A methodology for characterising nanoparticle size and shape using nanopores.

The discovery and characterisation of nanomaterials represents a multidisciplinary problem. Their properties and applications within biological, physical and medicinal sciences depend on their size, shape, concentration and surface charge. No single technology can currently measure all characteristics. Here we combine resistive pulse sensing with predictive logistic regression models, termed RPS-LRM, to rapidly characterise a nanomaterial's size, aspect ratio, shape and concentration when mixtures of nanorods and nanospheres are present in the same solution. We demonstrate that RPS-LRM can be applied to the characterisation of nanoparticles over a wide size range, and varying aspect ratios, and can distinguish between nanorods over nanospheres when they possess an aspect ratio grater then two. The RPS-LRM can rapidly measure the ratios of nanospheres to nanorods in solution within mixtures, regardless of their relative sizes and ratios i.e. many large nanospherical particles do not interfere with the characterisation of smaller nanorods. This was done with a 91% correct classification of nanospherical particles and 72% correct classification of nanorods even when the fraction of nanorods in solution is as low as 20%. The methodology here will enable the classification of nanomedicines, new nanomaterials and biological analytes in solution.

Determinants of epidermal nerve fibre density in antiretroviral-naïve HIV-infected individuals.

OBJECTIVES: Distal leg epidermal nerve fibre density (ENFD) is a validated predictor of small unmyelinated nerve fibre damage and neuropathy risk in HIV infection. As pre-existing damage may increase the risk of neuropathy following antiretroviral (ARV) therapy, particularly when the regimen contains stavudine (d4T), we assessed the relationship between ENFD and various parameters including mitochondrial factors in HIV-infected Thai individuals naïve to ARV therapy. METHODS: Distal leg and proximal thigh ENFDs were quantified in HIV-infected Thai individuals without neuropathy prior to randomization to a HIV clinical trial that focused on mitochondrial toxicity issues. We assessed their association with various clinical and immunovirological parameters as well as with peripheral blood mononuclear cell (PBMC) mitochondrial (mt) DNA copies/cell, oxidative phosphorylation (OXPHOS) complex I (CI) and complex IV (CIV) enzyme activities, and mt 8-oxo-deoxyguanine (8-oxo-dG) break frequencies. RESULTS: In 132 subjects, the median (interquartile range) ENFD (fibres/mm) values were 21.0 (16.2-26.6) for the distal leg and 31.7 (26.2-40.0) for the proximal thigh. By linear regression, lower CD4 count (P < 0.01), older age (P < 0.01), increased body mass index (BMI) (P = 0.04), increased height (P = 0.02), and higher PBMC OXPHOS activity as measured by CIV activity (P = 0.02) were associated with lower distal leg ENFD. CONCLUSIONS: Older age, increased height, higher BMI, poorer immunological status and higher PBMC OXPHOS activity are associated with lower distal leg ENFD in HIV-infected subjects free of neuropathy prior to initiation of first-time ARV therapy.

Longitudinal assessment of oxaliplatin-induced neuropathy.

OBJECTIVES: To characterize the natural history of oxaliplatin-associated neuropathy (ON) and determine whether intraepidermal nerve fiber density (IENFD) is a sensitive measure of neuropathy progression. In addition, we sought to assess the potential of ON as a neuroprotection model and gain insight into the relationship between axon loss and neuropathic symptoms. METHODS: Eight subjects receiving oxaliplatin for advanced colorectal cancer were prospectively followed prior to starting chemotherapy and at 30, 90, 180, and 360 days (180 days after completing treatment). Electrophysiology, punch biopsies, symptom assessment, and examinations with calculation of a reduced total neuropathy score (rTNS) were performed at each time point. Changes over time were assessed through Poisson regression for IENFD and a mixed effects model for rTNS and electrophysiology measures. RESULTS: The distal leg IENFD, rTNS, peroneal, and sural amplitudes were all significantly reduced over time, while conduction velocity (peroneal and sural) and distal thigh IENFD were not. Measures of axon loss continued to worsen following discontinuation of oxaliplatin. Five of 8 subjects reported prominent symptoms associated with oxaliplatin administration. CONCLUSIONS: This study demonstrates that oxaliplatin is associated with mild, sensory, and motor axon loss that may not be reversible. Axonal loss was detected by electrophysiology, rTNS, and distal leg IENFD. Several subjects reported prominent sensory symptoms that were not associated with axon loss, and that may or may not represent neuropathy. ON is an attractive paradigm for neuroprotection studies and the distal leg IENFD is an objective measure that requires minimal subject participation or study site expertise.

