Assessing disease severity in late infantile neuronal ceroid lipofuscinosis using quantitative MR diffusion-weighted imaging.

BACKGROUND AND PURPOSE: Late infantile neuronal ceroid lipofuscinosis (LINCL), a form of Batten disease, is a fatal neurodegenerative genetic disorder, diagnosed via DNA testing, that affects approximately 200 children in the United States at any one time. This study was conducted to evaluate whether quantitative data derived by diffusion-weighted MR imaging (DWI) techniques can supplement clinical disability scale information to provide a quantitative estimate of neurodegeneration, as well as disease progression and severity. MATERIALS AND METHODS: This study prospectively analyzed 32 DWI examinations from 18 patients having confirmed LINCL at various stages of disease. A whole-brain apparent diffusion coefficient (ADC) histogram was fitted with a dual Gaussian function combined with a function designed to model voxels containing a partial volume fraction of brain parenchyma versus CSF. Previously published whole-brain ADC values of age-matched control subjects were compared with those of the LINCL patients. Correlations were tested between the peak ADC of the fitted histogram and patient age, disease severity, and a CNS disability scale adapted for LINCL. RESULTS: ADC values assigned to brain parenchyma were higher than published ADC values for age-matched control subjects. ADC values between patients and control subjects began to differ at 5 years of age based on 95% confidence intervals. ADC values had a nearly equal correlation with patient age (R2=0.71) and disease duration (R2=0.68), whereas the correlation with the central nervous system disability scale (R2=0.27) was much weaker. CONCLUSION: This study indicates that brain ADC values acquired using DWI may be used as an independent measure of disease severity and duration in LINCL.

Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

Brain Region-Specific Degeneration with Disease Progression in Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2 Disease).

BACKGROUND AND PURPOSE: Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene. Our hypothesis was that regional analysis of cortical brain degeneration may identify brain regions that are affected earliest and most severely by the disease. MATERIALS AND METHODS: Fifty-two high-resolution 3T MR imaging datasets were prospectively acquired on 38 subjects with CLN2. A retrospective cohort of 52 disease-free children served as a control population. The FreeSurfer software suite was used for calculation of cortical thickness. RESULTS: An increased rate of global cortical thinning in CLN2 versus control subjects was the primary finding in this study. Three distinct patterns were observed across brain regions. In the first, subjects with CLN2 exhibited differing rates of cortical thinning versus age. This was true in 22 and 26 of 34 regions in the left and right hemispheres, respectively, and was also clearly discernable when considering brain lobes as a whole and Brodmann regions. The second pattern exhibited a difference in thickness from healthy controls but with no discernable change with age (9 left hemispheres, 5 right hemispheres). In the third pattern, there was no difference in either the rate of cortical thinning or the mean cortical thickness between groups (3 left hemispheres, 3 right hemispheres). CONCLUSIONS: This study demonstrates that CLN2 causes differential rates of degeneration across the brain. Anatomic and functional regions that degenerate sooner and more severely than others compared with those in healthy controls may offer targets for directed therapies. The information gained may also provide neurobiologic insights regarding the mechanisms underlying disease progression.

Chemical approaches to the study of protein tyrosine kinases and their implications for mechanism and inhibitor design.

Protein tyrosine kinases are critical enzymes for signal transduction. Using C-terminal Src kinase (Csk) as a model system, we discuss progress in three main areas. First, we describe our efforts to measure the transition state of the reaction using peptide substrates containing fluorotyrosine analogs. It is shown that the Brønsted nucleophile coefficient for the reaction is near zero (similar to the nonenzymatic reaction) and the required nucleophile is the neutral phenol (rather than the more chemically reactive phenoxide anion). By studying the kinase reaction in the reverse direction, a Brønsted leaving group coefficient of -0.3 was measured, indicative of protonation of the departing phenol in the transition state. Taken together, these results strongly support a dissociative transition state mechanism for the kinase. These findings set constraints on the design of transition state analog inhibitors. Second, we describe efforts toward defining the specificity of Csk for peptide and protein substrates. The main findings are that local amino acids surrounding a phosphorylated tyrosine can influence recognition, but that long-range interactions probably are more important in a physiologic protein substrate. These findings underscore the complexities in how protein kinases select protein substrates. Third, we describe a new method in protein engineering that has been applied to the study of protein kinases. The method, expressed protein ligation, allows a general approach for ligating synthetic peptides to recombinant proteins. Using expressed protein ligation, obtaining site-specifically phosphorylated proteins and proteins with the incorporation of biophysical probes becomes relatively straightforward. We have used this method to generate a tail phosphorylated, conformationally altered Csk that showed an unexpected increase in kinase activity.

Feasibility of gene therapy for late neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis is a progressive childhood neurodegenerative disorder characterized by intracellular accumulation of autofluorescent material resembling lipofuscin in neuronal cells. This report summarizes the new therapies under consideration for late infantile neuronal ceroid lipofuscinosis, with a focus on strategies for in vivo gene therapy for the retinal and central nervous system manifestations of the disease.

