Kavitha Yaddanapudi

Passive transfer of streptococcus-induced antibodies reproduces behavioral disturbances in a mouse model of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection

Streptococcal infections can induce obsessive-compulsive and tic disorders. In children, this syndrome, frequently associated with disturbances in attention, learning and mood, has been designated pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS). Autoantibodies recognizing central nervous system (CNS) epitopes are found in sera of most PANDAS subjects, but may not be unique to this neuropsychiatric subset. In support of a humoral immune mechanism, clinical improvement often follows plasmapheresis or intravenous immunoglobulin. We recently described a PANDAS mouse model wherein repetitive behaviors correlate with peripheral anti-CNS antibodies and immune deposits in brain following streptococcal immunization. These antibodies are directed against group A β-hemolytic streptococcus matrix (M) protein and cross-react with molecular targets complement C4 protein and α-2-macroglobulin in brain. Here we show additional deficits in motor coordination, learning/memory and social interaction in PANDAS mice, replicating more complex aspects of human disease. Furthermore, we demonstrate for the first time that humoral immunity is necessary and sufficient to induce the syndrome through experiments wherein naive mice are transfused with immunoglobulin G (IgG) from PANDAS mice. Depletion of IgG from donor sera abrogates behavior changes. These functional disturbances link to the autoimmunity-related IgG1 subclass but are not attributable to differences in cytokine profiles. The mode of disrupting blood–brain barrier integrity differentially affects the ultimate CNS distribution of these antibodies and is shown to be an additional important determinant of neuropsychiatric outcomes. This work provides insights into PANDAS pathogenesis and may lead to new strategies for identification and treatment of children at risk for autoimmune brain disorders.

A Murine Model for Neuropsychiatric Disorders Associated with Group A β-Hemolytic Streptococcal Infection

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A β-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freunds adjuvant, whereas controls received Freunds adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and α-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology.

Induction of Toll-Like Receptor 3-Mediated Immunity during Gestation Inhibits Cortical Neurogenesis and Causes Behavioral Disturbances

ABSTRACT Maternal infection during pregnancy with a wide range of RNA and DNA viruses is associated with increased risk for schizophrenia and autism in their offspring. A common feature in these exposures is that virus replication induces innate immunity through interaction with Toll-like receptors (TLRs). We employed a mouse model wherein pregnant mice were exposed to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA molecular mimic of replicating virus. Poly(I ⋅ C) inhibited embryonic neuronal stem cell replication and population of the superficial layers of the neocortex by neurons. Poly(I ⋅ C) also led to impaired neonatal locomotor development and abnormal sensorimotor gating responses in adult offspring. Using Toll-like receptor 3 (TLR3)-deficient mice, we established that these effects were dependent on TLR3. Inhibition of stem cell proliferation was also abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen, a cyclooxygenase (COX) inhibitor. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and behavioral dysfunction, and they may suggest strategies for reducing the risk of neuropsychiatric disorders subsequent to prenatal exposures to pathogens and other triggers of innate immunity. IMPORTANCE Maternal infection during gestation increases the risk of neuropsychiatric disorders in their offspring. Furthermore, work in animal models indicates that pre- or neonatal infections with a wide range of viruses results in similar neurodevelopmental outcomes. These observations are consistent with a mechanism whereby damage is mediated through common pathways. Exposure of pregnant mice to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA (dsRNA) molecular mimic of replicating virus, inhibited embryonic neuronal stem cell replication and led to behavioral abnormalities in their offspring. These effects were mediated through TLR3 and abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and may suggest strategies for reducing the risk of neuropsychiatric diseases following exposures to infectious agents and other triggers of innate immunity during gestation. Maternal infection during gestation increases the risk of neuropsychiatric disorders in their offspring. Furthermore, work in animal models indicates that pre- or neonatal infections with a wide range of viruses results in similar neurodevelopmental outcomes. These observations are consistent with a mechanism whereby damage is mediated through common pathways. Exposure of pregnant mice to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA (dsRNA) molecular mimic of replicating virus, inhibited embryonic neuronal stem cell replication and led to behavioral abnormalities in their offspring. These effects were mediated through TLR3 and abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and may suggest strategies for reducing the risk of neuropsychiatric diseases following exposures to infectious agents and other triggers of innate immunity during gestation.

Control of Tumor-Associated Macrophage Alternative Activation by Macrophage Migration Inhibitory Factor