Acetyl-l-carnitine and nucleoside reverse transcriptase inhibitor-associated neuropathy in HIV infection.

OBJECTIVES: Antiretroviral toxic neuropathy (ATN) is associated with dideoxynucleoside reverse transcriptase inhibitor use in patients infected with HIV, possibly as a result of mitochondrial toxicity. Acetyl-l-carnitine (ALC) has been linked to symptomatic improvement in ATN. We present an open-label single-arm pilot study to evaluate changes in intra-epidermal nerve fibre (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000 mg ALC daily. METHODS: Punch skin biopsies were examined at baseline and after 24 weeks of therapy. Participants reported neuropathic symptoms using the Gracely Pain Intensity Score. Neurological examinations were completed. RESULTS: Twenty-one subjects completed the study. ALC was generally well tolerated. The IENF density did not change in cases completing 24 weeks of ALC therapy, with median (90% confidence interval) IENF changes of -1.70 (-3.50, infinity) (P=0.98) and 2.15 (-0.10, infinity) (P=0.11) for the distal leg and proximal thigh, respectively. Fat mtDNA copies/cell did not change with therapy. Improvements in neuropathic pain (P<0.01), paresthesias (P=0.01), and symptoms of numbness (P<0.01) were noted. Similarly, improvement was noted on the Gracely Pain Intensity Score. CONCLUSIONS: ALC therapy coincided with improvements in subjective measures of pain in this open-label single-arm study. However, changes were not observed in objective measures of IENF density or mtDNA levels, providing little objective support for use of ALC in this setting.

Correlates of epidermal nerve fiber densities in HIV-associated distal sensory polyneuropathy.

OBJECTIVE: To demonstrate the relationship between epidermal nerve fiber density (ENFD) in the leg and the phenotype of HIV-associated distal sensory polyneuropathy (HIV-DSP) in a multicenter prospective study (ACTG A5117). METHODS: A total of 101 HIV-infected adults, with CD4 cell count <300 cells/mm(3) and who had received antiretroviral therapy (ART) for at least 15 consecutive weeks, underwent standardized clinical and electrophysiologic assessment. All 101 subjects were biopsied at the distal leg (DL) and 99 at the proximal thigh (PT) at baseline. ENFD was assessed by skin biopsy using PGP9.5 immunostaining. Associations of ENFD with demographics, ART treatment, Total Neuropathy Score (TNS), sural sensory nerve action potential (SNAP) amplitude and conduction velocity, quantitative sensory testing (QST) measures, and neuropathic pain were explored. RESULTS: ENFD at the DL site correlated with neuropathy severity as gauged by TNS (p < 0.01), the level of neuropathic pain quantified by the Gracely Pain Scale (GPS) (p = 0.01) and Visual Analogue Scale (VAS) (p = 0.01), sural SNAP amplitude (p < 0.01), and toe cooling (p < 0.01) and vibration (p = 0.02) detection thresholds. ENFD did not correlate with neurotoxic ART exposure, CD4 cell count, or plasma HIV-1 viral load. CONCLUSIONS: In subjects with advanced HIV-1 infection, epidermal nerve fiber density (ENFD) assessment correlates with the clinical and electrophysiologic severity of distal sensory polyneuropathy (DSP). ENFD did not correlate with previously established risk factors for HIV-DSP, including CD4 cell count, plasma HIV-1 viral load, and neurotoxic antiretroviral therapy exposure.

Impaired reinnervation in HIV infection following experimental denervation.

