Development of standardized insulin treatment protocols for spontaneous rodent models of type 1 diabetes.

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.

Rhesus cytomegalovirus encodes seventeen microRNAs that are differentially expressed in vitro and in vivo.

Human cytomegalovirus (HCMV) miRNAs are important for regulation of viral infection and evasion of host immune responses. Unfortunately, the importance of HCMV miRNAs cannot be addressed in vivo due to the species specificity of CMVs. Rhesus CMV (RhCMV) infection of rhesus macaques provides an important model system for HCMV pathogenesis due to the genetic similarity between the viruses. In this report, seventeen RhCMV miRNAs were identified using Next Generation Sequencing. In fibroblasts, RhCMV miRNAs associate with Argonaute proteins and display several patterns of expression, including an early peak in expression followed by decline and accumulation throughout infection. Additionally, RhCMV encodes an HCMV miR-US5-2 homologue that targets the 3' UTR of RhCMV US7. Finally, examination of salivary gland tissue from infected animals revealed the presence of a subset of viral miRNAs. This study highlights the importance of the RhCMV model system for evaluating the roles of CMV miRNAs during viral infection.

Cytomegalovirus microRNA expression is tissue specific and is associated with persistence.

MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3' and 5' ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection.

A mucosal vaccination approach for herpes simplex virus type 2.

An estimated 1 out of every 5 Americans is infected with herpes simplex virus type 2 (HSV-2). Efforts in developing a potent vaccine for HSV-2 have shown limited success. Here we describe a heterologous vaccination strategy for HSV-2 based on an intramuscular DNA prime followed by a liposome-encapsulated antigen boost delivered intranasally. Both portions of the vaccine express the immunogenic HSV-2 glycoprotein D. In female Balb/c mice, this heterologous immunisation regimen stimulated high titers of serum neutralising antibodies, a DNA priming dose dependent T helper type response, enhanced mucosal immune responses and potent protective immunity at the portal of entry for the virus: the vaginal cavity. A clear synergistic effect on immune responses and protection from infection was seen using this heterologous immunisation approach. Suboptimal DNA prime (0.5 µg) followed by the liposome boost resulted in an 80% survival rate when mice were infected 2 weeks after immunisation. A higher dose of DNA priming (5 µg) followed by the liposome boost resulted in sterilising immunity in 80% of mice. The vaccine induced durable protection in mice, demonstrated by a 60% survival rate when lethal infections were performed 20 weeks after the immunisation primed with 0.5 µg of DNA vaccine.

Infection with viruses from several families triggers autoimmune diabetes in LEW*1WR1 rats: prevention of diabetes by maternal immunization.

OBJECTIVE: The contribution of antecedent viral infection to the development of type 1 diabetes in humans is controversial. Using a newer rat model of the disease, we sought to 1) identify viruses capable of modulating diabetes penetrance, 2) identify conditions that increase or decrease the diabetogenicity of infection, and 3) determine whether maternal immunization would prevent diabetes. RESEARCH DESIGN AND METHODS: About 2% of LEW*1WR1 rats develop spontaneous autoimmune diabetes, but disease penetrance is much higher if weanling rats are exposed to environmental perturbants including Kilham rat virus (KRV). We compared KRV with other viruses for diabetogenic activity. RESULTS: Both KRV and rat cytomegalovirus (RCMV) induced diabetes in up to 60% of LEW*1WR1 rats, whereas H-1, vaccinia, and Coxsackie B4 viruses did not. Simultaneous inoculation of KRV and RCMV induced diabetes in 100% of animals. Pretreatment of rats with an activator of innate immunity increased the diabetogenicity of KRV but not RCMV and was associated with a moderate rate of diabetes after Coxsackie B4 and vaccinia virus infection. Inoculation of LEW*1WR1 dams with both KRV and RCMV prior to pregnancy protected weanling progeny from virus-induced diabetes in a virus-specific manner. CONCLUSIONS: Exposure to viruses can affect the penetrance of autoimmune diabetes in genetically susceptible animals. The diabetogenicity of infection is virus specific and is modified by immunomodulation prior to inoculation. Maternal immunization protects weanlings from virus-induced diabetes, suggesting that modification of immune responses to infection could provide a means of preventing islet autoimmunity.

A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection.

