Long-term liver disease in methylmalonic and propionic acidemias

Background and objectives

Patients affected with methylmalonic acidemia (MMA) and propionic acidemia (PA) exhibit diverse long-term complications and poor outcome. Liver disease is not a reported complication. The aim of this study was to characterize and extensively evaluate long-term liver involvement in MMA and PA patients.

The natural history of the inherited methylmalonic acidemias

Six biochemical and genetic forms of methylmalonic acidemia have been defined previously: two (mut degrees and mut-) resulting from defects in the mutase apoenzyme, and four (cbl A, cbl B, cbl C, and cbl D) resulting from deficient adenosylcobalamin synthesis. We retrospectively surveyed the clinical presentation, response to cobalamin supplementation, and long-term outcome in the four most prevalent mutant classes by collecting detailed information on 45 patients (15 mut degrees, 5 mut-, 14 cbl A, and 11 cbl B). Most patients presented acutely with a common set of clinical and laboratory findings; however, there were significant differences between mutant classes: mut degrees patients presented earlier in infancy than did cbl A and cbl B patients; in response to cobalamin supplements, marked decreases in the concentration of methylmalonic acid in blood or urine were reported in most cbl A patients and in nearly half the cbl B patients, but not in mut degrees or mut- patients; and finally, most cbl A, cbl B, and mut- patients were still living, whereas most mut degrees patients died during the first few months of life. Our data indicate that genotypic classification of the methylmalonic acidemias has prognostic and therapeutic use as well as diagnostic value.     

A critical reappraisal of dietary practices in methylmalonic acidemia raises concerns about the safety of medical foods. Part 1: isolated methylmalonic acidemias

PurposeMedical foods for methylmalonic acidemias (MMAs) and propionic acidemias contain minimal valine, isoleucine, methionine, and threonine but have been formulated with increased leucine. We aimed to assess the effects of imbalanced branched-chain amino acid intake on metabolic and growth parameters in a cohort of patients with MMA ascertained via a natural history study.MethodsCross-sectional anthropometric and body-composition measurements were correlated with diet content and disease-related biomarkers in 61 patients with isolated MMA (46 mut, 9 cblA, and 6 cblB).ResultsPatients with MMA tolerated close to the recommended daily allowance (RDA) of complete protein (mut⁰: 99.45 ± 32.05% RDA). However, 85% received medical foods, in which the protein equivalent often exceeded complete protein intake (35%). Medical food consumption resulted in low plasma valine and isoleucine concentrations, prompting paradoxical supplementation with these propiogenic amino acids. Weight- and height-for-age z-scores correlated negatively with the leucine-to-valine intake ratio (r = −0.453; P = 0.014; R² = 0.209 and r = −0.341; P = 0.05; R² = 0.123, respectively).ConclusionIncreased leucine intake in patients with MMA resulted in iatrogenic amino acid deficiencies and was associated with adverse growth outcomes. Medical foods for propionate oxidation disorders need to be redesigned and studied prospectively to ensure efficacy and safety.     

Feasibility of nonsense mutation readthrough as a novel therapeutical approach in propionic acidemia

Aminoglycosides and other compounds can promote premature termination codon (PTC) readthrough constituting a potential therapy for patients with nonsense mutations. In a cohort of 190 propionic acidemia (PA) patients, we have identified 12 different nonsense mutations, six of them novel, accounting for 10% of the mutant alleles. Using an in vitro system, we establish the proof-of-principle that nonsense mutations in the PCCA and PCCB genes encoding both subunits of the propionyl-CoA carboxylase (PCC) enzyme can be partially suppressed by aminoglycosides, with different efficiencies depending on the sequence context. To correct the metabolic defect, the amino acid incorporated at the PTC should support protein function, and this has been evaluated in silico and by in vitro expression analysis of the predicted missense changes, most of which retain partial activity, confirming the feasibility of the approach. In patients' fibroblasts cultured with readthrough drugs, we observe a fourfold to 50-fold increase in the PCC activity, reaching up to 10-15% level of treated control cells. The ability to partially correct nonsense PCCA and PCCB alleles represents a potential therapy or supplementary treatment for a number of propionic acidemia (PA) patients, encouraging further clinical trials with readthrough drugs without toxic effects such as PTC124 or other newly developed compounds. Hum Mutat 33:973-980, 2012. ? 2012 Wiley Periodicals, Inc.     

A novel approach for small sample size family-based association studies: sequential tests

In this paper, we propose a sequential probability ratio test (SPRT) to overcome the problem of limited samples in studies related to complex genetic diseases. The results of this novel approach are compared with the ones obtained from the traditional transmission disequilibrium test (TDT) on simulated data. Although TDT classifies single-nucleotide polymorphisms (SNPs) to only two groups (SNPs associated with the disease and the others), SPRT has the flexibility of assigning SNPs to a third group, that is, those for which we do not have enough evidence and should keep sampling. It is shown that SPRT results in smaller ratios of false positives and negatives, as well as better accuracy and sensitivity values for classifying SNPs when compared with TDT. By using SPRT, data with small sample size become usable for an accurate association analysis.     