OBJECTIVE: To determine whether abnormalities in peripheral nerve regeneration are present in HIV-infected individuals. DESIGN: We studied human epidermal nerve fiber reinnervation by collateral and regenerative sprouting using validated cutaneous nerve injury models in healthy control subjects and those infected with HIV. Characteristics associated with the degree of reinnervation were identified. METHODS: Seventy-one healthy volunteers and 22 HIV+ individuals underwent topical capsaicin treatment at the distal lateral thigh resulting in complete epidermal denervation. Regenerative sprouting was quantified in skin biopsies at intervals up to 100 days. Five healthy subjects and 5 HIV+ individuals underwent a 3-mm distal thigh punch skin biopsy to create an intracutaneous axotomy, followed 2 months later by a 4-mm overlapping biopsy. The collateral sprouting distance was measured. RESULTS: The mean rate of regenerative sprouting was highest for healthy control subjects (0.17 +/- 0.073 fibers/mm/day), followed by HIV+ subjects without neuropathy (0.13 +/- 0.06) and then HIV+ subjects with neuropathy (0.097 +/- 0.07) (p = 0.002). Regenerative sprouting was significantly associated with the baseline intraepidermal nerve fiber density (p < 0.001) but not with HIV viral load, HIV duration, CD4 cell count, or ApoE epsilon4 status. HIV+ individuals were also found to have a reduced collateral sprouting rate compared to controls (5.31 mum +/- 0.73 vs 9.78 mum +/- 1.5/day, p = 0.03). CONCLUSIONS: Abnormalities in both regenerative and collateral sprouting are present in people infected with HIV, and are detectable in subjects with and without evidence of peripheral nerve dysfunction. Our results indicate that abnormalities in nerve regeneration occur early in HIV infection and provide a rationale to include neuropathy-free HIV subjects in regenerative peripheral nerve trials.

Antiretroviral use and other risks for HIV-associated neuropathies in an international cohort.

OBJECTIVE: To explore the association between specific nucleoside reverse transcriptase inhibitors and sensory neuropathies (SNs) and define the modifying roles of hepatitis C (HCV), vitamin B12 deficiency, and impaired glucose tolerance. METHODS: The authors conducted a prospective cohort study of 147 HIV-infected adults at two sites chosen to emphasize demographic differences. Standardized assessments included detailed antiretroviral histories, neurologic examinations, skin biopsies for epidermal nerve quantitation, and quantitative sensory testing. RESULTS: There were significant differences between subjects at Johns Hopkins University (JHU) and Monash University (MU) in gender, race, HIV transmission route, and HCV seroprevalence. Symptomatic SN was present in 49% at JHU and 55% at MU (chi2 = 4.02, p = 0.134) and was significantly more common in those at least age 40 than younger patients (odds ratio [OR] = 2.87, 95% CI = 1.27, 6.49). After adjusting for site, age, and CD4 cell count, exposure to didanosine (ddI) or stavudine (d4T) was associated with an significantly increased likelihood of symptomatic SN (ddI: OR = 3.21, 95% CI: 1.56, 6.60; d4T: OR = 7.66, 95% CI: 2.89, 20.33). Plasma HIV RNA, lactate, and HCV were not associated with SN. Quantitative vibratory testing identified neuropathy with a positive predictive value of 76% and epidermal nerve fiber densities 59%. CONCLUSIONS: Exposure to stavudine and didanosine was significantly associated with a heightened risk for symptomatic sensory neuropathy. Reduced vibration thresholds and epidermal nerve fiber densities had the highest diagnostic efficiency of the laboratory indicators of neuropathy examined, but were relatively insensitive in isolation.

The utility of skin biopsy for prediction of progression in suspected small fiber neuropathy.

Twenty-eight patients with sensory complaints of unknown etiology had repeated skin biopsies. Patients with large nerve fiber swellings on initial biopsy showed a decline in epidermal nerve fiber density on repeated biopsies (p < 0.05 within group; p < 0.05 vs those without swellings). Patients without nerve fiber swellings did not have changes in nerve fiber density between biopsies. Patients with large nerve fiber swellings were most likely to present clinically with paresthesias (p < 0.05).

Factors influencing nerve regeneration in a trial of timcodar dimesylate.

