Upregulation of NADPH Oxidase 2 Contributes to Renal Fibrosis in pcy Mice: An Experimental Model of Nephronophthisis

<b><i>Background:</i></b> DBA/2FG-<i>pcy</i> (<i>pcy</i>) mice harbor a homozygous <i>Nphp3</i> missense mutation and develop nephronophthisis with renal interstitial fibrosis. Previous studies have shown that aberrant oxygen homeostasis contributes to the renal pathology in <i>pcy</i> mice, but the underlying molecular mechanism remains largely unknown. <b><i>Methods:</i></b> <i>pcy</i> mice and a control strain, DBA/2N (DBA) mice, were used. Renal levels of 62 mRNAs involved in oxygen homeostasis were investigated by real-time PCR, and the resulting data were used for extraction of pathological pathways. On the basis of the genes found to be upregulated and pathway analysis, further studies were performed using immunoblotting, immunohistochemistry, and pharmacological intervention. <b><i>Results:</i></b> In comparison with DBA mice, the levels of 18 mRNAs were altered by &#x3e;2-fold in <i>pcy</i> mice. Pathway analysis extracted molecular pathways related to oxidative stress, inflammation, and cell adhesion. As the levels of mRNAs relevant to the NADPH oxidase 2 (NOX2) pathway were prominently (4 genes &#x3e;5-fold) increased in <i>pcy</i> mice, we further analyzed the molecules related to this pathway. A time course study suggested that the pathway was gradually activated in <i>pcy</i> mice from at least 5 weeks of age. Immunohistochemistry study revealed that NOX2 protein was colocalized with a macrophage marker protein in the renal interstitium. Moreover, treatment of <i>pcy</i> mice with apocynin, an inhibitor of the NOX2 pathway, ameliorated the renal fibrosis. <b><i>Conclusion:</i></b> Our findings suggest that the activation of the NOX2 pathway, possibly mediated by macrophage infiltration, plays a pivotal role in progressive renal fibrosis in <i>pcy</i> mice.

Factor IX Delivery to the Skin Primes Inhibitor Formation and Sensitizes Hemophilia B Mice to Systemic Factor IX Administration