A novel approach to the psychopharmacologic treatment of insomnia in depression

BACKGROUND: Depressed patients who respond to anti-depressant drugs but have persisting insomnia can be hypothesized as having one foot on the bipolar spectrum. In this hypothesis, the insomnia would respond to augmentation of the anti-depressants with anti-kindling agents (i.e., anti-convulsant drugs for the purposes of this paper) in the same way that many anti-convulsants are effective in bipolar disorders as mood-stabilizers from above (anti-manic agents) or below (anti-depressants). CASE REPORTS: Cases supporting this augmentation strategy are presented. The patients met DSM-IV criteria for recurrent major depression, depression with psychotic features, and depression NOS. There was no discernible history of mania or hypomania and all had insomnia that did not respond or partially responded to anti-depressant drugs singly or in combinations. All responded to the addition of anti-convulsant drugs. DISCUSSION: This augmentation strategy seems to be an effective treatment for chronic insomnia in depression, but controlled studies are needed to quantify and qualify these effects. This strategy's effectiveness may be due to the anti-kindling or inhibitory effects of anti-convulsant drugs, which are often GABAergic. Since it is becoming clear that bipolar spectrum disorders are misdiagnosed as unipolar depression at an alarming rate, and since anti-depressants often have deleterious effects on the course of bipolar illness, perhaps most depressed patients should be protected with an anti-convulsant whenever they are started on an anti-depressant.     

JAK/STAT inhibitors and other small molecule cytokine antagonists for the treatment of allergic disease

Objective

To provide an overview of janus kinase (JAK), chemoattractant receptor homologous molecule expressed on T_H2 cells (CRTH2), and phosphodiesterase 4 (PDE4) inhibitors in allergic disorders.

Cancer immunotherapeutic potential of novel small molecule TLR7 and TLR8 agonists

Toll-like receptor (TLR)-mediated signaling is proposed as an immunotherapeutic target against tumorigenesis. Natural killer (NK) cells play a critical role in host defense against tumors. Specifically, formation of tumor metastasis in various organs can be suppressed by the local activity of NK cells. In this study, we present a novel TLR7 agonist (termed SC-1) that induces pro-inflammatory cytokines in human blood cells, activates NK cell function, and is highly efficient in preventing lung metastases in a pulmonary metastatic Renca model. Furthermore, a second compound (termed SC-2), acting as dual-specific TLR7 and TLR8 agonist, was evaluated with respect to its immunostimulatory and NK cell–activating capacities. The release of pro-inflammatory cytokines was shown to be even more pronounced with this compound. Additional experiments showed a significant up-regulation of activation marker CD69 on NK cells and increased cytolytic activity of peripheral blood cells compared to the effect of a monospecific TLR7 agonist SC-1. Normally, TLR7 and TLR8 are expressed on different immune cell subpopulations. TLR7 expression on antigen-presenting cells is detected in plasmacytoid dendritic cells, CD34⁺-derived dendritic cells, and B-cells, whereas TLR8 is mainly expressed on cells of the myeloid lineage, such as monocytes, macrophages, and myeloid dendritic cells. Therefore, a compound that activates both TLR7 and TLR8 would result in a highly efficient immune system activation and may give rise to an enhanced anti-tumor activity in vivo compared to that elicited by a monospecific TLR7 agonist.     

A novel thymic epithelial adhesion molecule

Interactions between thymocytes and thymic stromal cells are responsible for positive and negative selection of T cell, their differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. The identification of specific adhesion molecules in this context, is, therefore, a vital first step in determining the nature of the signal they mediate or facilitate at a given stage of differentiation. In the present work we identify, isolate and partially characterize a ligand present on thymic medullary epithelial cells which selectively binds CD4+ CD8+ thymocytes found primarily in the thymic cortex. This adhesion molecule is composed of two non-covalently associated glycoproteins of 23 kDa and 45 kDa, respectively, both of which are needed to bind to thymocytes. The importance of the finding is that the ligand, in isolated immobilized form, binds the same thymocyte subset as the original epithelial cell line from which it was isolated. The CD4+ CD8+ thymocyte subset is the precursor of single-positive mature T cells; hence the putative biological activity of the ligand in question takes place at a pivotal stage of T cell differentiation.     