Tumor stromal alternatively activated macrophages are important determinants of antitumor T lymphocyte responses, intratumoral neovascularization, and metastatic dissemination. Our recent efforts to investigate the mechanism of macrophage migration inhibitory factor (MIF) in antagonizing antimelanoma immune responses reveal that macrophage-derived MIF participates in macrophage alternative activation in melanoma-bearing mice. Both peripheral and tumor-associated macrophages (TAMs) isolated from melanoma bearing MIF-deficient mice display elevated proinflammatory cytokine expression and reduced anti-inflammatory, immunosuppressive, and proangiogenic gene products compared with macrophages from tumor-bearing MIF wild-type mice. Moreover, TAMs and myeloid-derived suppressor cells from MIF-deficient mice exhibit reduced T lymphocyte immunosuppressive activities compared with those from their wild-type littermates. Corresponding with reduced tumor immunosuppression and neo-angiogenic potential by TAMs, MIF deficiency confers protection against transplantable s.c. melanoma outgrowth and melanoma lung metastatic colonization. Finally, we report for the first time, to our knowledge, that our previously discovered MIF small molecule antagonist, 4-iodo-6-phenylpyrimidine, recapitulates MIF deficiency in vitro and in vivo, and attenuates tumor-polarized macrophage alternative activation, immunosuppression, neoangiogenesis, and melanoma tumor outgrowth. These studies describe an important functional contribution by MIF to TAM alternative activation and provide justification for immunotherapeutic targeting of MIF in melanoma patients.

Astrocytes recognize intracellular polyinosinic-polycytidylic acid via MDA-5

RNA virus replication results in expression of double‐stranded RNA (ds‐RNA) molecules that trigger innate immune responses through interactions with both intracellular and extracellular receptors. We investigated the contributions of the extracellular and intracellular pathways to innate immunity in murine astrocyte primary cultures using polyinosinic‐polycytidylic acid (poly I:C), a synthetic ds‐RNA molecule designed to mimic RNA virus infection. Whereas extracellular poly I:C (naked poly I:C) mainly induced the expression of regulated on activation normal T‐cell expressed and secreted (RANTES), interleukin‐8 (IL 8), and tumor necrosis factor a (TNF‐α), intracellular delivery of poly I:C (complexed poly I:C) chiefly induced expression of IFN‐β and IL‐6. Experiments with astrocytes from Toll‐like receptor 3 (TLR‐3) knockout mice indicated that naked poly I:C signals via a TLR‐3‐dependent NF‐κB pathway. Complexed poly I:C induced the expression of the intracellular ds‐RNA sensor proteins, retinoic acid inducible gene I (RIG‐I), and melanoma differentiation‐associated gene 5 (MDA‐5). However, transfection of astrocytes with dominant negative forms of the helicases implicated MDA‐5, but not RIG‐I, as the intracellular sensor of poly I:C. Complexed poly I:C‐mediated MDA‐5 stimulation transmitted “downstream” signals, resulting in activation of the transcription factors NF‐κB and IRF‐3. Our results illustrate the intricacy of extracellular and intracellular ds‐RNA recognition in viral infections of the central nervous system and indicate the importance of MDA‐5 helicase as an intracellular ds‐RNAsensor in astrocytes. De Miranda, J., Yaddanapudi, K., Hornig, M., Lipkin, W. I. Astrocytes recognize intracellular polyinosinic‐polycytidylic acid via MDA‐5. FASEB J. 23, 1064–1071 (2009)

Hippocampal Poly(ADP-Ribose) Polymerase 1 and Caspase 3 Activation in Neonatal Bornavirus Infection

ABSTRACT Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.

Metallothioneins and Zinc Dysregulation Contribute to Neurodevelopmental Damage in a Model of Perinatal Viral Infection

Neonatal Borna disease (NBD) virus infection in the Lewis rat results in life‐long viral persistence and causes behavioral and neurodevelopmental abnormalities. A hallmark of the disorder is progressive loss of cerebellar Purkinje and dentate gyrus granule cells. Findings of increased brain metallothionein‐I and‐II (MT‐I/‐II) mRNA expression in cDNA microarray experiments led us to investigate MT isoforms and their relationship to brain zinc metabolism, cellular toxicity, and neurodevelopmental abnormalities in this model. Real‐time PCR confirmed marked induction of MT‐I/‐II mRNA expression in the brains of NBD rats (40.5‐fold increase in cerebellum, p<0.0001; 6.8‐fold increase in hippocampus, p = 0.003; and 9.5‐fold increase in striatum, p = 0.0012), whereas a trend toward decreased MT‐III mRNA was found in hippocampus (1.25‐fold decrease, p = 0.0841). Double label immunofluorescence revealed prominent MT‐I/‐II expression in astrocytes throughout the brain; MT‐III protein was decreased in granule cell neurons and increased in astrocytes, with differential subcellular distribution from cytoplasmic to nuclear compartments in NBD rat hippocampus. Modified Timm staining of hippocampus revealed reduced zinc in mossy fiber projections to the hilus and CA3, accumulation of zinc in glial cells and degenerating granule cell somata, and robust mossy fiber sprouting into the inner molecular layer of the dentate gyrus. Zinc Transporter 3 (ZnT‐3) mRNA expression was decreased in hippocampus (2.3‐fold decrease, p = 0.0065); staining for its correlate protein was reduced in hippocampal mossy fibers. Furthermore, 2 molecules implicated in axonal pathfinding and mossy fiber sprouting, the extracellular matrix glycoprotein, tenascin‐R (TN‐R), and the hyaluronan receptor CD44, were increased in NBD hippocampal neuropil. Abnormal zinc metabolism and mechanisms of neuroplasticity may contribute to the pathogenesis of disease in this model, raising more general implications for neurodevelopmental damage following viral infections in early life.