A variety of DNA vaccine prime and recombinant viral boost immunization strategies have been developed to enhance immune responses in humans, but inherent limitations to these strategies exist. There is still an overwhelming need to develop safe and effective approaches that raise broad humoral and T cell-mediated immune responses systemically and on mucosal surfaces. We have developed a novel mucosal immunization regimen that precludes the use of viral vectors yet induces potent T cell responses. Using hepatitis B surface Ag (HBsAg), we observed that vaccination of BALB/c mice with an i.m. HBsAg-DNA vaccine prime followed by an intranasal boost with HBsAg protein encapsulated in biologically inert liposomes enhanced humoral and T cell immune responses, particularly on mucosal surfaces. Intranasal live virus challenge with a recombinant vaccinia virus expressing HBsAg revealed a correlation between T cell immune responses and protection of immunized mice. A shortened immunization protocol was developed that was successful in both adult and neonatal mice. These results support the conclusion that this new approach is capable of generating a Th-type-1-biased, broad spectrum immune response, specifically at mucosal surfaces. The success of this design may provide a safe and effective vaccination alternative for human use.

LEW.1WR1 rats develop autoimmune diabetes spontaneously and in response to environmental perturbation.

We describe a new rat model of autoimmune diabetes that arose in a major histocompatibility complex congenic LEW rat. Spontaneous diabetes in LEW.1WR1 rats (RT1(u/u/a)) occurs with a cumulative frequency of approximately 2% at a median age of 59 days. The disease is characterized by hyperglycemia, glycosuria, ketonuria, and polyuria. Both sexes are affected, and islets of acutely diabetic rats are devoid of beta-cells, whereas alpha- and delta-cell populations are spared. The peripheral lymphoid phenotype is normal, including the fraction of ART2(+) regulatory T-cells. We tested the hypothesis that the expression of diabetes would be increased by immunological perturbation of innate or adaptive immunity. Treatment of young rats with depleting anti-ART2.1 monoclonal antibody increased the frequency of diabetes to 50%. Treatment with the toll-like receptor 3 ligand polyinosinic:polycytidylic acid increased the frequency of diabetes to 100%. All diabetic rats exhibited end-stage islets. The LEW.1WR1 rat is also susceptible to collagen-induced arthritis but is free of spontaneous thyroiditis. The LEW.1WR1 rat provides a new model for studying autoimmune diabetes and arthritis in an animal with a genetic predisposition to both disorders that can be amplified by environmental perturbation.

The BBZDR/Wor rat model for investigating the complications of type 2 diabetes mellitus.

Congenic and inbred strains of rats offer researchers invaluable insight into the etiopathogenesis of diabetes and associated complications. The inbred Bio-Breeding Zucker diabetic rat (BBZDR)/Wor rat strain is a relatively new and emerging model of type 2 diabetes. This strain was created by classical breeding methods used to introgress the defective leptin receptor gene (Lepr(fa)) from insulin-resistant Zucker fatty rats into the inbred BBDR/Wor strain background. The diabetic male BBZDR/Wor rat is homozygous for the fatty mutation and shares the genetic background of the original BB strain. Although lean littermates are phenotypically normal, obese juvenile BBZDR/Wor rats are hyperlipidemic and hyperleptinemic, become insulin resistant, and ultimately develop hyperglycemia. Furthermore, the BBZDR/Wor rat is immune competent and does not develop autoimmunity. Similar to patients with clinical diabetes, the BBZDR/Wor rat develops complications associated with hyperglycemia. The BBZDR/Wor rat is a model system that fully encompasses the ability to study the complications that affect human type 2 diabetic patients. In this review, recent work that has evaluated type 2 diabetic complications in BBZDR/Wor rats is discussed, including the authors' preliminary unpublished studies on cardiovascular disease.

Biochemical and functional analysis of smallpox growth factor (SPGF) and anti-SPGF monoclonal antibodies.

Variola, the causative agent of smallpox, is a highly infectious double-stranded DNA virus of the orthopox genus that replicates within the cytoplasm of infected cells. For unknown reasons prominent skin manifestations, including "pox," mark the course of this systemic human disease. Here we characterized smallpox growth factor (SPGF), a protein containing an epidermal growth factor (EGF)-like domain that is conserved among orthopox viral genomes, and investigated its possible mechanistic link. We show that after recombinant expression, refolding, and purification, the EGF domain of SPGF binds exclusively to the broadly expressed cellular receptor, erb-B1 (EGF receptor), with subnanomolar affinity, stimulating the growth of primary human keratinocytes and fibroblasts. High affinity monoclonal antibodies specific for SPGF reveal in vivo immunoprotection in a murine vaccinia pneumonia model by a mechanism distinct from viral neutralization. These findings suggest that blockade of pathogenic factor actions, in general, may be advantageous to the infected host.

The human cytomegalovirus US8 glycoprotein binds to major histocompatibility complex class I products.

Human cytomegalovirus US8 is a type I membrane protein that partially colocalizes with cellular endosomal and lysosomal proteins. Although US8 does not have discernible effects on the processing and cell surface distribution of major histocompatibility complex (MHC) class I products, we have demonstrated that US8 binds to MHC class I heavy chains in the endoplasmic reticulum.