The authors assessed the ability of the neurophilin compound, timcodar dimesylate, to accelerate the return of epidermal nerve fiber density (ENFD) after a standardized nerve injury in a randomized double blind, placebo controlled trial. While there was no difference in the regeneration rate between the treatment and placebo arms, the baseline ENFD (p = 0.006), height (p = 0.02), and race (p = 0.03) were associated with the regeneration rate.

Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy.

OBJECTIVE: To explore the relationship between intraepidermal nerve fiber (IENF) density in HIV-associated sensory neuropathy (HIV-SN) to measurements of neuropathy severity and progression of HIV disease. BACKGROUND: SN affects 30% of individuals with AIDS, and treatment is often ineffective. Recombinant human nerve growth factor (rhNGF) has been proposed as a trophic factor for unmyelinated nerve fibers injured in HIV-SN, and a clinical trial has recently concluded. Skin biopsy with IENF density determination has emerged as a diagnostic test for patients with small-fiber sensory neuropathy. METHODS: Sixty-two of the 270 patients with HIV-SN who participated in the trial of rhNGF were included in a substudy examining epidermal nerve fibers. IENF density was compared with neuropathic pain intensity (measured with the Gracely Pain Scale), patient and physician global pain assessments, quantitative sensory testing, CD4 counts, and plasma HIV RNA levels both at baseline and at conclusion of the placebo-controlled phase. RESULTS: IENF density was inversely correlated with neuropathic pain as measured by patient (p = 0.004) and physician (p = 0.05) global pain assessments, but not using the Gracely Pain Scale. Decreased IENF density at the distal leg was associated with lower CD4 counts and higher plasma HIV RNA levels. IENF density measurements were stable over time. CONCLUSIONS: IENF loss at the distal leg is associated with increased neuropathic pain, lower CD4 counts, and higher plasma viral load in HIV-SN. The robustness of the longitudinal measurement of IENF density supports its use in future longitudinal studies and clinical trials.

Subclinical sensory neuropathy in late-onset restless legs syndrome.

OBJECTIVE: To determine the prevalence of different forms of peripheral neuropathy in patients with restless legs syndrome (RLS) and correlate the findings with other clinical characteristics. BACKGROUND: RLS is characterized by a desire to move the extremities, often associated with paresthesias or dysesthesias, motor restlessness, worsening of symptoms with rest with relief by activity, and worsening of symptoms in the evening or night. The association between RLS and peripheral neuropathy remains controversial. The observation that many patients with small-fiber neuropathy also complain of RLS prompted this prospective case series. METHODS: Twenty-two consecutive patients with RLS were evaluated for evidence of large-fiber neuropathy (LFN) and small sensory fiber loss (SSFL). RESULTS: In eight of the 22 (36%) patients, neuropathy was identified. Three patients had pure LFN; two had mixed LFN and SSFL; and three had isolated SSFL. The SSFL group had a later onset of RLS (p < 0.009), reported pain in their feet with RLS more frequently (p < 0.001), and tended to have no family history of RLS (p < 0.078). Patients with LFN did not have similar associations with age at onset, family history status, or presence of pain. CONCLUSION: The results suggest that two forms of RLS exist: one is triggered by painful dysesthesias associated with SSFL, has later onset, and no family history; and one without involvement of SSF, with an earlier onset age, positive family history for RLS, and no pain. The authors hypothesize that patients with the SSFL subtype of RLS will preferentially respond to neuropathic pain medications.

Development of monoclonal antibodies suitable for use in antigen quantification potency tests for clostridial veterinary vaccines.

The quality control testing of clostridial veterinary vaccines currently requires large numbers of animals. Alternative in vitro test methods are being investigated by researchers in industry and by regulatory authorities in many countries. Monoclonal antibodies that neutralize Clostridium perfringens alpha toxin, C. perfringens beta toxin, C. perfringens epsilon toxin, and C. sordellii lethal toxin as well as a monoclonal antibody directed against C. chauvoei flagellar antigen have been developed by the Center for Veterinary Biologics-Laboratory for use in antigen quantification assays. A proposal to create an international standard collection of clostridial-specific monoclonal antibodies is made.

