Correlation of collagen fibril properties and inner limiting membrane thickness with vitreoretinal adhesion in human eyes.

Vitreoretinal adhesive strength is thought to play a mechanical role in various retinal diseases; however, collagen fibril properties and inner limiting membrane (ILM) thickness have not been quantitatively correlated to adhesive strength. In this work, we quantified the relationship between collagen fibril density, angle, length, and ILM thickness with vitreoretinal adhesive strength to advance our understanding of structure-function relationships in vitreoretinal adhesion. Following mechanical peel tests, human retinal sections from the equator and posterior pole of donors 42-89 years of age were extracted and processed for transmission electron microscopy. Collagen fibrils at the vitreoretinal interface were segmented and fibril density, angle, length, and ILM thickness quantified. Morphological measurements were correlated with vitreoretinal adhesion measured in the same location. We found that collagen fibril density was 1.6 times greater in the equator compared to the posterior pole across all ages (p=0.0305). Steady-state peel force showed a slight positive correlation with increasing density in both the equator and posterior pole, but was only statistically significant in the equator (p<0.05). Collagen fibril angle and length did not significantly vary with age or region. ILM thickness was 3.8 times thicker in the posterior pole compared to the equator (p<0.0001). ILM thickness was 1.8 times greater in eyes ≤60 years of age compared to eyes >60 of age (p=0.0136). Maximum peel force was significantly correlated with increasing ILM thickness in the equator (p=0.015). A similar trend was seen in the posterior pole, but this was not significant. These data suggest that collagen contributes to adhesion at the vitreoretinal interface, but the structure of the ILM is more influential on the initiation of separation between the vitreous and retina.

Haplotype variations in glutathione transferase zeta 1 influence the kinetics and dynamics of chronic dichloroacetate in children.

Dichloroacetate (DCA) is biotransformed by glutathione transferase zeta 1 (GSTZ1), a bifunctional enzyme that, as maleylacetoacetate isomerase (MAAI), catalyzes the penultimate step in tyrosine catabolism. DCA inhibits GSTZ1/MAAI, leading to delayed plasma drug clearance and to accumulation of potentially toxic tyrosine intermediates. Haplotype variability in GSTZ1 influences short-term DCA kinetics in healthy adults, but the impact of genotype in children treated chronically with DCA is unknown. Drug kinetics was studied in 17 children and adolescents with congenital mitochondrial diseases administered 1,2-(13) C-DCA. Plasma drug half-life and trough levels varied 3-6-fold, depending on GSTZ1/MAAI haplotype and correlated directly with urinary maleylacetone, a substrate for MAAI. However, chronic DCA exposure did not lead to progressive accumulation of plasma drug concentration; instead, kinetics parameters plateaued, consistent with the hypothesis that equipoise is established between the inhibitory effect of DCA on GSTZ1/MAAI and new enzyme synthesis. GSTZ1/MAAI haplotype variability affects DCA kinetics and biotransformation. However, these differences appear to be stable in most individuals and are not associated with DCA plasma accumulation or drug-associated toxicity in young children.

Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors.

BACKGROUND: Recurrent malignant brain tumors (RMBTs) carry a poor prognosis. Dichloroacetate (DCA) activates mitochondrial oxidative metabolism and has shown activity against several human cancers. DESIGN: We conducted an open-label study of oral DCA in 15 adults with recurrent WHO grade III - IV gliomas or metastases from a primary cancer outside the central nervous system. The primary objective was detection of a dose limiting toxicity for RMBTs at 4 weeks of treatment, defined as any grade 4 or 5 toxicity, or grade 3 toxicity directly attributable to DCA, based on the National Cancer Institute's Common Toxicity Criteria for Adverse Events, version 4.0. Secondary objectives involved safety, tolerability and hypothesis-generating data on disease status. Dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism. RESULTS: Eight patients completed at least 1 four week cycle. During this time, no dose-limiting toxicities occurred. No patient withdrew because of lack of tolerance to DCA, although 2 subjects experienced grade 0-1 distal parasthesias that led to elective withdrawal and/or dose-adjustment. All subjects completing at least 1 four week cycle remained clinically stable during this time and remained on DCA for an average of 75.5 days (range 26-312). CONCLUSIONS: Chronic, oral DCA is feasible and well-tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases. The importance of genetic-based dosing is confirmed and should be incorporated into future trials of chronic DCA administration.

Possible involvement of toll-like receptor 4/myeloid differentiation factor-2 activity of opioid inactive isomers causes spinal proinflammation and related behavioral consequences.