Improved growth and nutrition status in children with methylmalonic or propionic acidemia fed an elemental medical food

BACKGROUND: Failure-to-thrive (FTT) has been described in patients with organic acidemias treated with low protein diets. OBJECTIVE: To determine if patients with methylmalonic (MMA) or propionic acidemia (PA) can achieve normal growth and nutrition status. METHODS: A 6-month multicenter outpatient study was conducted with infants and toddlers treated with Propimex-1 Amino Acid-Modified Medical Food With Iron (Ross Products Division, Abbott Laboratories, Columbus, OH). Main outcome measures were anthropometrics, protein status indices, plasma retinol, and alpha-tocopherol. RESULTS: Sixteen patients completed the study. Mean baseline age was 0.54 +/- 0.02 years (range 0.03-3.00 years). By study end, mean National Center for Health Statistics (NCHS) weight centile increased from 26 to 49%; mean crown-heel length centile from 25 to 33%; and mean head circumference centile from 43 to 54%. Mean (+/- SE) protein and energy intakes by <6-month-old, 6<12-month-old, and 1<4-year-old patients were 15.3 +/- 0.9 g and 645 +/- 10 kcal; 18.3 +/- 1.1 g and 741 +/- 92 kcal; and 25.1 +/- 2.46 g and 1062 +/- 100 kcal, respectively. Plasma glycine concentrations were significantly and negatively correlated with energy intake (r=-0.77, p<0.0005). No correlation was found between dietary protein intakes and plasma ammonia concentrations. Protein status indices, retinol and alpha-tocopherol concentrations were within reference ranges at study end. CONCLUSIONS: Propimex-1 improved growth and nutrition status in patients with MMA or PA in just 6 months when fed in sufficient amounts. Providing energy and protein for patients with FTT at intakes recommended for catch-up growth may have resulted in even better growth.     

MDR-selective microbial-based therapy: A novel approach to cancer treatment

Microbial-based therapy of cancer is one of the earliest non-surgical anticancer therapies. The main limitation of such therapies is the toxicity of the therapeutic dose. This article discusses a novel approach that exploits cancer multidrug resistance (MDR) to provide a safer microbial-based therapy. As multidrug resistant cells can only contain limited amounts of a variety of susceptible drugs including certain antibiotics, we can take advantage of MDR to create a micro-environment (antibiotic free) that favors growth of intracellular bacteria within cancer cells. Thus, this approach targets cancer cells and spares normal cells (shielded by antibiotic): providing a more selective thus safer anticancer treatment. This article also explores the potentials of Chlamydia pneumoniae as an anti-cancer agent in this MDR-selective microbial-based therapy: its unique life cycle and the immune response to its infection suggest that it could be used directly, in the proposed approach, without any pre-requirements.     

A novel molecule expressing HLA-DR antigenic determinants

Our previous studies suggested that the polymorphism of HLA-DR antigens (the human equivalent of murine I-E antigens) was a result of structural variation in the small (beta) subunit. In order to more accurately define this polymorphism we have expanded these studies to include HLA-DR antigens isolated with monoclonal cells derived from genotypically HLA-homozygous DRw2, DR2w5, and DRw7 lymphoblastoid cells derived from offspring of consanguineous relationships. Our results indicate the large (alpha) subunits of DRw2 and DRw7 antigens are nearly identical, while their beta subunits show many differences. In contrast, both the alpha and beta subunits of the DRw5 antigen differ strikingly from the respective subunits of the DRw2 and DRw7 antigens. The significance of the variability of the DRw5 alpha subunit is in question at this point. One intriguing possibility is that DRw5 actually represents the human counterpart of the mouse I-A subregion antigen and that the monoclonal antibody is reacting with a determinant which is shared by the human equivalents of murine I-A and I-E antigens.     

Targeting neurotensin as a potential novel approach for the treatment of autism

The pathophysiology of autism remains obscure. Recently, serum neurotensin levels in children with autistic disorder have been found to be higher than those of normal children. Neurotensin is known to intensify neuronal NMDA-mediated glutamate signaling, which may cause apoptosis in autism. Further, an imbalance of glutamate/GABAergic system in autism has been described. These observations lead to a postulate that neurotensin may accentuate the hyperglutaminergic state in autism, leading to apoptosis. Targeting neurotensin might be a possible novel approach for the treatment of autism.     

ICG-001, a novel small molecule regulator of TCF/beta-catenin transcription

Inherited and somatic mutations in the APC gene, a human tumor-suppressor, occur in a large percentage of colon cancers, leading to elevated levels of nuclear beta-Catenin, and to activation of TCF/beta-Catenin-responsive genes including cyclin D1 and c-myc. To identify small molecule antagonists of this pathway, we screened transformed colorectal cells with a secondary structure-templated chemical library, in search of compounds that attenuated a TCF/beta-Catenin-responsive reporter gene. From this library we selected ICG-001 (IC50=3 microM) as a lead compound. Design and synthesis of the chemical library and some preliminary biological evaluation is described.     

