Synergistic Effects of GDNF and VEGF on Lifespan and Disease Progression in a Familial ALS Rat Model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.     

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca²⁺-coordinating residues. The data explain the Ca²⁺-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.     

A phase I trial of immunostimulatory CpG 7909 oligodeoxynucleotide and 90yttrium ibritumomab tiuxetan radioimmunotherapy for relapsed B‐cell non‐Hodgkin lymphoma

Radioimmunotherapy (RIT) for relapsed indolent non‐Hodgkin lymphoma produces overall response rates (ORR) of 80% with mostly partial remissions. Synthetic CpG oligonucleotides change the phenotype of malignant B‐cells, are immunostimulatory, and can produce responses when injected intratumorally and combined with conventional radiation. In this phase I trial, we tested systemic administration of both CpG and RIT. Eligible patients had biopsy‐proven previously treated CD20+ B‐cell NHL and met criteria for RIT. Patients received rituximab 250 mg/m² days 1,8, and 15; ¹¹¹In‐ibritumomab tiuxetan days 1, 8; CpG 7909 days 6, 13, 20, 27; and 0.4 mCi/kg of ⁹⁰Y‐ibritumomab tiuxetan day 15. The doses of CpG 7909 tested were 0.08, 0.16, 0.32 (six patients each) and 0.48 mg/kg (12 patients) IV over 2 hr without dose limiting toxicity. The ORR was 93% (28/30) with 63% (19/30) complete remission (CR); median progression free survival of 42.7 months (95% CI 18‐NR); and median duration of response (DR) of 35 months (4.6–76+). Correlative studies demonstrated a decrease in IL10 and TNFα, and an increase in IL1β, in response to therapy. CpG 7909 at a dose of 0.48 mg/kg is safe with standard RIT and produces a high CR rate and long DR; these results warrant confirmation. Am. J. Hematol. 88:589–593, 2013. © 2013 Wiley Periodicals, Inc.     

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.The identification of the genetic causes of rare Mendelian diseases is becoming increasingly important following some success with gene-based therapy, as recently reported for patients with a form of Leber congenital amaurosis (LCA), a severe autosomal recessive hereditary retinal dystrophy (1–3). The evidence that restoring a gene in the diseased retina could yield therapeutic effects has stimulated the pursuit of the genetic causes of other retinal dystrophies, including retinitis pigmentosa (RP).RP is the name given to a group of hereditary retinal conditions in which degeneration of rod photoreceptors, responsible for vision under starlight or moonlight conditions, is more pronounced than that of cone photoreceptors, which mediate daylight vision. Individuals with RP typically experience night blindness at first, followed by progressive and unstoppable visual impairment in daytime conditions as well (4). Their visual fields become reduced gradually and sight is lost from the midperiphery to the periphery and then from the midperiphery to the center, resulting eventually in complete or near-complete blindness if left untreated. Most patients show intraretinal pigment in a bone spicule configuration around the fundus periphery, for which this condition was named. In addition, they typically show retinal arteriolar attenuation, elevated final dark adapted thresholds, and reduced and delayed electroretinograms (ERGs) (4). Vitamin A supplementation in combination with an omega-3 rich diet can slow the course of retinal degeneration and preserve visual acuity among adults with this condition (5, 6). Autosomal, recessively inherited RP (ARRP) is the most common form of hereditary retinal degeneration in humans. To date, over 50 genes have been associated with ARRP and allied disorders, among patients who are predominantly of European ancestry (RetNet; www.sph.uth.tmc.edu/retnet/home.htm). However, despite this high number of identified disease genes, ∼40–50% of all diagnosed cases have no mutations in recognized loci (7). Furthermore, genetic defects in RP are also population specific. For example, a screening of 193 unrelated Japanese patients with isolate or autosomal recessive RP for 30 disease genes identified commonly within North American or European patients revealed candidate pathogenic mutations in only 14% of the cohort (8).Recent advances in massively parallel sequencing have enabled the analysis of large amounts of sequences (genes) at reasonable costs, revolutionizing the traditional approach of exon-by-exon Sanger sequencing (9). The two major forms of sequencing strategies allowing large-scale analyses are whole genome sequencing (WGS) and whole exome sequencing (WES). The former reads the entire genome with no distinction between exons and nonexonic regions. It allows the detection of intergenic variants, copy number variations (CNVs), and other structural rearrangements, as well as unrecognized exonic sequences. The latter technique relies on targeted DNA capture and focuses on the analysis of the known exonic content of the genome, performed according to the genomic annotation available at a given point in time.In this study, we performed WGS as a method for mutation discovery in a highly genetically heterogeneous Mendelian disease; to this end, we evaluated 16 unrelated RP patients from diverse ethnic backgrounds.     

