cells from mice sensitized with OVA. Alloreactive CD4+ T cell clones also were derived in limiting dilution cultures prepared with naive BALB/c-H-2dm2 lymph node cells stimulated with irradiated BALB/c splenocytes. A total of 24 days elapsed between establishment of cultures and analysis of lymphokine production and cytolytic activity. Cytolytic capacity was assessed by using target cells that had been pulsed with Ag or coated with anti-CD3 mAb. We observed that: 1) the frequency of OVA-reactive T cells from various mouse strains was approximately the same; 2) both Th1 and Th2 cells as well as cells not encompassed within these categories could be lytic if derived from DBA/2, B10.D2, B10.A, C57BL/10, or C57BL/6 mice; and 3) the vast majority of CD4+ cloned T cells derived from BALB/c, BALB/c-H-2dm2, BALB.B, or BALB.K that did not produce IFN-gamma (including Th2 cells) did not exhibit cytolytic activity, whereas most clones derived from these strains that produced IFN-gamma were cytolytic. These observations indicate that both Th1 and Th2 cells from several mouse strains express cytolytic activity. Such cytolytic activity was not restricted to clones maintained in long term cultures. However, genes outside the MHC appeared to regulate the cytolytic activity of T cells. In particular, CD4+ T cell clones which did not produce IFN-gamma were not cytolytic when they were derived from BALB/c mice and mutant or MHC congenic inbred mice having a BALB background. Cytolytic activity of CD4+ T cells, in addition to the pattern of lymphokine production, may be important in graft rejection and in immune responses to infectious diseases.

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.

Cutting edge: T cell-mediated pathology in murine Lyme borreliosis.

Even in the absence of an appropriate model or direct evidence, T cells have been hypothesized to exacerbate the manifestations of Lyme disease. To define definitely the role of T cells in Lyme disease, the course of disease in immunocompetent and B cell-deficient mice was compared. By 8 wk postinoculation, immunocompetent mice resolved both carditis and arthritis, whereas foci of myocarditis and severe destructive arthritis characterized disease of B cell-deficient mice. Cell transfer experiments using infected B6-Rag1 knock out mice demonstrated that: 1) innate immunity mediated the initial sequelae of infection, 2) transferring both naive T cells and B cells induced resolution of carditis and arthritis, 3) infected mice reconstituted with T cells developed myocarditis and severe destructive arthritis, and 4) CD4+ T cells were responsible for the observed immune-mediated pathology. These data demonstrate directly the deleterious effect of T cells in Lyme disease.

Cytolytic activity of murine IL-2-producing CD4+ and CD8+ T cell clones cycles in response to IL-2.

Alloreactive or OVA-reactive cloned murine CD4+ or CD8+ T cells that produce IL-2 exhibit greatly reduced cytolytic activity after being cultured with high concentrations of rIL-2. Furthermore, such cells fail to produce lymphokines or proliferate when stimulated with Ag. The duration of this unresponsiveness to Ag correlates with the concentration of rIL-2 to which the cells were exposed; higher concentrations of rIL-2 prolong the period of unresponsiveness. The presence of ionomycin during the cytolytic assay restores lytic activity to cells rendered unresponsive by exposure to rIL-2. These results suggest that rIL-2-induced unresponsiveness to Ag is a consequence of impairment of a calcium-dependent signal important for cytolysis, proliferation, and lymphokine production. Thus, IL-2 appears to be an important lymphokine that regulates T cell responses downward as well as upward.

Differential regulation of murine T lymphocyte subsets.

Signaling pathways in T lymphocytes have been incompletely characterized. It is evident that differences exist among the T cell subsets. We have defined several distinct mechanisms that affect differentially the activities of murine T lymphocyte clones representing various CD4+ and CD8+ subsets: Interferon-gamma (IFN-gamma) inhibits proliferation of but not lymphokine production by TH2 cells. IL-10 inhibits antigen-presenting cell (APC)-induced lymphokine production by TH1 cells but not by TH2 cells. Murine TH1 and TH2 clones proliferate optimally in response to distinct APC populations. TH1 and TH2 clones utilize different TCR-associated signaling pathways. High concentrations of antigen (or anti-TCR mAb) inhibit IL-2-induced proliferation (but not lymphokine production) by TH1 and cytolytic T lymphocyte (CTL) clones only. Exposure of TH1 clones (but not TH2 clones or CD8+ CTL clones) to IL-2 induces unresponsiveness to antigen. TH1 and TH2 clones as well as CD8+ clones can be cytolytic, but not all T cells use the same cytolytic mechanisms. CD4+ clones from some mouse strains are not cytolytic if they do not secrete IFN-gamma. Understanding the mechanisms that differentially regulate the various kinds of T cells, in addition to providing insights into the molecular events associated with activation of those subsets, should facilitate modulation of their activities in vivo, making it possible to influence favorably the outcome of disease processes.

