Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis

Autoimmune diseases such as systemic lupus erythematosus are complex genetic traits with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Studies of animal models of lupus have provided important insight into the immunopathogenesis of disease, and genetic analyses of these models overcome certain obstacles encountered when studying human patients. Genome-wide scans of different genetic crosses have been used to map several disease-linked loci in New Zealand hybrid mice. Although some consensus exists among studies mapping the New Zealand Black (NZB) and New Zealand White (NZW) loci that contribute to lupus-like disease, considerable variability is also apparent. A variable in these studies is the genetic background of the non-autoimmune strain, which could influence genetic contributions from the affected strain. A direct examination of this question was undertaken in the present study by mapping NZB nephritis-linked loci in backcrosses involving different non-autoimmune backgrounds. In a backcross with MHC-congenic C57BL/6J mice, H2z appeared to be the strongest genetic determinant of severe lupus nephritis, whereas in a backcross with congenic BALB/cJ mice, H2z showed no influence on disease expression. NZB loci on chromosomes 1, 4, 11, and 14 appeared to segregate with disease in the BALB/cJ cross, but only the influence of the chromosome 1 locus spanned both crosses and showed linkage with disease when all mice were considered. Thus, the results indicate that contributions from disease-susceptibility loci, including MHC, may vary markedly depending on the non-autoimmune strain used in a backcross analysis. These studies provide insight into variables that affect genetic heterogeneity and add an important dimension of complexity for linkage analyses of human autoimmune disease.

Localization of non-Mhc collagen-induced arthritis susceptibility loci in DBA/1j mice

One approach to understanding common human diseases is to determine the genetic defects responsible for similar diseases in animal models and place those defective genes in their corresponding biochemical pathways. Our laboratory is working with an animal model for human rheumatoid arthritis called collagen-induced arthritis (CIA). We are particularly interested in determining the location of disease-predisposing loci. To that end, we performed experiments to localize susceptibility loci for CIA in an F2 cross between the highly susceptible mouse strain DBA/1j and the highly resistant mouse strain SWR/j. Specifically, a quantitative trait locus analysis was performed to localize regions of the mouse genome responsible for susceptibility/severity to CIA. One susceptibility locus, Cia1 in the major histocompatibility locus, had been identified previously. Two additional loci were detected in our analysis that contribute to CIA severity (Cia2, Cia3) on chromosomes 2 and 6. A third locus was detected that contributes to the age of onset of the disease. This locus (Cia4) was located on chromosome 2 and was linked to the same region as Cia2. Determining the identity of these loci may provide insights into the etiology of human rheumatoid arthritis.

An initiator element mediates autologous downregulation of the human type A γ-aminobutyric acid receptor β1 subunit gene

The regulated expression of type A γ-aminobutyric acid receptor (GABAAR) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABAAR subunit gene expression, including decreased β1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABAAR number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABAAR β1 subunit gene that is neuron specific and autologously down-regulated by GABA. β1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABAAR activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3′ flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of β1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of β1 subunit GABAAR gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.

Modifications of retinal neurons in a mouse model of retinitis pigmentosa

Animal models of retinitis pigmentosa include the rd mouse, in which a mutation of a rod-specific phosphodiesterase leads to the rapid loss of photoreceptors during the early postnatal life. Very little is known about changes occurring in inner retinal neurons after photoreceptor loss. These changes are important in view of the possibility of restoring vision in retinas with photoreceptor degeneration by means of cell transplantation or direct stimulation of inner layers. In this paper, we show that bipolar and horizontal cells of the rd mouse retina undergo dramatic morphological modifications accompanying photoreceptor loss, demonstrating a dependence of second order neurons on these cells. While describing modifications of the rd retina, we also provide quantitative information about neurons of the wild-type mouse retina, useful for future studies on genetically altered animals.

Increased anxiety of mice lacking the serotonin1A receptor

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT1A receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT1A receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.

Repetitive transcranial magnetic stimulation activates specific regions in rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.

Decreased ability of HIV-1 Tat protein-treated accessory cells to organize cellular clusters is associated with partial activation of T cells

It has been shown in several animal models that HIV infection of accessory cells (ACs) plays an important role in development of AIDS. Here, we report that ACs treated with HIV-1 Tat protein (Tat-ACs) have a decreased ability to organize cellular aggregates as compared with untreated ACs, resulting in incomplete activation of T cells in responses to anti-CD3 mAb or staphylococcal enterotoxin B stimulation. The T cells failed to up-regulate adhesion molecules CD11a and CD2 on the cell surface and had reduced proliferative responses, as determined by [3H]thymidine incorporation, but they obtained lymphoblast-like morphology and expressed early activation antigens on the cell surface such as Fas and CD69 and interleukin 2 receptor, at comparable levels as those T cells undergoing a maximal proliferation. These results suggest that the Tat-AC-induced defect occurs in the late, but not in the early, phases of T cell activation. Normal expression of cell surface Fas antigen accompanied by defects in late activation thus may result in the susceptibility of these T cells to apoptosis. Our studies suggest that dysfunction, hyperactivation, and susceptibility to apoptosis, as observed with T cells isolated from HIV-infected individuals, may be, at least in part, a consequence of abnormal functions of ACs.

Intralymphatic immunization enhances DNA vaccination

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8+ cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, α-myosin heavy chain promoter (α-MHC). In unstressed mice, forced expression of hMCIP1 resulted in a 5–10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy that otherwise result from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibited hypertrophic responses to β-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure.

Unbalanced expression of the different subunits of elongation factor 1 in diabetic skeletal muscle

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1α. With Northern blot analysis, EF-1α expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1β or -γ were noted. We observed similar results in animal models of Type 1 diabetes. EF-1α expression, but not EF-1β or -γ expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1α mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Regional and cellular fractionation of working memory

This chapter recounts efforts to dissect the cellular and circuit basis of a memory system in the primate cortex with the goal of extending the insights gained from the study of normal brain organization in animal models to an understanding of human cognition and related memory disorders. Primates and humans have developed an extraordinary capacity to process information “on line,” a capacity that is widely considered to underlay comprehension, thinking, and so-called executive functions. Understanding the interactions between the major cellular constituents of cortical circuits—pyramidal and nonpyramidal cells—is considered a necessary step in unraveling the cellular mechanisms subserving working memory mechanisms and, ultimately, cognitive processes. Evidence from a variety of sources is accumulating to indicate that dopamine has a major role in regulating the excitability of the cortical circuitry upon which the working memory function of prefrontal cortex depends. Here, I describe several direct and indirect intercellular mechanisms for modulating working memory function in prefrontal cortex based on the localization of dopamine receptors on the distal dendrites and spines of pyramidal cells and on interneurons in the prefrontal cortex. Interactions between monoamines and a compromised cortical circuitry may hold the key to understanding the variety of memory disorders associated with aging and disease.

Reconsidering targeted toxins to eliminate HIV infection: You gotta have HAART

The success of highly active anti-retroviral therapy (HAART) has inspired new concepts for eliminating HIV from infected individuals. A major obstacle is the persistence of long-lived reservoirs of latently infected cells that might become activated at some time after cessation of therapy. We propose that, in the context of treatment strategies to deliberately activate and eliminate these reservoirs, hybrid toxins targeted to kill HIV-infected cells be reconsidered in combination with HAART. Such combinations might also prove valuable in protocols aimed at preventing mother-to-child transmission and establishment of infection immediately after exposure to HIV. We suggest experimental approaches in vitro and in animal models to test various issues related to safety and efficacy of this concept.