N-Acetylcysteine amide (NACA) and diNACA inhibit H2O2-induced cataract formation ex vivo in pig and rat lenses.

Oxidative stress plays a central role in cataract formation suggesting that antioxidants might slow cataract progression. The anticataract activity of N-acetylcysteine amide (NACA) and (2 R, 2 R')-3,3'-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and/or N-acetylcysteine (NAC), were evaluated in porcine and rat lens models. Cataractogenesis via oxidation was induced with H2O2 and/or glucose oxidase (GO). Porcine lenses were incubated in 0.1 mM, 1 mM, or 10 mM NAC, NACA or diNACA for 24 h. Lenses were then transferred to media containing 0.75 mM H2O2 and 4.63U of GO in order to maintain a constant H2O2 level for an additional 8 h. At the end of incubation, lenses were imaged under darkfield microscopy. Separately, rat lenses were extracted from 3-week-old Wistar rats and incubated with either 10 mM NACA or 10 mM diNACA for 24 h prior to treatment with 0.2U GO to generate a steady source of ∼0.6 mM H2O2. Rat lenses were analyzed by LC-MS/MS to quantify changes in cysteine, cystine, glutathione (GSH) or oxidised glutathione (GSSG) levels in the lens epithelium, cortex or core. Pre-treatment with NACA or diNACA followed by oxidation with H2O2 and/or GO to stimulate cataract formation afforded rapid assessment in ex vivo porcine (32 h) and rat (48 h) lens models. Pre-treatment of isolated porcine lenses with 0.1 mM, 1 mM or 10 mM of either NAC, NACA or diNACA followed by H2O2/GO treatment resulted in reduced lens opacity relative to the lenses exposed to H2O2/GO, with NACA and diNACA reducing opacities to a greater extent than NAC. Rat lenses incubated with 10 mM NACA or 10 mM diNACA without exposure to H2O2 showed no signs of opacities. Pre-treatment of rat lenses with 10 mM NACA or 10 mM diNACA, followed by GO cataract induction resulted in reduced opacities compared to control (GO alone). LC-MS/MS analyses revealed that NACA, but not diNACA, increased cysteine, cystine and GSH levels in rat lens epithelium and cortex regions. Taken together, both NACA and diNACA inhibited cataract formation to a greater extent than NAC (all at 1-10 mM) in an ex vivo porcine lens model. Both NACA and diNACA (both at 10 mM) reduced cataract formation in rat lenses. Based on LC-MS/MS analyses, NACA-induced reduction in opacity observed in rat lenses was attributed to enhanced cysteine and GSH levels while the diNACA-induced reduction in opacity induced did not consistently increase cysteine, cystine and GSH levels and, therefore, appears to involve a different antioxidant mechanism. These screening studies warrant further testing of NACA and diNACA as anticataract agents.

Protocol for a Randomised controlled trial to Evaluate the effectiveness and cost benefit of prescribing high dose FLuoride toothpaste in preventing and treating dEntal Caries in high-risk older adulTs (reflect trial).

BACKGROUND: Dental caries in the expanding elderly, predominantly-dentate population is an emerging public health concern. Elderly individuals with heavily restored dentitions represent a clinical challenge and significant financial burden for healthcare systems, especially when their physical and cognitive abilities are in decline. Prescription of higher concentration fluoride toothpaste to prevent caries in older populations is expanding in the UK, significantly increasing costs for the National Health Services (NHS) but the effectiveness and cost benefit of this intervention are uncertain. The Reflect trial will evaluate the effectiveness and cost benefit of General Dental Practitioner (GDP) prescribing of 5000 ppm fluoride toothpaste and usual care compared to usual care alone in individuals 50 years and over with high-risk of caries. METHODS/DESIGN: A pragmatic, open-label, randomised controlled trial involving adults aged 50 years and above attending NHS dental practices identified by their dentist as having high risk of dental caries. Participants will be randomised to prescription of 5000 ppm fluoride toothpaste (frequency, amount and duration decided by GDP) and usual care only. 1200 participants will be recruited from approximately 60 dental practices in England, Scotland and Northern Ireland and followed up for 3 years. The primary outcome will be the proportion of participants receiving any dental treatment due to caries. Secondary outcomes will include coronal and root caries increments measured by independent, blinded examiners, patient reported quality of life measures, and economic outcomes; NHS and patient perspective costs, willingness to pay, net benefit (analysed over the trial follow-up period and modelled lifetime horizon). A parallel qualitative study will investigate GDPs' practises of and beliefs about prescribing the toothpaste and patients' beliefs and experiences of the toothpaste and perceived impacts on their oral health-related behaviours. DISCUSSION: The Reflect trial will provide valuable information to patients, policy makers and clinicians on the costs and benefits of an expensive, but evidence-deficient caries prevention intervention delivered to older adults in general dental practice. TRIAL REGISTRATION: ISRCTN: 2017-002402-13 registered 02/06/2017, first participant recruited 03/05/2018. Ethics Reference No: 17/NE/0329/233335. Funding Body: Health Technology Assessment funding stream of National Institute for Health Research. Funder number: HTA project 16/23/01. Trial Sponsor: Manchester University NHS Foundation Trust, Oxford Road, Manchester, M13 9WL. The Trial was prospectively registered.

