Immuno- and genetic therapy in autoimmune diseases

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.     

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

Human mini-chromosomes in mouse embryonal stem cells

We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini- chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re- arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini- chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.      

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Survival outcomes of patients with giant cell myocarditis bridged by ventricular assist devices

Giant cell myocarditis is a highly lethal disorder characterized by rapidly progressive congestive heart failure. The aim of this study was to describe the clinical course of patients with giant cell myocarditis who received a ventricular assist device. Patients with giant cell myocarditis were identified from the Multicenter Giant cell Myocarditis Registry. Bridging to cardiac transplantation in the giant cell myocarditis patients who received a ventricular assist device was compared with bridging in the general population of heart failure patients, as reported in the literature. Median posttransplantation survival for patients with giant cell myocarditis who received and did not receive ventricular assist devices was calculated by the Kaplan-Meier method and compared with use of the log-rank test. Nine patients with giant cell myocarditis who received ventricular assist devices were identified. Seven patients survived to transplantation, four were alive 30 days posttransplantation, and two survived to 1 year. The rate of successful bridging to transplantation in seven of nine patients (78%) is similar to that reported for other ventricular assist device recipients. Posttransplantation survival of 57% (4 of 7) at 30 days and 29% (2 of 7) at 1 year was significantly lower compared with 93% 1-year survival of the 30 patients with giant cell myocarditis who did not receive ventricular assist devices before transplantation (p<0.001). Ventricular assist devices can be an effective bridge to transplantation for patients with heart failure caused by giant cell myocarditis. Although their posttransplantation survival was poor in our series, a few patients had long-term survival.     

High-sensitivity PCR detection of parvovirus B19 in plasma

Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10(4) genome equivalents/ml may not be infectious, while those greater than 10(7)/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10(4) IU/ml (6.5 x 10(3) genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 x 10(3) IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 x 10(3) to 2 x 10(5) IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 +/- 17.5 IU/ml (mean +/- standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.     

Do H2-receptor antagonists inhibit gastric secretion induced by cholinergic agonists?

The effect of experimental design on the ability of metiamide to inhibit gastric secretion induced by histamine, bethanechol and carbachol has been investigated in the Heidenhain pouch dog. All three secretagogues can be inhibited by metiamide to an extent depending on the experimental design.     

Essential tremor, nystagmus and duodenal ulceration. A "new" dominantly inherited condition.

The familial occurrence of essential tremor combined with (congenital) nystagmus, duodenal ulceration and a narcolepsy-like sleep disturbance caused by an autosomal dominant gene with high penetrance and fairly uniform expressivity is reported in a family of Swedish-Finnish ancestry. Twelve of 17 affected family members had essential tremor which began between 30-40 years of age and which could be controlled temporarily by alcohol; this resulted in alcoholism in several affected individuals. The most severly affected persons showed cerebellar signs which may reflect a possible pathogenetic relationship of the syndrome to the genetic cerebellar atrophies. Nystagmus, observed in 12 of 17 affected family members (eight of whom were also affected with tremor) usually was congenital and accompanied by refractive errors. Duodenal ulcers occurred almost exclusively in individuals with the neurological syndrome, and preceded its onset in some cases. The ulcer disease therefore seems to be a component manifestation of the syndrome and is interpreted as a pleiotropic effect of the gene which also causes the nystagmus, tremor and sleep disturbance.