SUMMARY Acute Trypanosoma cruzi infections can be asymptomatic, but chronically infected individuals can die of Chagas' disease. The transfer of the parasite mitochondrial kinetoplast DNA (kDNA) minicircle to the genome of chagasic patients can explain the pathogenesis of the disease; in cases of Chagas' disease with evident cardiomyopathy, the kDNA minicircles integrate mainly into retrotransposons at several chromosomes, but the minicircles are also detected in coding regions of genes that regulate cell growth, differentiation, and immune responses. An accurate evaluation of the role played by the genotype alterations in the autoimmune rejection of self-tissues in Chagas' disease is achieved with the cross-kingdom chicken model system, which is refractory to T. cruzi infections. The inoculation of T. cruzi into embryonated eggs prior to incubation generates parasite-free chicks, which retain the kDNA minicircle sequence mainly in the macrochromosome coding genes. Crossbreeding transfers the kDNA mutations to the chicken progeny. The kDNA-mutated chickens develop severe cardiomyopathy in adult life and die of heart failure. The phenotyping of the lesions revealed that cytotoxic CD45, CD8 + γδ, and CD8α + T lymphocytes carry out the rejection of the chicken heart. These results suggest that the inflammatory cardiomyopathy of Chagas' disease is a genetically driven autoimmune disease.

Chagas disease is the clinical condition triggered by infection with the protozoan Trypanosoma cruzi. The infection is transmitted by triatomine insects while blood feeding on a human host. Field studies predict that one third of an estimated 18 million T cruzi-infected humans in Latin America will die of Chagas disease. Acute infections are usually asymptomatic, but the ensuing chronic T cruzi infections have been associated with high ratios of morbidity and mortality: Chagas heart disease leads to unexpected death in 37.5% of patients, 58% develop heart failure and die and megacolon or megaoesophagus has been associated with death in 4.5%. The pathogenesis of Chagas disease appears to be related to a parasite-induced mutation of the vertebrate genome. Currently, treatment is unsatisfactory.

BackgroundThe administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis.Methodology/Principal FindingsTo understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45+, CD8γδ+, and CD8α lymphocytes.Conclusions/SignificanceThese results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.

BackgroundInfection with the protozoan Trypanosoma cruzi manifests in mammals as Chagas heart disease. The treatment available for chagasic cardiomyopathy is unsatisfactory.Methods/Principal FindingsTo study the disease pathology and its inhibition, we employed a syngeneic chicken model refractory to T. cruzi in which chickens hatched from T. cruzi inoculated eggs retained parasite kDNA (1.4 kb) minicircles. Southern blotting with EcoRI genomic DNA digests revealed main 18 and 20 kb bands by hybridization with a radiolabeled minicircle sequence. Breeding these chickens generated kDNA-mutated F1, F2, and F3 progeny. A targeted-primer TAIL-PCR (tpTAIL-PCR) technique was employed to detect the kDNA integrations. Histocompatible reporter heart grafts were used to detect ongoing inflammatory cardiomyopathy in kDNA-mutated chickens. Fluorochromes were used to label bone marrow CD3+, CD28+, and CD45+ precursors of the thymus-dependent CD8α+ and CD8β+ effector cells that expressed TCRγδ, vβ1 and vβ2 receptors, which infiltrated the adult hearts and the reporter heart grafts.Conclusions/SignificanceGenome modifications in kDNA-mutated chickens can be associated with disruption of immune tolerance to compatible heart grafts and with rejection of the adult host's heart and reporter graft, as well as tissue destruction by effector lymphocytes. Autoimmune heart rejection was largely observed in chickens with kDNA mutations in retrotransposons and in coding genes with roles in cell structure, metabolism, growth, and differentiation. Moreover, killing the sick kDNA-mutated bone marrow cells with cytostatic and anti-folate drugs and transplanting healthy marrow cells inhibited heart rejection. We report here for the first time that healthy bone marrow cells inhibited heart pathology in kDNA+ chickens and thus prevented the genetically driven clinical manifestations of the disease.

