Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

Pharmacodynamics of recombinant human DNase I in serum.

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 +/- 1.4 ng/ml (mean +/- s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50-100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC50 approximately 100-200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50-100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes.

Improved potency of hyperactive and actin-resistant human DNase I variants for treatment of cystic fibrosis and systemic lupus erythematosus.

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a approximately 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was approximately 100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of approximately 400-fold over wild type for the +2 variant E13R/N74K. The +3 enzymes have approximately 4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the +6 enzyme has approximately 30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

Endogenous NGF and CNTF levels in human peripheral nerve injury.

Nerve growth factor (NGF) is trophic to sensory and sympathetic fibres, and ciliary neurotrophic factor (CNTF) to motoneurones, in animal models of peripheral nerve injury: NGF excess produces hyperalgesia. In this first study of injured human nerves and sensory ganglia, we quantified and localized endogenous NGF and CNTF in 59 neonate and adult patients with brachial plexus and peripheral nerve injury. NGF levels were generally depleted in injured nerves, but relatively preserved acutely in nerve segments distal to injury. NGF immunostaining was observed in Schwann cells in distal nerve segments with pockets of high levels in some neuromas. CNTF levels and immunostaining in Schwann cells were markedly decreased distally within days of injury. We propose that early local administration of NGF and CNTF-like agents may help prevent degenerative changes in injured nerves, while at later stages local anti-NGF treatment (e.g. of some neuromas) may ameliorate chronic pain.

Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis.

OBJECTIVES: To determine whether nerve growth factor (NGF) is elevated in painful conditions of the urinary bladder (idiopathic sensory urgency, interstitial cystitis and painful chronic cystitis). PATIENTS AND METHODS: Sixteen women patients were recruited from the Urodynamic Clinic at The Elizabeth Garrett Anderson Hospital, London. Four each had idiopathic sensory urgency (mean age 34 years, range 24-51), chronic cystitis (mean age 51 years, range 40-79) and interstitial cystitis (mean age 41 years, range 29-53). Four women who had genuine stress incontinence on cystometry but with no irritative symptoms were used as controls (mean age 45 years, range 35-54). The levels of NGF were determined in bladder biopsies from all women and biopsy sections were immunostained to detect NGF. RESULTS: The levels of NGF were higher in samples from all three painful bladder conditions than in samples from controls. Immunostaining showed increased NGF expression in the urothelium, most marked in patients with idiopathic sensory urgency. CONCLUSIONS: The increased level of NGF may explain several clinical and pathological features in these conditions, including sensitization of nociceptor fibres and increased numbers of mast cells. We propose that anti-NGF treatment may be a rational and effective treatment in intractable bladder pain.

The role of endogenous nerve growth factor in human diabetic neuropathy.

Nerve growth factor (NGF) is trophic to sensory and sympathetic fibers. In animal models, NGF is depleted in diabetic nerves and NGF deprivation produces hypoalgesia. Exogenous NGF can reverse some of the pathological changes in diabetic nerves and NGF excess leads to hyperalgesia. We have quantified sensory and autonomic function in early diabetic polyneuropathy and correlated changes with levels of NGF and neuropeptides in affected skin. We describe an early length-dependent dysfunction of sensory small-diameter fibers, prior to dysfunction of sympathetic fibers, with depletion of skin NGF and the sensory neuropeptide substance P. We describe a significant correlation between NGF depletion and decreased skin axon-reflex vasodilation, mediated by small sensory fibers partly via substance P release. Immunostaining shows depletion of NGF in keratinocytes in diabetic skin. We propose that a decrease in endogenous skin-derived NGF influences the presentation of diabetic polyneuropathy, although metabolic or vascular abnormalities may be the cause of the neuropathy. As loss of nociception and axon-reflex vasodilation contribute to diabetic foot ulceration, early and prolonged NGF treatment at an appropriate dose may provide rational prophylaxis for this condition.

Compaction assay: a rapid and simple in vitro method to assess the responsiveness of a biopolymer matrix to enzymatic modification.

A rapid and simple in vitro method is described which measures the extent of unrecoiled solids compression when a complex biopolymer is subjected to a centrifugal force. This method, termed the compaction assay, was used to assess the response of purulent cystic fibrosis (CF) sputum samples to the addition of recombinant human deoxyribonuclease I (rhDNase). Enzyme treatment resulted in a dramatic decrease in DNA size, a redistribution of total DNA content from the pellet to supernatant, a significant decrease in that pellet volume and a decrease in elastic modulus. Sample elasticity, measured by a dynamic cone and plate viscometer, could be related to compaction assay results. These results suggest that the compaction assay may be a useful in vitro method for rapidly assessing the actions of enzymatic disruption of a complex biopolymer, such as that observed for the actions of rhDNase on purulent airway secretions.

An antibody capture bioassay (ACB) for DNase in human serum samples.