Cancer vaccines: Looking to the future.

These are exciting times for the field of cancer immunotherapy. Although the clinical efficacy of monoclonal antibodies has been demonstrated since the early 1990s, the therapeutic profile of other immunotherapeutic approaches—especially vaccines—has not yet been formally clarified. However, the recent success of several immunotherapeutic regimens in cancer patients has boosted the development of this treatment modality. These achievements stemmed from recent scientific advances demonstrating the tolerogenic nature of cancer and the fundamental role of the tumor immune microenvironment in the suppression of antitumor immunity. New immunotherapeutic strategies against cancer attempt to promote protective antitumor immunity while disrupting the immunoregulatory circuits that contribute to tumor tolerance. Cancer vaccines differ from other anticancer immunotherapeutics in that they initiate the dynamic process of activating the immune system so as to successfully re-establish a state of equilibrium between tumor cells and the host. This article reviews recent clinical trials involving several different cancer vaccines and describes some of the most promising immunotherapeutic approaches that harness antitumor T-cell responses. In addition, we describe strategies whereby cancer vaccines can be exploited in combination with other therapeutic approach to overcome—in a synergistic fashion—tumor immunoevasion. Finally, we discuss prospects for the future development of broad spectrum prophylactic anticancer vaccines.

Spatiotemporal Analysis of Purkinje Cell Degeneration Relative to Parasagittal Expression Domains in a Model of Neonatal Viral Infection

ABSTRACT Infection of newborn Lewis rats with Borna disease virus (neonatal Borna disease [NBD]) results in cerebellar damage without the cellular inflammation associated with infections in later life. Purkinje cell (PC) damage has been reported for several models of early-life viral infection, including NBD; however, the time course and distribution of PC pathology have not been investigated rigorously. This study examined the spatiotemporal relationship between PC death and zonal organization in NBD cerebella. Real-time PCR at postnatal day 28 (PND28) revealed decreased cerebellar levels of mRNAs encoding the glycolytic enzymes aldolase C (AldoC, also known as zebrin II) and phosphofructokinase C and the excitatory amino acid transporter 4 (EAAT4). Zebrin II and EAAT4 immunofluorescence analysis in PND21, PND28, PND42, and PND84 NBD rat cerebella revealed a complex pattern of PC degeneration. Early cell loss (PND28) was characterized by preferential apoptotic loss of zebrin II/EAAT4-negative PC subsets in the anterior vermis. Consistent with early preferential loss of zebrin II/EAAT4-negative PCs in the vermis, the densities of microglia and the Bergmann glial expression of metallothionein I/II and the hyaluronan receptor CD44 were higher in zebrin II/EAAT4-negative zones. In contrast, early loss in lateral cerebellar lobules did not reflect a similar discrimination between PC phenotypes. Patterns of vermal PC loss became more heterogeneous at PND42, with the loss of both zebrin II/EAAT4-negative and zebrin II/EAAT4-positive neurons. At PND84, zebrin II/EAAT4 patterning was abolished in the anterior cerebellum, with preferential PC survival in lobule X. Our investigation reveals regional discrimination between patterns of PC subset loss, defined by zebrin II/EAAT4 expression domains, following neonatal viral infection. These findings suggest a differential vulnerability of PC subsets during the early stages of virus-induced neurodegeneration.

Vaccination with Embryonic Stem Cells Protects against Lung Cancer: Is a Broad-Spectrum Prophylactic Vaccine against Cancer Possible?

The antigenic similarity between tumors and embryos has been appreciated for many years and reflects the expression of embryonic gene products by cancer cells and/or cancer-initiating stem cells. Taking advantage of this similarity, we have tested a prophylactic lung cancer vaccine composed of allogeneic murine embryonic stem cells (ESC). Naïve C57BL/6 mice were vaccinated with ESC along with a source of granulocyte macrophage-colony stimulating factor (GM-CSF) in order to provide immunostimulatory adjuvant activity. Vaccinated mice were protected against subsequent challenge with implantable Lewis lung carcinoma (LLC). ESC-induced anti-tumor immunity was not due to a non-specific “allo-response” as vaccination with allogeneic murine embryonic fibroblasts did not protect against tumor outgrowth. Vaccine efficacy was associated with robust tumor-reactive primary and memory CD8+ T effector responses, Th1 cytokine response, higher intratumoral CD8+ T effector/CD4+CD25+Foxp3+ T regulatory cell ratio, and reduced myeloid derived suppressor cells in the spleen. Prevention of tumorigenesis was found to require a CD8-mediated cytotoxic T lymphocyte (CTL) response because in vivo depletion of CD8+ T lymphocytes completely abrogated the protective effect of vaccination. Importantly, this vaccination strategy also suppressed the development of lung cancer induced by the combination of carcinogen administration and chronic pulmonary inflammation. Further refinement of this novel vaccine strategy and identification of shared ESC/tumor antigens may lead to immunotherapeutic options for lung cancer patients and, perhaps more importantly, could represent a first step toward the development of prophylactic cancer vaccines.