Nicotinic cholinergic antagonists: a novel approach for the treatment of autism

Evidence supports the hypothesis that normalization of cholinergic tone by selective antagonism of neuronal nicotinic acetylcholine receptors (NNRs) may ameliorate the core symptoms of autism. As often is the case, epidemiology has provided the first important clues. It is well recognized that psychiatric patients are significantly more often smokers than the general population. The only known exceptions are obsessive-compulsive disorder (OCD), catatonic schizophrenia and interestingly, autism. In this regard, clinical studies with nicotine have demonstrated amelioration of symptoms of a number of diseases and disorders, including Alzheimer's disease, Parkinson's disease, ADHD and Tourette's syndrome. Nicotine's agonist properties at CNS NNRs have been implicated in these effects and support the concept of self-medication as a strong motivation for smoking in cognitively compromised individuals. On the other hand, the inverse correlation between autism and smoking suggests that smoking does not provide symptomatic relief and may actually be indicative of an active avoidance of nicotine's agonist effects in this disorder. Neuroanatomical evidence is consistent with this idea based on the presence of hypercholinergic architecture in the autistic brain, particularly during the first few years of development, making the avoidance of further stimulation of an already hyperactive cholinergic system plausible. This may also explain why stimulants (known to increase dopamine levels as do NNR agonists) appear to aggravate autistic symptoms and why studies with cholinesterase inhibitors that increase acetylcholine levels in the brain have yielded variable effects in autism. Taken together, the evidence suggests the possibility that nicotinic cholinergic antagonism may in fact be palliative. Pharmacological evidence supports this hypothesis. For example, antidepressants, many of which are now known to be non-competitive NNR antagonists, have been used successfully to treat a number of autistic symptoms. More specifically, there is anecdotal evidence from at least one medical practitioner that mecamylamine, a non-selective NNR antagonist, is effective in treating many autistic symptoms, particularly those that are refractory to most other treatments. Clearly there is a need for carefully controlled clinical studies with novel selective NNR antagonists to explore their potential as a new and exciting approach for the treatment of autism.     

A robust method for production of MHC tetramers with small molecule fluorophores

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.     

A novel approach to the treatment of ascites associated with ovarian hyperstimulation syndrome

Ascites is a clinical manifestation of severe ovarian hyperstimulation syndrome (OHSS) which may complicate the induction of ovulation using exogenous gonadotrophins. In severe OHSS severe ascites may occur and can lead to dyspnoea, abdominal discomfort and oliguria. To relieve ascites paracentesis is performed two to three times weekly as needed. We report three cases where an indwelling peritoneal catheter was used to decrease the need for repeated paracentesis. Under ultrasound guidance a closed system Dawson-Mueller catheter with 'simp-loc' locking design was inserted to allow continuous drainage of the ascitic fluid. A total of 23 l of the ascitic fluid were drained from the first, 20 l from the second and 28 l from the third patient with significant decrease in abdominal discomfort and improvement in the urine output. No complications or adverse reactions were noted. Continuous drainage of the ascitic fluid is efficient. It quickly decreases the abdominal discomfort, improves the urine output and prevents the need for multiple abdominal paracenteses which some patients may require.      

A novel approach for treating cerebellar ataxias

The terminology of cerebellar ataxias encompasses a variety of sporadic and inherited debilitating diseases. Patients exhibit disabling deficits such as dysmetria, kinetic tremor and ataxia of stance/gait. We are currently lacking effective treatments in degenerative cerebellar ataxias. Animal models of cerebellar disorders and studies in ataxic patients have demonstrated that the excitability of the sensorimotor cortex is severely depressed in case of cerebellar lesion. These reduced levels of excitability are associated with learning deficits. Recent experimental data show that transcranial direct current stimulation (tDCS) of the premotor cortex and low-frequency repetitive stimulation of the motor cortex (LFRSM1) restore the excitability of the motor cortex in hemicerebellectomized rats, reinstating the ability of the motor cortex to adapt to sustained peripheral stimulation. The hypothesis is based on the possibility that the combination of tDCS and contralateral LFRSM1 can improve human cerebellar ataxias. The proposed treatment consists of delivering trains of tDCS either in conjunction or in alternance with contralateral LFRSM1, in addition to application of peripheral nerve stimulation to sensitize the sensorimotor cortex. This hypothesis is to be tested in a procedure made of 3 steps in patients exhibiting a sporadic or inherited cerebellar disorder. First, patients are assessed clinically using validated scales of cerebellar ataxias and performing accepted quantified tests. Second, trains of tDCS and LFRSM1 are delivered, using a sham procedure in a cross-over design. Trains of peripheral stimulation are applied at peripheral nerves. Third, patients are re-assessed clinically and with quantified tests. Although grafting of stem cells and gene therapy are being developed, they will not be available soon. A successful treatment of combined neurostimulation would lead to a new and readily available approach in the management of cerebellar ataxias. This new therapy is safe, feasible and may bring symptomatic improvement.