Inhibitor formation is the most serious complication of factor (F)IX replacement therapy for hemophilia B, exacerbated by anaphylactoid reactions occurring in up to 50% of inhibitor patients. Low success rates and a high burden of immune tolerance induction (ITI) therapy necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues (SALT) have been successfully targeted in allergen-specific immunotherapies. In this study, we aimed to develop a prophylactic immune tolerance protocol based on intradermal (ID) administration of rFIX. In a dose-finding experiment, hemophilia B mice on C3H/HeJ genetic background (C3H/HeJ-F9tm1Dws) received twice weekly ID injections of 0.01, 0.1 or 1 IU rFIX only for four weeks, followed by one intraperitoneal (IP) and four weekly intravenous (IV) injections of 1 IU FIX co-injected with anti-allergic agents (triprolidine and ABT-491). Control animals received the IP/IV doses only. One week after the last injection, plasma and/or lymph nodes (LNs) were collected for Bethesda assay, ELISA and flow cytometry analyses. Unexpectedly, all animals developed significantly (8.9-17.6-fold, p&lt;0.05) higher FIX inhibitor titers than the control group (mean 4.4, 3.9, 8.5 and 0.6 BU/ml in the 0.01, 0.1, 1 IU and control group, respectively). Anti-FIX IgG1 antibody levels were also significantly higher (2.7-3.4-fold, p&lt;0.001) in all ID treated groups (mean 54.8, 68.8, 69.3 and 20.1 µg/ml in the 0.01, 0.1, 1 IU and control group, respectively). This apparent enhancement of inhibitor formation prompted analyses of plasma samples collected after four weeks of ID FIX injections only to find out whether ID treatment alone could elicit FIX inhibitor formation. Two (0.1 and 1 IU) of the three evaluated ID FIX doses led to inhibitor formation of a similar magnitude to the full-length ID/IP/IV regimen. Although most (seven out of nine) mice treated with the lowest ID dose of FIX (0.01 IU) had no detectable inhibitors, the same dose, when followed by the IP/IV treatment, resulted in a similar inhibitor response to the two higher doses (0.1 and 1 IU), suggesting that the lowest dose primed the immune responses, which were further amplified by the IP/IV regimen. Also, all ID treated animals had readily detectable anti-FIX IgG1, but the levels were smaller than after the full-length treatment (mean 17.8, 35.6 and 33 µg/ml in the 0.01, 0.1 and 1 IU groups, respectively). This suggests that the IP/IV regimen following the ID treatment enhanced mainly the total anti-FIX IgG1 response, with a lesser impact on the neutralizing antibody development. Inhibitor formation following ID treatment seemed to be driven by T follicular helper (PD1 +CXCR5 +CD4 +) and Germinal Center B cell (GL7 +CD95 +CD19 +) responses in inguinal LNs, the frequencies of which were 1.75-fold and 4-fold (p&lt;0.05) elevated in animals treated with 1 IU FIX ID compared to control mice that received PBS only. Notably, none of the ID treated mice died of anaphylaxis despite receiving eight ID injections of FIX without anti-allergic agents. IV administration of FIX alone in C3H/HeJ-F9tm1Dws hemophilia B mice results in fatal IgE-dependent anaphylaxis beginning after fourth injection with ~20% mortality and rising with subsequent injections in the surviving mice, which necessitates the use of anti-allergic agents. Interestingly, the ID treated mice had elevated levels of anti-FIX IgE antibodies. Animals that received ID injections only or followed by IP/IV treatment had 1.5- and 3.3-fold higher (p&lt;0.01) anti-FIX IgE levels than mice that received the IP/IV treatment only. The absence of mortality in the ID treated animals suggested that ID FIX delivery might have a neutral or protective effect against anaphylaxis despite anti-FIX IgE development. To test this hypothesis, hemophilia B mice received eight twice weekly ID doses of 1 IU FIX followed by IV or IV only, with neither group receiving anti-allergic agents at any time (n=15/group). After the first IV injection, 33.3% and 0% animals died in the ID treated and the IV only group, respectively, prompting early termination of the experiment for humane reasons. In conclusion, ID administration of FIX primes or produces strong inhibitor responses in a wide range of doses and sensitizes hemophilia B mice to systemic delivery of FIX, suggesting that the skin-associated lymphoid tissue may not be amenable to FIX tolerance induction. Disclosures No relevant conflicts of interest to declare.

Recombinant NAGLU-IGF2 prevents physical and neurological disease and improves survival in Sanfilippo B syndrome

Recombinant human alpha-N-acetylglucosaminidase-insulin-like growth factor-2 (rhNAGLU-IGF2) is an investigational enzyme replacement therapy for Sanfilippo B, a lysosomal storage disease. The fusion protein with IGF2 permits binding to the cation-independent mannose 6-phosphate receptor, because recombinant human NAGLU (rhNAGLU) is poorly mannose 6-phosphorylated. We previously administered rhNAGLU-IGF2 intracerebroventricularly to Sanfilippo B mice. We demonstrated therapeutic restoration of NAGLU, normalization of lysosomal storage, and improvement in markers of neurodegeneration and inflammation. Here, we studied intracerebroventricular rhNAGLU-IGF2 in murine and canine Sanfilippo B to determine potential effects on the behavioral phenotype and survival. Heparan sulfate (HS) levels in brain and heart were reduced following treatment with rhNAGLU-IGF2 or rhNAGLU. Treated mice showed improvement in disease markers such as beta-hexosaminidase, CD68, and LAMP1. We found a more normal number of stretch attend postures, a fear pose, in mice treated with either rhNAGLU or rhNAGLU-IGF2 vs vehicle-treated Sanfilippo B mice on elevated plus maze testing (p<0.001). We found a more normal dark/light activity pattern in Sanfilippo B mice treated with rhNAGLU-IGF2 compared to vehicle-treated Sanfilippo B mice (p=0.025). We found a 61% increase in survival in Sanfilippo B mice treated with rhNAGLU-IGF2 (mean 53d, median 48d) compared to vehicle-treated Sanfilippo B mice (mean 33d, median 37d; p<0.001). In canine Sanfilippo B, we found that rhNAGLU-IGF2 administered into cerebrospinal fluid normalized heparan sulfate and beta-hexosaminidase activity in gray and white matter brain regions. Proteomics analysis of cerebral cortex showed restoration of protein concentrations in pathways relevant to cognitive function, synapse, and the lysosome. Treatment with rhNAGLU-IGF2 may improve the phenotype of Sanfilippo B disease.

Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity

Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)–mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.

Constitutive activation of Lyn kinase enhances BCR responsiveness, but not the development of CLL in Eµ-TCL1 mice

The treatment of chronic lymphocytic leukemia (CLL) has been improved dramatically by inhibitors targeting B-cell receptor (BCR)–associated kinases. The tyrosine kinase Lyn is a key modulator of BCR signaling and shows increased expression and activity in CLL. To evaluate the functional relevance of Lyn for CLL, we generated a conditional knockin mouse model harboring a gain-of-function mutation of the Lyn gene (LynY508F), which was specifically expressed in the B-cell lineage (Lynup-B). Kinase activity profiling revealed an enhanced responsiveness to BCR stimulation in Lynup-B B cells. When crossing Lynup-B mice with Eµ-TCL1 mice (TCL1tg/wt), a transgenic mouse model for CLL, the resulting TCL1tg/wt Lynup-B mice showed no significant change of hepatomegaly, splenomegaly, bone marrow infiltration, or overall survival when compared with TCL1tg/wt mice. Our data also suggested that TCL1 expression has partially masked the effect of the Lynup-B mutation, because the BCR response was only slightly increased in TCL1tg/wt Lynup-B compared with TCL1tg/wt. In contrast, TCL1tg/wt Lynup-B were protected at various degrees against spontaneous apoptosis in vitro and upon treatment with kinase inhibitors targeting the BCR. Collectively, and consistent with our previous data in a Lyn-deficient CLL model, these data lend further suggest that an increased activation of Lyn kinase in B cells does not appear to be a major driver of leukemia progression and the level of increased BCR responsiveness induced by Lynup-B is insufficient to induce clear changes to CLL pathogenesis in vivo.

Ameliorative Effects of Kolaviron on Behavioural Deficits and Oxidative Damage in Prefrontal Cortex and Hippocampus of Cuprizone-Induced Demyelinated Mice

Cuprizone neurotoxicity is commonly induced to mimic demyelinating disorders of the central nervous system, especially multiple sclerosis. This study assessed the role of kolaviron, a Garcinia kola biflavonoid, in restoring behavioural functions in cuprizone-induced neurotoxicity after termination of cuprizone treatment. Eighteen adult male Swiss albino mice aged between 6-8 weeks were randomly divided into 3 equal groups (A to C). Group A (control) mice were fed with normal rodent diet while groups B and C received 0.2% cuprizone diet for 5 weeks to induce demyelination; thereafter group B mice with cuprizone-induced demyelination were administered corn oil (0.5 mL), while group C mice with cuprizone-induced demyelination were administered kolaviron (200 mg/kg/d) for 14 days. The mice were assessed for learning, memory, anxiety and exploratory drive, and thereafter the concentration of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the prefrontal cortex and hippocampus of the mice were evaluated. Findings revealed improved behavioural outcomes in mice treated with kolaviron compared to control and the untreated cuprizone-induced demyelinated mice. There was significant reduction in SOD and GPx activities with significant increase in MDA concentration in the untreated cuprizone-induced mice compared to controls. However, there was significant increase in SOD and GPx activities with significant reduction in MDA concentration in the kolaviron-treated cuprizone-induced mice compared to those of untreated cuprizone-induced mice. The results suggest that kolaviron may effectively ameliorate the oxidative damage and behavioural deficits associated with cuprizone-induced neurotoxicity.