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Currently, no effective treatments exist for Sjögren’s syndrome, and there is a limited understanding of the physiological mechanisms associated with xerostomia and hyposalivation. The present work revealed that aquaporin 5 expression, a water channel critical for salivary gland fluid secretion, is regulated by bone morphogenetic protein 6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjögren’s syndrome, the loss of salivary gland function. This finding led us to develop a therapy in the treatment of Sjögren’s syndrome by increasing the water permeability of the gland to restore saliva flow. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjögren’s syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease, with only palliative treatments available. Recent work has suggested that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction. We examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models. Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that occurs predominately in women, with a female to male ratio of 9-16:1 1,2 . Hallmarks of the disease include a loss of lacrimal and salivary gland (SG) function, the presence of lymphocytic infiltrates in SGs, and increased levels of proinflammatory cytokines and circulating autoantibodies. Patients report significant fatigue and a decreased quality of life. The etiology of pSS is unknown and treatment is limited to symptomatic care 2 .Previous studies reported that expression of bone morphogenetic protein 6 (BMP6) is increased in the SGs of some pSS patients and that this overexpression is linked to a decrease in SG function and increased lymphocytic infiltration of the gland 3,4 . Overexpression of BMP6 in the SGs of C57BL/6 mice results in a Sjögren's syndrome-like phenotype 3 . However, the downstream signals that are activated by BMP6 and induce SG dysfunction and autoimmunity, are still unknown. Gene therapy experiments in these mice have shown that reengineering the glandular epithelia to express aquaporin 1 (AQP1), to restore membrane water permeability, recovers some of the secretory function of the SG epithilia 4 . However, AQP1 gene therapy, does not correct all of the effects of increased expression of the BMP6 ligand that likely affects the activity of other cells in the gland, such as infiltrating lymphocytes and bone marrow mesenchymal stem cells 5,6 . Therefore, a systemic treatment that targets the fundamental signaling problem may be useful in correcting the loss of immune homeostasis associated with the autoimmunity seen in SS.The TGF-β cytokine superfamily is a group of 33 proteins, which include bone morphogenetic protein (BMP) and TGF members. Members of this family bind to and signal through specific type I and type II transmembrane serine/threonine kinase receptor complexes. The particular ligand determines the downstream signaling, which then activates specific intracellular...

Adeno-associated virus (AAV) vector technology is rapidly advancing and becoming not only the leading vector platform in the field of gene therapy but also a useful tool for functional genomic studies of novel proteins. As most vectors utilize constitutive promoters, this results in transgene expression during production. Depending on the transgene product, this could induce proapoptotic, cytostatic, or other unknown effects that interfere with producer cell function and, therefore, reduce viral vector yield. This can be a major limitation when trying to characterize poorly described genes. We describe the novel use of shRNA encoding plasmids cotransfected during packaging to limit the expression of the cytotoxic transgene product. This allowed the production of an otherwise unpackageable vector. The approach is simple, versatile, does not require modification of the vector plasmid, and should be easily adaptable to almost any transgene with minimal cost.

ObjectiveSjögren's disease (SjD) has a strong sex bias, suggesting an association with sex hormones. Male SjD represents a distinct subset of the disease, but the pathogenic mechanisms of male SjD is poorly characterized. The aim of this study is to identify initiating events related to the development of gland hypofunction and autoimmunity in male SjD patients.Materials and methodsHuman minor salivary glands were transcriptomically analyzed with microarrays to detect differentially expressed genes in male SjD patients. Identified genes were tested on their involvement in the disease using conditional transgenic mice and gene‐overexpressing cells.ResultsGPR78, an orphan G protein–coupled receptor, was overexpressed in the salivary glands of male SjD patients compared with male healthy controls and female SjD patients. Male GPR78 transgenic mice developed salivary gland hypofunction with increased epithelial apoptosis, which was not seen in control or female transgenic mice. In cell culture, GPR78 overexpression decreased lysosomal integrity, leading to caspase‐dependent apoptotic cell death. GPR78‐induced cell death in vitro was inhibited by treatment with estradiol.ConclusionGPR78 overexpression can induce apoptosis and salivary gland hypofunction in male mice through lysosomal dysfunction and increased caspase‐dependent apoptosis in salivary gland epithelium, which may drive disease in humans.