Correlation of interferon‐inducible chemokine plasma levels with disease severity in systemic sclerosis

Objective

To measure interferon (IFN)–inducible chemokines in the plasma of patients with systemic sclerosis (SSc) and investigate whether the chemokine levels are correlated with disease severity.

Methods

Plasma levels of the IFN‐inducible chemokines IFNγ‐inducible protein 10 (IP‐10/CXCL10), IFN‐inducible T cell α chemoattractant (I‐TAC/CXCL11), and monocyte chemoattractant protein 1 (CCL2) were measured in SSc patients and examined for correlation with the IFN gene expression signature. A composite IFN‐inducible chemokine score was generated for chemokines showing a correlation with the IFN gene signature (IP‐10 and I‐TAC), and this score was compared between 266 patients with SSc enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 97 matched control subjects. Subsequently, the correlation between the IFN‐inducible chemokine score at baseline and markers of disease severity was assessed. In addition, the course of the IFN‐inducible chemokine score over time was examined.

Results

The plasma IFN‐inducible chemokine score correlated with the IFN gene expression signature, and this score was higher in SSc patients compared to controls. The IFN‐inducible chemokine score was also associated with the absence of anti–RNA polymerase III antibodies and presence of anti–U1 RNP antibodies, but not with disease duration, disease type, or other autoantibodies. The chemokine score correlated with concomitantly obtained scores on the Medsger Severity Index for muscle, skin, and lung involvement in SSc, as well as the forced vital capacity, diffusing capacity for carbon monoxide, and creatine kinase levels. The association of the chemokine score with disease severity was independent of the presence of anti–U1 RNP or other potential confounders (age, sex, ethnicity, disease duration, and treatment with immunosuppressive agents). Finally, there was not a significant change in the IFN‐inducible chemokine score over time.

Conclusion

The IFN‐inducible chemokine score is a stable serologic marker of a more severe form of SSc and may be useful for risk stratification of patients, regardless of disease type (limited or diffuse) or duration of disease.

     

Evidence for volume regulatory increase by Na-K-Cl co-transport in rat arterial smooth muscle.

A component of K efflux (86Rb as tracer) from rat femoral arterial smooth muscle was blocked by loop diuretics. Following hypertonic challenge with 100 mM-sucrose, K efflux increased but not in the presence of loop diuretics. This is taken as evidence for a volume regulatory increase mediated by Na-K-Cl co-transport.     

Inhibition of arachidonic acid-induced ear oedema as a model for assessing topical anti-inflammatory compounds

We have found that mouse ear oedema induced by the topical application of arachidonic acid is not a specific screen for compounds inhibiting the lipoxygenase or cyclo-oxygenase pathways of arachidonic acid metabolism. Although such compounds are able to reduce the oedema substantially, pharmacological agents such as histamine antagonists, phosphodiesterase inhibitors, free radical scavengers, and also various compounds not normally considered to have anti-inflammatory properties, can equally effectively reduce the oedema. A mutual potentiation of the effects of prostaglandins, leukotrienes and mast cell-derived histamine would allow many, but not all, of the active agents to be rationalised. The ability of compounds not influencing these three types of inflammatory mediators to reduce the oedematous response means the model is of limited value for directed screening.     

Vasopressin-resistant diabetes insipidus, liver dysfunction, hyperuricemia and decreased renal function. A case report

A case of transient nephrogenic diabetes insipidus occurred in late pregnancy, the first associated with biopsy-proven hepatitis. Five previously reported cases occurred in pregnancy. All six patients demonstrated similar but transient signs, symptoms and laboratory abnormalities suggesting a syndrome peculiar to pregnancy. The characteristics of this syndrome are elevated liver function studies, decreased renal function, hyperuricemia and transient vasopressin-resistant diabetes insipidus.