Epidermal nerve fiber density and sural nerve morphometry in peripheral neuropathies.

OBJECTIVE: To study intraepidermal nerve fiber (IENF) density in distal leg skin biopsies, sural nerve morphometry, electrophysiology, and clinical features in patients with peripheral neuropathies. METHODS: We studied 26 patients with neuropathic complaints who had undergone clinical evaluation, nerve conduction studies, distal leg skin biopsy, and sural nerve biopsy. We quantified densities of IENF and of myelinated and unmyelinated fibers in the sural nerve. Associations among skin and sural nerve morphometric measures and sensory nerve action potential (SNAP) amplitudes were examined nonparametrically. Morphometric measures were examined with respect to diagnostic category of neuropathy. RESULTS: IENF density correlated with the densities of sural nerve total myelinated (r = 0.57, p = 0.0011), small myelinated (r = 0.53, p = 0.0029), and large myelinated fibers (r = 0.49, p = 0.0054). There was a trend toward an association between IENF and sural nerve unmyelinated fiber densities (r = 0.32, p = 0.054). Sural SNAP amplitude and large myelinated fiber densities were highly correlated (r = 0.87, p < 0.0001). IENF density and sural nerve small fiber measures were concordant in 73% of patients. Reduced IENF density was the only indicator of small fiber depletion in 23% of cases. It was usually normal in acquired demyelinating neuropathies and where clinical suspicion for neuropathy was low. CONCLUSIONS: Distal leg Intraepidermal nerve (IENF) density may be more sensitive than sural nerve biopsy in identifying small fiber sensory neuropathies. Assessments of IENF density and large fiber measures on biopsy and electrophysiology are both useful for characterizing sensory and sensorimotor neuropathies.

Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.

In previous work we demonstrated little effect of aging on the density and spatial pattern of epidermal innervation, however, this was restricted to two sites proximal and distal in the leg. To expand on these observations, we used punch skin biopsy in ten healthy controls to examine the variation in intra-epidermal nerve fiber (IENF) density at multiple specific sites in the leg. There was a consistent gradient in IENF from proximal to distal sites in all subjects, but minimal effect of age was noted. In the older age group (> or =70 years), the IENF densities ranged from 28.6+/-1.9 IENF/mm at the trunk to 15.5+/-1.5 at the distal leg. In a group of six patients with painful sensory neuropathy, we confirmed a length-dependent reduction in IENF. We observed what may be a predegenerative change, namely increased branching of epidermal nerve fibers at clinically unaffected sites. These data suggest little age-related change in IENF, at least up to age 75 years, in healthy normals. The increased branching complexity noted in unaffected sites in patients with sensory neuropathies implies that this may be a predegenerative change, preceding the actual loss of nerve fibers. Skin biopsy may be a useful tool for assessing the topographic extent and degree of nerve fiber damage in sensory neuropathies and its quantitative interpretation should be little affected by aging changes.

Epidermal nerve fiber density: normative reference range and diagnostic efficiency.

