David L. Hirschberg

Autoantigen microarrays for multiplex characterization of autoantibody responses

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005.

Suppressive vaccination with DNA encoding a variable region gene of the T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immunity.

A variable region gene of the T–cell receptor, Vβ8.2, is rearranged, and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) in H–2u mice after immunization with myelin basic protein (MBP). Vaccination of these mice with naked DNA encoding Vβ8.2 protected mice from EAE. Analysis of T cells reacting to the pathogenic portion of the MBP molecule indicated that in the vaccinated mice there was a reduction in the Thl cytokines interleukin–2 (IL–2) and interferon–γ. In parallel, there was an elevation in the production of IL–4, a Th2 cytokine associated with suppression of disease. A novel feature of DNA immunization for autoimmune disease, reversal of the autoimmune response from Thl to Th2, may make this approach attractive for treatment of Thl–mediated diseases like multiple sclerosis, juvenile diabetes and rheumatoid arthritis.

Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.

Characterization of a canine homolog of hepatitis C virus

An estimated 3% of the worlds population is chronically infected with hepatitis C virus (HCV). Although HCV was discovered more than 20 y ago, its origin remains obscure largely because no closely related animal virus homolog has been identified; furthermore, efforts to understand HCV pathogenesis have been hampered by the absence of animal models other than chimpanzees for human disease. Here we report the identification in domestic dogs of a nonprimate hepacivirus. Comparative phylogenetic analysis of the canine hepacivirus (CHV) confirmed it to be the most genetically similar animal virus homolog of HCV. Bayesian Markov chains Monte Carlo and associated time to most recent common ancestor analyses suggest a mean recent divergence time of CHV and HCV clades within the past 500–1,000 y, well after the domestication of canines. The discovery of CHV may provide new insights into the origin and evolution of HCV and a tractable model system with which to probe the pathogenesis, prevention, and treatment of diseases caused by hepacivirus infection.

Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Kochs postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.

Combination of gene delivery and DNA vaccination to protect from and reverse Th1 autoimmune disease via deviation to the Th2 pathway

Using a combination of local gene delivery and tolerizing DNA vaccination, we demonstrate that codelivery of the interleukin-4 (IL-4) gene and a DNA vaccine encoding the self-peptide proteolipid protein 139-151 (PLP139-151) provides protective immunity against experimental autoimmune encephalomyelitis (EAE). We provide evidence for a mechanism whereby IL-4 expressed from the naked DNA is secreted and acts locally on autoreactive T cells via activation of STAT6 to shift their cytokine profile to T helper 2. We also show that DNA vaccines can be used to reverse established EAE by covaccination with the genes for myelin oligodendrocyte glycoprotein and IL-4. This treatment strategy combines the antigen-specific effects of DNA vaccination and the beneficial effects of local gene delivery.

Detection of Respiratory Viruses and Subtype Identification of Influenza A Viruses by GreeneChipResp Oligonucleotide Microarray

ABSTRACT Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4+ T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4+ Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-γ-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-γ activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.

Novel Picornavirus in Turkey Poults with Hepatitis, California, USA

To identify a candidate etiologic agent for turkey viral hepatitis, we analyzed samples from diseased turkey poults from 8 commercial flocks in California, USA, that were collected during 2008–2010. High-throughput pyrosequencing of RNA from livers of poults with turkey viral hepatitis (TVH) revealed picornavirus sequences. Subsequent cloning of the ≈9-kb genome showed an organization similar to that of picornaviruses with conservation of motifs within the P1, P2, and P3 genome regions, but also unique features, including a 1.2-kb sequence of unknown function at the junction of P1 and P2 regions. Real-time PCR confirmed viral RNA in liver, bile, intestine, serum, and cloacal swab specimens from diseased poults. Analysis of liver by in situ hybridization with viral probes and immunohistochemical testing of serum demonstrated viral nucleic acid and protein in livers of diseased poults. Molecular, anatomic, and immunologic evidence suggests that TVH is caused by a novel picornavirus, tentatively named turkey hepatitis virus.