Autonomic impairment in painful neuropathy

OBJECTIVES: 1) To determine the degree and distribution and quantitate the severity of autonomic impairment in painful neuropathy (PN). 2) To assess the role of autonomic testing in evaluating PN. METHODS: The authors studied 92 patients with PN (60 women and 32 men, age 56.9 +/- 12.4 years) using: 1) autonomic reflex testing (ART), Quantitative Sudomotor Axon Reflex Test (QSART), cardiac-vagal, head-up tilt, and surface skin temperature; 2) autonomic symptoms questionnaire; 3) nerve conduction (NCS) and laboratory studies; 4) quantitative sensory testing; 5) skin biopsy; and 6) Composite Autonomic Symptoms Score (CASS) scale to grade ART results from 0 (normal) to 10 (autonomic failure). RESULTS: Autonomic involvement in PN had characteristic features. Main symptoms were pain, secretory and skin vasomotor signs, hypertension, and impotence. ART results were abnormal in 86 (93.5%) (CASS < 4), QSART in 67 (72.8%), cardiac-vagal index in 58 (63%), skin temperature in 51 (55.4%), orthostatic hypertension in 39 (42.3%), and family history of PN in 26 (21%) of patients. Group 1 (abnormal NCS) (n = 45) had more severe ART and sensory abnormalities than the Group 2 (normal NCS) (n = 47): 1) CASS 2.0 +/- 0.96 vs 1.55 +/- 0.88 (p < 0.01), cardiac-vagal index (p < 0.02), skin temperature (p < 0.02), hypertension (p < 0.03), cooling (p < 0.002), and vibration (p < 0.0005) thresholds. CONCLUSIONS: Autonomic symptoms in painful neuropathy are predominantly cholinergic and form a unique constellation of features that are distinct from other autonomic neuropathies.     

Treatment of painful sensory neuropathy with tiagabine: A pilot study

To evaluate the effect of tiagabine hydrochloride in painful neuropathy in a pilot, open-label study. Painful neuropathy is characterized by preferential involvement of small sensory and autonomic fibers. Tiagabine increases -aminobutyric acid and might enhance the central pain-control mechanisms. Seventeen patients (10 men, 7 women; mean age 51.4±7.7 y) with chronic painful neuropathy (>6 months) were enrolled in this study. Week 0:All pain medications were discontinued. Weeks 1–4: Dose of tiagabine was increased weekly by 4 mg orally up to 16 mg in week 4. Quantitative sensory testing for vibration, cooling, and heat-pain, and quantitative sudomotor axon reflex test (QSART) were done at week 0 and week 4. The McGill Pain Questionnaire was administered weekly.Nine patients completed the study; 8 patients discontinued the treatment. Baseline pain intensity was 6.2±3.1 on the McGill Pain Questionnaire scale (0–10 range). Low doses (4–8 mg) of tiagabine reduced pain symptoms by 16–38%, improving surface pain (37.5%), skin sensitivity (32.8%), burning (38.6%), cold (25.4%) and pain sharpness (29%; p<0.03). Dull and deep pain did not improve. Quantitative sensory testing abnormalities diminished with treatment (p<0.02). Autonomic test results did not change.This pilot study evaluated the potential of tiagabine hydrochloride (Gabitril) in treatment of painful sensory neuropathy. Pain symptoms and quantitative sensory test results improved with treatment, especially at low doses of tiagabine (4–8 mg). Higher doses (12–16 mg) were associated with increased number of adverse events.Tiagabine may have potential benefits for treatment of painful neuropathy; however, assessment of its efficacy in a larger study is needed.This study was sponsored by a grant from Abbott Laboratories and Cephalon, Inc., Westchester, PA, U.S.A.     

The Structure of PEG-Modified Poly(Ethylene Imines) Influences Biodistribution and Pharmacokinetics of Their Complexes with NF-κB Decoy in Mice

Purpose. To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-B decoy. Methods. A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. Results. Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (–2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76 % of injected dose), kidney (3 - 22 %) and spleen (2 - 16 %) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. Conclusions. A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.     

Intracellular Processing of Poly(Ethylene Imine)/Ribozyme Complexes Can Be Observed in Living Cells by Using Confocal Laser Scanning Microscopy and Inhibitor Experiments

Purpose. Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Methods. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin A1, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Results. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Conclusions. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.     

Effect of chitosan structure properties and molecular weight on the intranasal absorption of tetramethylpyrazine phosphate in rats

The objective of this work was to assess and compare the absorption promoting effect of different molecular-weight chitosans, trimethyl chitosans and thiolated chitosans for intranasal absorption of 2,3,5,6-tetramethylpyrazine phosphate (TMPP). An in situ nasal perfusion technique in rats was utilized to test the rate and extent of TMPP absorption in situ. In vivo studies were carried out in rats and the pharmacokinetic parameters were calculated and compared with that of intravenous injection. All the chitosan derivatives investigated could enhance the intranasal absorption of TMPP significantly. However, thiolation could not improve the absorption-enhancing capacity of chitosan remarkably even when the thiolation ratio was as high as 152 μmol/g. In contrast, trimethylated chitosan exhibited stronger absorption-enhancing ability than the homopolymer chitosan. The permeation enhancing effect of chitosan increased with increasing molecular weight up to M_w 100 kDa. In vivo studies indicated that chitosan 100 kDa and TMC 50 kDa had comparable absorption-enhancing effect but chitosan 100 kDa functioned for more than 120 min versus 90 min for TMC. A good correlation was found between the in situ absorption data and plasma concentration in vivo for the polymers investigated. This study demonstrated that both chitosan structural features and chitosan molecular weight play a key role on promoting the intranasal absorption of TMPP. Taking safety reason into account, chitosan 100 kDa is the most promising as an intranasal absorption enhancer.     

