Immunization and Host Responses to MB-1, a Live Hatchery Vaccine against Infectious Bursal Disease.

MB-1 is an attenuated infectious bursal disease virus vaccine. Previously, we observed a temporal delay of vaccine virus replication in the bursae of chicks due to maternally derived antibodies (MDAs). The mechanism that allowed its survival despite MDA neutralization remained unclear. We hypothesized that after vaccination at 1 day of age (DOA), the MB-1 virus penetrates and resides in local macrophages that are then distributed to lymphoid organs. Furthermore, MB-1's ability to survive within macrophages ensures its survival during effective MDA protection. PCR analysis of lymphoid organs from chicks with MDA, vaccinated on 1 DOA, demonstrated that the MB-1 virus was identified at low levels solely in the spleen pre-14 days of age. Fourteen days after vaccination, the virus was identified using PCR in the bursa, with viral levels increasing with time. The possible delay in viral colonization of the bursa was attributed to the presence of anti-IBDV capsid VP2 maternal IgA and IgY in the bursa interstitium. These indicate that during the period of high MDA levels, a small but viable MB-1 viral reservoir was maintained in the spleen, which might have served to colonize the bursa after MDA levels declined. Thereafter, individual immunization of chicks against Gumboro disease was achieved.

A Humanized Monoclonal Antibody against Heat Shock Protein 60 Suppresses Murine Arthritis and Colitis and Skews the Cytokine Balance toward an Anti-Inflammatory Response.

We have previously shown that naturally occurring as well as acquired Abs against the Mycobacterium tuberculosis heat shock protein (HSP)65 protect against the induction of murine autoimmune inflammatory arthritis. In the present work, we have studied the anti-inflammatory effect of prozumab, a humanized anti-HSP mAb in murine inflammatory arthritis and colitis, and its effects on cytokine secretion. Prozumab was shown to bind to HSP60, the highly conserved mammalian homolog of the bacterial protein, and it was found to be effective in protecting and suppressing autoimmune arthritis in the models of adjuvant arthritis and collagen-induced arthritis in rats and mice, respectively, as well as in acute hapten-mediated colitis and chronic, spontaneous colitis models. Mechanistically, prozumab induces IL-10 secretion from naive human PBMCs and suppresses the secretion of IFN-γ and IL-6 from anti-CD3-activated human PBMCs. These findings make prozumab a promising potential drug for treating human rheumatoid arthritis and inflammatory bowel disease, as well as a wide range of autoimmune inflammatory diseases.

Α-synuclein neuropathology is controlled by nuclear hormone receptors and enhanced by docosahexaenoic acid in a mouse model for Parkinson's disease.

α-Synuclein (α-Syn) is a neuronal protein that accumulates progressively in Parkinson's disease (PD) and related synucleinopathies. Attempting to identify cellular factors that affect α-Syn neuropathology, we previously reported that polyunsaturated fatty acids (PUFAs) promote α-Syn oligomerization and aggregation in cultured cells. We now report that docosahexaenoic acid (DHA), a 22:6 PUFA, affects α-Syn oligomerization by activating retinoic X receptor (RXR) and peroxisome proliferator-activated receptor γ2 (PPARγ2). In addition, we show that dietary changes in brain DHA levels affect α-Syn cytopathology in mice transgenic for the PD-causing A53T mutation in human α-Syn. A diet enriched in DHA, an activating ligand of RXR, increased the accumulation of soluble and insoluble neuronal α-Syn, neuritic injury and astrocytosis. Conversely, abnormal accumulations of α-Syn and its deleterious effects were significantly attenuated by low dietary DHA levels. Our results suggest a role for activated RXR/PPARγ 2, obtained by elevated brain PUFA levels, in α-Syn neuropathology.

α-Synuclein expression selectively affects tumorigenesis in mice modeling Parkinson's disease.

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn-dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over-expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.

The transgenic overexpression of alpha-synuclein and not its related pathology associates with complex I inhibition.

Alpha-synuclein (alphaS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance alphaS pathogenesis. Here we show that alphaS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T alphaS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I activity is not associated with alphaS-related pathology. Specifically, complex I activity measured in purified mitochondria from A53T alphaS transgenic mouse brains was not affected by mouse age; Parkinson disease-like symptoms; levels of alphaS soluble oligomers; levels of insoluble, lipid-associated alphaS; or alphaS intraneuronal depositions in vivo. Likewise, no correlation was found between complex I activity and polyunsaturated fatty acid-induced alphaS depositions in Lewy body-like inclusions in cultured dopaminergic cells. We further show that the effect of alphaS on complex I activity is not due to altered mitochondrial protein levels or affected complex I assembly. Based on the results herein, we suggest that alphaS expression negatively regulates complex I activity as part of its normal, physiological role.

alpha-Synuclein abnormalities in mouse models of peroxisome biogenesis disorders.

alpha-Synuclein (alphaS) is a presynaptic protein implicated in Parkinson's disease (PD). Growing evidence implicates mitochondrial dysfunction, oxidative stress, and alphaS-lipid interactions in the gradual accumulation of alphaS in pathogenic forms and its deposition in Lewy bodies, the pathological hallmark of PD and related synucleinopathies. The peroxisomal biogenesis disorders (PBD), with Zellweger syndrome serving as the prototype of this group, are characterized by malformed and functionally impaired peroxisomes. Here we utilized the PBD mouse models Pex2-/-, Pex5-/-, and Pex13-/- to study the potential effects of peroxisomal dysfunction on alphaS-related pathogenesis. We found increased alphaS oligomerization and phosphorylation and its increased deposition in cytoplasmic inclusions in these PBD mouse models. Furthermore, we show that alphaS abnormalities correlate with the altered lipid metabolism and, specifically, with accumulation of long chain, n-6 polyunsaturated fatty acids that occurs in the PBD models.

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling.

Alpha-synuclein (alphaS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, alphaS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in alphaS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that alphaS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, alphaS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, alphaS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted alphaS mouse brains. We suggest that alphaS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.

Polyunsaturated fatty acids induce alpha-synuclein-related pathogenic changes in neuronal cells.

The misfolding and aggregation of normally soluble proteins has emerged as a key feature of several neurodegenerative diseases. In Parkinson's disease, progressive loss of dopaminergic neurons is accompanied by polymerization of the cytoplasmic protein alpha-synuclein (alphaS) into filamentous inclusions found in neuronal somata (Lewy bodies) and dendrites (Lewy neurites). Similar alphaS aggregates occur in cortical neurons in dementia with Lewy bodies. Numerous reports now indicate that alphaS can interact with lipids. We previously found that treating dopaminergic cells expressing alphaS with polyunsaturated fatty acids (PUFAs) induced the formation of soluble, sodium dodecyl sulfate-stable oligomers whereas treatment with saturated fatty acids did not. Here, we examine the relevance of alphaS-PUFA interactions to the development of Parkinson's disease-like cytopathology. Exposure of alphaS-overexpressing dopaminergic or neuronal cell lines to physiological levels of a PUFA induced the formation of proteinaceous inclusions in the cytoplasm. Kinetic experiments indicated that PUFA-induced soluble oligomers of alphaS precede these Lewy-like inclusions. Importantly, we found that alphaS oligomers were associated with cyto-toxicity, whereas the development of Lewy-like inclusions appeared to be protective. We conclude that alterations in PUFA levels can lead to aggregation of alphaS and subsequent deposition into potentially cyto-toxic oligomers that precede inclusions in dopaminergic cells.