Antivector and antitransgene host responses in gene therapy.

Current viral gene therapy vectors effectively transfer genes in vivo at the price of eliciting innate and acquired host responses against the vector and/or transgene. Antigens present in the viral vector and the expression of the transgene both cause cellular and humoral immune responses dependent on the viral vector, the route of administration, and the genotype and infection history of the host. In general, adenoviral vectors cause strong immune responses, which result in only transient expression of the therapeutic gene. Adeno-associated virus and retrovirus vectors elicit weaker immune responses and can therefore result in long-term gene transfer and expression. Methods to avoid host responses, including modification of viral vector and immunosuppression of the host, can increase the longevity and efficiency of gene transfer.

Wernicke's encephalopathy in a non-alcoholic man: case report and brief review.

Wernicke's encephalopathy, a serious neurological disorder caused by thiamine deficiency, is most commonly found in chronic alcoholics. We present a typical case of Wernicke's encephalopathy in a non-alcoholic man. Our patient presented with altered mental status, slurred speech, fever, vomiting and headache of one-week duration. An infectious etiology of the symptoms was ruled out by spinal fluid cultures. The patient improved dramatically within 24 hours of administration of thiamine.

Castleman's disease and superior vena cava thrombi: a rare presentation and a review of the literature.

Castleman's disease is a clinicopathological entity in which growth of lymphoid tissue is unregulated. It may present as asymptomatic involvement of one lymph node group or as a multicentric disease with systemic symptoms. Unlike localized disease, for which surgical excision is curative regardless of the histological type, multicentric disease often necessitates aggressive systemic therapy and portends a poor outcome. Superior vena caval thrombosis is an uncommon manifestation associated with Castleman's disease.

Microscopic polyangiitis presenting as idiopathic pulmonary fibrosis: is anti-neutrophilic cytoplasmic antibody testing indicated?

We report a 55-year old woman with microscopic polyangiitis who presented with idiopathic pulmonary fibrosis and 1 year later developed hematuria and proteinuria. She had a high serum level of perinuclear anti-neutrophilic cytoplasmic antibodies. Renal angiogram was normal. The diagnosis of microscopic polyangiitis was confirmed by renal biopsy, which showed pauci-immune crescentic glomerulonephritis. The patient received immunosuppressive therapy and improved markedly. Consideration of small vessel vasculitis is important in the differential diagnosis of idiopathic pulmonary fibrosis.

Assessment of disease severity in late infantile neuronal ceroid lipofuscinosis using multiparametric MR imaging.

BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. METHODS: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). RESULTS: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. CONCLUSION: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.

Expressed protein ligation: a general method for protein engineering.

A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Oro-dental pattern in mentally retarded.

The study was carried out in 25 mentally retarded children and compared with equal number of normal children. They were subjected to detailed psychiatric evaluation and dental examination. The dental anomalies were corroborated with cephalometric analysis of lateral cephalograms. It was concluded that all mentally retarded children had some dental abnormality in them in form of dental malocclusion, wide inter dental spaces, absence of teeth etc. We suggest early dental management for such patients for reinforcing their neuromuscular coordination modifying the mastication power, swallowing, speech, stomatognathic function and above all their facial profile for better social acceptance.

Peptide and protein phosphorylation by protein tyrosine kinase Csk: insights into specificity and mechanism.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in down-regulation of Src family members. The molecular basis of Csk's substrate specificity and catalytic mechanism with a protein substrate was investigated. Using a peptide library approach, preferential amino acids which are unrelated to the conserved Src C-terminal sequence were identified. The validity of these preferences was confirmed by synthesizing a short consensus peptide and demonstrating its high catalytic efficiency with Csk. These results underscore the difficulties of relying on amino acids neighboring tyrosine in protein sequences as predictors of protein kinase substrate specificity for in vivo analysis. In addition, a catalytically inactive version of the Src family member, Lck (lymphoid cell kinase), was expressed, purified, and evaluated as a Csk substrate. It was proven to be the most catalytically efficient substrate yet identified for Csk. The high efficiency of purified Csk phosphorylating a pure, unphosphorylated Src family member argues against the importance of an SH2-phosphotyrosine docking interaction or the involvement of extra recruitment proteins in facilitating Csk phosphorylation of Src family members. Kinetic studies revealed that the chemical step is at least partially rate-determining in Csk-mediated phosphoryl transfer to the Lck protein. Other properties including preferences for Mn over Mg, thio effects, and Km's for ATP also correlate fairly well between protein and peptide phosphorylation. The lack of a significant impact of increased salt on the Km for Lck phosphorylation differs from Csk-mediated poly(Glu,Tyr) phosphorylation, and argues against the importance of electrostatic effects in the Csk-Lck binding interaction. The failure of the Lck phosphorylation product (phosphotyrosine-505) to significantly inhibit Csk phosphorylation of Lck is consistent with a catalytic model involving multidomain structural interactions between substrate and enzyme.