A novel assay for antibody captured bioactivity (ACB) has been developed to quantitate deoxyribonuclease I (DNase) in human serum samples. The procedure is simple, sensitive, reproducible and has a high throughput. Serum samples are diluted a minimum of 1/4 and assayed in 96-well microtiter plates coated with polyclonal antibodies specific to DNase. The serum is removed from the wells, the plates are washed and the antibody bound DNase is incubated at 37 degrees C with a DNA-methyl green substrate. The assay is sensitive to 0.8 ng/ml with a range to 10 +/- 2 ng/ml, depending upon the time of incubation (48 +/- 2 h). The recovery of rhDNase spiked into human serum samples averaged 84.4% +/- 6.7% in sera diluted 1/4 and 97.8% +/- 7.2% at a 1/8 serum dilution. Intra-assay precision ranged from 3.0 to 7.5% coefficient of variation (% CV) and interassay precision ranged from 5.0 to 10.2% CV for spiked serum controls. Endogenous DNase concentrations in 27 normal human sera were found to range from < 2.0 to 11.4 ng/ml. Endogenous DNase-like activity was found in Cynomolgus and Rhesus monkey sera; this activity diluted linearly and did not interfere with accurate quantitation of added rh DNase. No endogenous DNase-like activity could be detected in ten Sprague-Dawley rat sera. Bovine pancreatic DNase was found to have only very low cross-reactivity in this assay system. The ACB assay format can potentially be applied to the quantitation of other enzymes in serum and other biological samples.

Quantitation of E. coli protein impurities in recombinant human interferon-gamma.

A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.

Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.

Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.

Isolation of motoneuron cell bodies from spinal cord stored at -70 degrees C in ethylene glycol.

Pretreatment of spinal cord with ethylene glycol permits long-term storage of the tissue at -70 degrees C prior to isolation and biochemical analysis of the cell bodies of spinal motoneurons. The method is useful for storing spinal tissue from laboratory animals, as well as from human post mortem specimens, where aliquots of tissue may then be used for motoneuron isolation over an indefinitely long period. In addition to inhibiting the loss of soluble proteins from the neurons during freezing and thawing, cryoprotection increases the yield and improves the appearance of the isolated cell bodies. The method should aid biochemical studies of many kinds of neuronal subpopulations isolated from small amounts of starting material.

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Changes in the amounts of tubulin, actin, and neurofilament polypeptides were found in regenerating motoneurons of grass frogs during the period of axonal elongation. Ventral roots 9 and 10 were transected unilaterally about 7 mm from the spinal cord. 35 d later, [3H]colchicine binding had decreased in the proximal stumps to approximately one-half of contralateral control values, well before the regenerating motor axons had reinnervated skeletal muscles of the hind limb. [3H]colchicine binding did not change significantly in the operated halves of the 9th and 10th spinal cord segments over a 75-d period. The relative amounts of actin, tubulin, and neurofilament polypeptides in the operated ventral roots were measured by quantitative densitometry of stained two-dimensional electrophoretic gels. Alpha-tubulin, beta-tubulin, and the 68,000 molecular weight subunit of neurofilaments (NF68) decreased within the transected ventral roots to 78%, 57%, and less than 15% of control values, respectively. The amount of actin increased to 132% of control values within the operated ventral roots, although this change was not statistically significant. Opposite changes were found within motoneuronal cell bodies isolated from the spinal cord. The relative amounts of alpha-tubulin, beta-tubulin and NF68 within axotomized perikarya increased, respectively, to 191%, 146%, and 144% of that in control perikarya isolated from the contralateral side of the spinal cord. Thus, the changes in NF68 and tubulin did not occur uniformly throughout the injured cells. The possible structural and functional consequences of these changes are discussed.

Acetylcholinesterase distribution in axotomized frog motoneurons.

The distribution of acetylcholinesterase (AChE; EC 3.1.1.7) activity was examined in the perikarya and proximal axonal stumps of frog motoneurons injured by ventral root transection. Based upon measurements of net AChE accumulation in the proximal stumps of transected ventral roots, and upon orthograde clearances of AChE reported by others, it was determined that an amount of AChE equivalent to at least 0.7-2 times the perikaryal content of this enzyme enters the motor axon each day. A progressive decrease in the rate of AChE accumulation in transected axons during the first 3 days after ventral rhizotomy raised the possibility that excess enzyme might accumulate elsewhere within the axotomized motoneurons. However, AChE accumulation was detected only near the cut ends of the ventral roots and was not appreciably increased within injured motoneuronal cell bodies and proximal dendrites, which were isolated by a new method combining bulk and single-cell isolation techniques. These data suggest that AChE turnover is altered rapidly in response to axonal injury, thereby avoiding large perikaryal accumulations of this enzyme.

Retrograde alteration of 6-phosphogluconate dehydrogenase in axotomized superior cervical ganglia of the rat.

Mechanisms underlying increased activity of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase [decarboxylating] EC 1.1.1.44) in axotomized rat superior cervical ganglia were explored using a highly sensitive micro-immunochemical assay employing antibodies raised in rabbits against the purified enzyme. 6-Phosphogluconate dehydrogenase was purified from rat brain more than 1700-fold by salt fractionation, anion exchange, and immunoaffinity chromatography. The purified enzyme consisted of identical subunits having molecular weights of about 48,800 which could aggregate to catalytically active isomers of various sizes; however, only one form of the enzyme was detected in freshly prepared homogenates of rat neural tissue. Physical and immunological properties of the enzyme from rat brain were similar to those from superior cervical ganglia and liver. Augmented 6-phosphogluconate dehydrogenase activity noted in superior cervical ganglia 2 days after transection of major postganglionic nerve trunks was accompanied by a parallel increase in immunoreactive protein. Michaelis constants of the enzyme were the same in control and axotomized ganglia, and the presence of activators and inhibitors was not detected. It is concluded that increases in 6-phosphogluconate dehydrogenase subsequent to axotomy can be accounted for entirely by an increase in the steady state concentration of this protein.