A nonlethal rat parvovirus infection suppresses rat T lymphocyte effector functions.

Inoculation of the UMass strain of rat virus (RV-UMass) into adult immunocompetent rats results in a prolonged subclinical infection that is resolved in 4 to 8 wk. Co-labeling studies, using in situ hybridization (ISH) and immunohistochemistry (IHC), confirmed that RV-UMass was lymphocytotropic and capable of infecting CD4+ and CD8+ T cells as well as B cells. ISH studies also revealed that virus replication was restricted in unstimulated cells but was productive in concanavalin A-stimulated lymphocytes. A corollary of productive infection of lymphocytes was the suppression of lymphocyte functions. Although RV-UMass did not appear to induce phenotypic changes during the course of infection, cells from infected rats had diminished proliferation and cytolytic responses. Both peripheral and mesenteric lymph node cells exhibited only partial recovery of their proliferative and cytolytic capacities one month after infection. Furthermore, RV-UMass-infected tissue culture maintained alloreactive CD4+ T cells in vitro, and a nonlethal infection of this T cell line inhibited Ag- and IL-2-induced proliferation. Because parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infection in rodents and in cultures of rat T lymphocytes.

Characterization of mouse parvovirus infection by in situ hybridization.

Infection of young adult BALB/cByJ mice with mouse parvovirus-1, a newly recognized, lymphocytotropic, nonpathogenic parvovirus, was examined by in situ hybridization. Virus appeared to enter through the small intestine and was disseminated to the liver and lymphoid tissues. Strand-specific probes detected virion DNA in a consistently larger number of cells than replicative forms of viral DNA and/or viral mRNA. The number of signal-positive cells in the intestinal mucosa, lymph nodes, spleen, and thymus increased through day 10 after oral inoculation but decreased after seroconversion. Positive cells were still detected, however, in peripheral lymphoid tissues of mice examined at 9 weeks postinoculation. The results underscore the need to assess potential effects of persistent mouse parvovirus-1 infection on immune function in mice.

Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells.

A putative parvovirus related to minute virus of mice (MVM), but distinct from MVM-prototype and MVM-immunosuppressive, was identified, using serologic techniques and Southern blot analysis, in maintenance cultures of established T cell clones. This putative viral agent resulted in a lytic infection of cloned L3 cytotoxic T cells but was unable to produce a productive infection in BHK.21 or EL-4(G) cells. Moreover, maintenance cultures of several distinct subsets of cloned T cells apparently contaminated with this putative viral agent contained poorly growing cells and erythrocyte aggregates. The aggregation of mouse erythrocytes appeared to be a reliable indicator of infection with this putative virus and may be related to the ability of this agent to agglutinate mouse erythrocytes. This putative virus also was found to inhibit the proliferative response of certain cloned T cells to IL-2 and Ag. Viremic mice and secondary MLC supernatant were identified as two potential sources of contamination and represent ways of propagating this agent in vitro. The finding that this agent interferes with the ability of T cell clones to thrive and, therefore has the potential to alter immune responses, emphasizes the importance of identifying and excluding parvoviral infections in cultures of murine T lymphocytes.

Rodent parvovirus infections.

Parvoviruses are among the most common infectious agents of laboratory rodents and major impediments to rodent-based research. The original prototypic rodent parvoviruses-minute virus of mice, rat virus, and H-1 virus-have recently been joined by biologically and antigenically distinct parvoviruses in mice, rats, and hamsters. Recognition of the increased diversity of rodent parvoviruses presents new challenges for determining the impact of parvovirus infection on research and for detecting, preventing, and eliminating infection. This review summarizes current knowledge about rodent parvoviruses and parvovirus infections, highlighting recent research on newly isolated virus strains.