Human alpha defensin 5 expression in the human kidney and urinary tract.

BACKGROUND: The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection. CONCLUSIONS/SIGNIFICANCE: DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.

Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract.

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood-brain barrier gene delivery.

The greatest challenge in developing therapies for mucopolysaccharidosis (MPS) IIIB is to achieve efficient central nervous system (CNS) delivery across the blood-brain barrier (BBB). In this study, we used the novel ability of adeno-associated virus serotype 9 (AAV9) to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMV-hNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to the correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system (PNS) targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 months) neurological benefits in MPS IIIB mice, resulting in significant improvement in behavioral performance, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases.

Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice.

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.

Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response.

BACKGROUND: Recently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic. METHODS: Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system. RESULTS: Our results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFalpha, IFNalpha and Ifi30, and intraparenchymal lymphocyte infiltration. CONCLUSIONS: Our data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis.

Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

BACKGROUND: Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. METHODS: In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. RESULTS: We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. CONCLUSIONS: A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

Innate and adaptive immune activation in the brain of MPS IIIB mouse model.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.