Opioid-induced glial activation and its proinflammatory consequences have been associated with both reduced acute opioid analgesia and the enhanced development of tolerance, hyperalgesia and allodynia following chronic opioid administration. Intriguingly, recent evidence demonstrates that these effects can result independently from the activation of classical, stereoselective opioid receptors. Here, a structurally disparate range of opioids cause activation of signaling by the innate immune receptor toll like receptor 4 (TLR4), resulting in proinflammatory glial activation. In the present series of studies, we demonstrate that the (+)-isomers of methadone and morphine, which bind with negligible affinity to classical opioid receptors, induced upregulation of proinflammatory cytokine and chemokine production in rat isolated dorsal spinal cord. Chronic intrathecal (+)-methadone produced hyperalgesia and allodynia, which were associated with significantly increased spinal glial activation (TLR4 mRNA and protein) and the expression of multiple chemokines and cytokines. Statistical analysis suggests that a cluster of cytokines and chemokines may contribute to these nociceptive behavioral changes. Acute intrathecal (+)-methadone and (+)-morphine were also found to induce microglial, interleukin-1 and TLR4/myeloid differentiation factor-2 (MD-2) dependent enhancement of pain responsivity. In silico docking analysis demonstrated (+)-naloxone sensitive docking of (+)-methadone and (+)-morphine to human MD-2. Collectively, these data provide the first evidence of the pro-nociceptive consequences of small molecule xenobiotic activation of spinal TLR4 signaling independent of classical opioid receptor involvement.

Immunological priming potentiates non-viral anti-inflammatory gene therapy treatment of neuropathic pain.

We recently described a non-viral gene therapy paradigm offering long-term resolution of established neuropathic pain in several animal models. Here, the requirements for long-term therapeutic effects are described, and evidence is provided for a mechanism of action based on immunological priming of the intrathecal (i.t.) space. Long-term pain reversal was achieved when two i.t. injections of various naked plasmid DNA doses were separated by 5 h to 3 days. We show that an initial DNA injection, regardless of whether a transgene is included, leads to an accumulation of phagocytic innate immune cells. This accumulation coincides with the time in which subsequent DNA injection efficacy is potentiated. We show the ability of non-coding DNA to induce short-term pain reversal that is dependent on endogenous interleukin-10 (IL-10) signaling. Long-term efficacy requires the inclusion of an IL-10(F129S) transgene in the second injection. Blockade of IL-10, by a neutralizing antibody, either between the two injections or after the second injection induces therapeutic failure. These results show that this gene therapy paradigm uses an initial 'priming' injection of DNA to induce accumulation of phagocytic immune cells, allowing for potentiated efficacy of a subsequent 'therapeutic' DNA injection in a time- and dose-dependent manner.

Anti-inflammatory cytokine gene therapy decreases sensory and motor dysfunction in experimental Multiple Sclerosis: MOG-EAE behavioral and anatomical symptom treatment with cytokine gene therapy.

Multiple Sclerosis (MS) is an autoimmune inflammatory disease that presents clinically with a range of symptoms including motor, sensory, and cognitive dysfunction as well as demyelination and lesion formation in brain and spinal cord. A variety of animal models of MS have been developed that share many of the pathological hallmarks of MS including motor deficits (ascending paralysis), demyelination and axonal damage of central nervous system (CNS) tissue. In recent years, neuropathic pain has been recognized as a prevalent symptom of MS in a majority of patients. To date, there have been very few investigations into sensory disturbances in animal models of MS. The current work contains the first assessment of hind paw mechanical allodynia (von Frey test) over the course of a relapsing-remitting myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE) rat model of MS and establishes the utility of this model in examining autoimmune induced sensory dysfunction. We demonstrate periods of both decreased responsiveness to touch that precedes the onset of hind limb paralysis, and increased responsiveness (allodynia) that occurs during the period of motor deficit amelioration traditionally referred to as symptom remission. Furthermore, we tested the ability of our recently characterized anti-inflammatory IL-10 gene therapy to treat the autoimmune inflammation induced behavioral symptoms and tissue histopathological changes. This therapy is shown here to reverse inflammation induced paralysis, to reduce disease associated reduction in sensitivity to touch, to prevent the onset of allodynia, to reverse disease associated loss of body weight, and to suppress CNS glial activation associated with disease progression in this model.

Familial absorptive hypercalciuria and renal tubular acidosis.

Hypercalciuria was considered as a secondary condition when associated with familial renal tubular acidosis. Later studies suggested that hypercalciuria could lead to renal tubular acidosis and nephrocalcinosis. Selected members of a family spanning five generations were studied. Renal tubular acidosis was present in eight subjects in three consecutive generations. Increased 24-hour urinary calcium excretion was present in nine subjects in three consecutive generations, alone in the younger generation, and in combination with renal tubular acidosis and nephrocalcinosis in the older generation. Calcium loading tests showed the absorptive nature of hypercalciuria in nine of 18 subjects studied. This report suggests that in this family the absorptive hypercalciuria is an autosomal dominant genetic defect with complete penetrance and variable expressivity which leads to renal tubular acidosis and nephrocalcinosis.