BACKGROUND: The sensitivity of neuron-specific antibodies permit the identification of the small unmyelinated nerve fibers within the skin. OBJECTIVES: To develop a reference range of epidermal nerve fiber density in humans, and to evaluate their diagnostic efficiency for sensory neuropathies. METHODS: Ninety-eight normal controls (age range, 13-82 years) were examined with both directed neurologic examinations and quantitative sensory testing. The diagnostic utility was examined in 20 patients with sensory neuropathies. Each subject had 2 punch biopsies performed at each site in the thigh and distal part of the leg (total of 392 biopsies). After formalin fixation, 50-microm-thick free-floating sections were stained with a polyclonal antibody to neuron-specific ubiquitin hydrolase, anti-protein gene product 9.5. We enumerated intraepidermal nerve fibers per millimeter to derive a "linear density." The linear density technique was validated against a stereological technique that used the fractionator to measure the total length of intraepidermal nerve fibers per 3-mm punch. RESULTS: The biopsy technique was well tolerated, with no notable complications. The linear density quantitation was rapid and had high intraobserver and interobserver reliability. We determined that the density of intraepidermal fibers in normal controls was 21.1+/-10.4 per millimeter (mean +/- SD) in the thigh (fifth percentile, 5.2 per millimeter), and was 13.8+/-6.7 per millimeter at the distal part of the leg (fifth percentile, 3.8 per millimeter). Significantly higher intraepidermal fiber densities were seen in the youngest group (P = .004), and we observed no significant effect of race, sex, height, or weight. The density at the thigh was significantly correlated with that at the distal part of the leg (P = .01) and was consistently higher by about 60%, a reflection of the normal proximal-distal gradient. The results obtained with stereology and the linear density correlated significantly (P=.001), providing internal validation for the technique. Epidermal nerve fiber density was significantly reduced (P = .001) in patients with sensory neuropathies. With a cutoff derived from the fifth percentile of the normative range for the distal part of the leg, the technique had a positive predictive value of 75%, a negative predictive value of 90%, and a diagnostic efficiency of 88%. CONCLUSIONS: We have established a reference range for intraepidermal nerve fiber density in normal humans by means of a simple quantitation method based on enumeration of individual intraepidermal nerve fibers on vertical sections of punch skin biopsy specimens stained with the sensitive panaxonal marker anti-protein gene product 9.5. The utility of the density measurement was confirmed for sensory neuropathy with a diagnostic efficiency of 88%. Skin biopsies may be useful to assess the spatial distribution of involvement in peripheral nerve disease and the response to neurotrophic and other restorative therapies.

Unilateral postherpetic neuralgia is associated with bilateral sensory neuron damage.

Shingles can cause chronic neuropathic pain (postherpetic neuralgia) long after skin lesions heal. To investigate its causes, we quantitated immunolabeled sensory neurites in skin biopsies from 18 subjects with and 16 subjects without postherpetic neuralgia after unilateral shingles. Subjects rated the intensity of their pain. Punch skin biopsies were evaluated from the site of maximum pain or shingles involvement, the homologous contralateral location, and a site on the back, distant from shingles involvement. Sections were immunostained with anti-PGP9.5 antibody, a pan-axonal marker, and the density of epidermal and dermal neurites determined. The group with postherpetic neuralgia had a mean density of 339 +/- 97 neurites/mm2 in shingles-affected epidermis compared with a density of 1,661 +/- 262 neurites/mm2 for subjects without pain. Neurite loss was more severe in epidermis than dermis. Unexpectedly, the group with pain had also lost half of the neurites in contralateral epidermis. Contralateral damage occurred despite the lack of contralateral shingles eruptions or pain, correlated with the presence and severity of ongoing pain at the shingles site, and did not extend to the distant site. Thus, the pathophysiology of postherpetic neuralgia pain may involve a new bilateral mechanism.

Neuropathological alterations in diabetic truncal neuropathy: evaluation by skin biopsy.

OBJECTIVES: To describe the neuropathological features in skin biopsies from patients with diabetic truncal neuropathy. METHODS: Three patients with diabetic truncal neuropathy underwent skin biopsies from both symptomatic and asymptomatic regions of the chest and trunk. After local anaesthesia, biopsies were performed using a 3 mm diameter punch device (Acupunch). Intraepidermal nerve fibres (IENFs), the most distal processes of small myelinated and unmyelinated nerve fibres, were identified after staining with PGP 9.5 as previously described. RESULTS: Diabetes was diagnosed at the time of the neurological presentation in two, and one was a known diabetic patient. All three had associated sensory-motor polyneuropathy. In all, skin biopsies showed a marked reduction of both epidermal and dermal nerve fibres in the symptomatic dermatomes, compared with skin from asymptomatic truncal areas. In one patient, a follow up skin biopsy when symptoms had improved showed a return of IENFs. CONCLUSIONS: In diabetic truncal neuropathy, skin biopsies from symptomatic regions show a loss of IENFs. After clinical recovery, there is a return of the IENF population, suggesting that improvement occurs by nerve regeneration. These findings suggest that sensory nerve fibre injury in diabetic truncal neuropathy is distal to or within the sensory ganglia. Skin biopsy provides a possible tool for understanding the pathophysiology of the disease.