Virulence-related genes in ColV plasmids of Escherichia coli isolated from human blood and intestines.

DNA probes for the colicin V, traT, iss, and iu genes were used in this study of four representative ColV plasmids together with 200 Escherichia coli strains isolated from the stools of patients with diarrhea and 146 E. coli strains isolated from the blood of patients with bacteremia. The study indicated that the ColV plasmids are heterogeneous. Southern and colony hybridization analyses showed that in most of the colicin V-producing intestinal E. coli strains, the colicin V genes are located in the chromosome (14 of 16); in most of the colicin V-producing E. coli strains isolated from the blood, they are located in plasmids (18 of 22). In both intestinal and blood E. coli isolates, the traT, iss, and aerobactin receptor genes were present at similar frequencies, but the frequency of the aerobactin synthesis genes was significantly different. The aerobactin receptor gene was present in 25% of the intestinal E. coli strains that lack the aerobactin synthesis gene. In the blood isolates, the aerobactin synthesis and receptor genes were present at almost equal frequencies. Among the colicin V-producing isolates, the iss, traT, and iu genes were present in 95.5, 86.4, and 90.9% of the blood isolates and in only 68.8, 43.8, and 81.3% of the intestinal isolates, respectively. The ColV plasmids from blood isolates that were tested for the presence of traT, iss, and iu genes were homogeneous and had DNA sequences that hybridized with each of the probes. On the other hand, the two intestinal strains containing ColV genes in a plasmid were heterogeneous in regard to the carriage of these genes. The presence of ColV is not restricted to specific O types.     

Definitive molecular diagnosis of facioscapulohumeral dystrophy.

OBJECTIVE: To establish the usefulness of a molecular diagnostic protocol for the autosomal dominant disease facioscapulohumeral dystrophy (FSHD). BACKGROUND: The genetic defect underlying the majority of cases is a deletion on chromosome 4q35 that is not associated with the coding sequence of any known gene. Molecular diagnosis of FSHD involves the visualization of this deletion as a "small" EcoRI restriction fragment. However, molecular diagnostics are complicated because of the homology of the telomeric regions of chromosomes 4q and 10q; the homologous 10q26 EcoRI fragments are also detected, and can fall into the size range considered to be diagnostic for FSHD. It is therefore important to distinguish the 4q35 and 10q26 EcoRI fragments, taking advantage of the presence of additional restriction sites (BlnI) in the alleles of chromosome 10q origin. METHODS: Paired digests of genomic DNA (EcoRI only and EcoRI/BlnI double digest), followed by pulsed field gel electrophoresis (PFGE), were used to establish the molecular diagnosis of FSHD in 82 unrelated index cases (46 familial, 24 proven sporadic with de novo mutations, and 12 with uncertain family history). RESULTS: In all cases fulfilling FSHD diagnostic criteria, a 4q35 EcoRI allele size of < or = 38 kb was present. The smallest 4q35 EcoRI allele in 205 normal control subjects was 41 kb. EcoRI alleles < or = 38 kb of chromosome 10q26 origin were present in 11.2% of this control group. In problematic cases, it was possible to resolve the diagnostic question. CONCLUSIONS: The combination of double digestion with EcoRI and BlnI followed by PFGE is the most reliable molecular protocol for distinguishing patients with FSHD.     

Muscular dystrophy: historical overview and classification in the genetic era

Despite recent advances in molecular genetics, it has proven very difficult to arrive at an accurate and clinically useful classification of the muscular dystrophies. Much of this difficulty arises from confusion related to the term "muscular dystrophy" itself, as well as a general reluctance on the part of the neuromuscular community to abandon traditional, clinically based classifications. Nevertheless, advances in the understanding of the molecular defects of these disorders have permitted a foundation for classification based on molecular biology. This review presents a historical perspective on the classification of the muscular dystrophies, and furnishes the underpinnings of a genetic classification that can be used both at the bedside and in the research laboratory.     

Sex ratio associated with timing of insemination and length of the follicular phase in planned and unplanned pregnancies during use of natural family planning

This was a multicentred, prospective study of pregnancies among women using natural family planning. The women maintained natural family planning charts of the conception cycle, recording acts of intercourse and signs of ovulation (cervical mucus changes, including peak day and basal body temperature). Charts were used to assess the most probable day of insemination relative to the day of ovulation and length of the follicular phase of the cycle. The sex ratio (males per 100 females) for 947 singleton births was 101.5, not significantly different from the expected value of 105. The sex ratio did not vary consistently or significantly with the estimated timing of insemination relative to the day of ovulation, with the estimated length of the follicular phase or with the planned or unplanned status of the pregnancy. Although these findings may be affected by imprecision of the data, the study suggests that manipulation of the timing of insemination during the cycle cannot be used to affect the sex of offspring.