A 100 kHz Pulse Shaping 2D-IR Spectrometer Based on Dual Yb:KGW Amplifiers.

A high-speed, high-sensitivity and compact two-dimensional infrared (2D-IR) spectrometer based on 100 kHz Yb:KGW regenerative amplifier technology is described and demonstrated. The setup is three color, using an independent pump OPA and two separately tunable probe OPAs. The spectrometer uses 100 kHz acousto-optic pulse shaping on the pump beam for rapid 2D-IR acquisitions. The shot-to-shot stability of the laser system yields excellent signal-to-noise figures (∼10 µOD noise on 5000 laser shots). We show that the reduced bandwidth of the Yb:KGW amplifiers in comparison with conventional Ti:sapphire systems does not compromise the ability of the setup to generate high-quality 2D-IR data. Instrument responses of <300 fs are demonstrated and 2D-IR data presented for several systems of interest to physical chemists, showing spectral diffusion in NaSCN, amide I and II bands of a ß sheet protein and DNA base-pair-backbone couplings. Overall, the increased data acquisition speed, intrinsic stability, and robustness of the Yb:KGW lasers are a significant step forward for 2D-IR spectroscopy.

Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.

PURPOSE: To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. RESULTS: Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. CONCLUSIONS: We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.

Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels.

Gap junctions contain numerous channels that are clustered in apposed membrane patches of adjacent cells. These cell-to-cell channels are formed by pairing of two hemichannels or connexons, and are also referred to as connexon pairs. We have investigated various detergents for their ability to separately solubilize hemichannels or connexon pairs from isolated ovine lens fiber membranes. The solubilized preparations were reconstituted with lipids with the aim to reassemble native-type gap junctions and to provide a model system for the characterization of the molecular interactions involved in this process. While small gap junction structures were obtained under a variety of conditions, large native-type gap junctions were assembled using a novel two-step procedure: in the first step, hemichannels that had been solubilized with octylpolyoxyethylene formed connexon pairs by dialysis against n-decyl-beta-D-maltopyranoside. In the second step, connexon pairs were reconstituted with phosphatidylcholines by dialysis against buffer containing Mg2+. This way, double-layered gap junctions with diameter < or = 300 nm were obtained. Up to several hundred channels were packed in a noncrystalline arrangement, giving these reconstituted gap junctions an appearance that was indistinguishable from that of the gap junctions in the lens fiber membranes.

A quantitative study of potassium channel kinetics in rat skeletal muscle from 1 to 37 degrees C.

Potassium currents were measured using the three-microelectrode voltage-clamp technique in rat omohyoid muscle at temperatures from 1 to 37 degrees C. The currents were fitted according to the Hodgkin-Huxley equations as modified for K currents in frog skeletal muscle (Adrian et al., 1970a). The equations provided an approximate description of the time course of activation, the voltage dependence of the time constant of activation (tau n), and the voltage dependence of gK infinity. At higher temperatures the relationship between gK infinity and voltage was shifted in the hyperpolarizing direction. The effect of temperature on tau n was much greater in the cold than in the warm: tau n had a Q10 of nearly 6 at temperatures below 10 degrees C, but a Q10 of only approximately 2 over the range of 30-38 degrees C. The decreasing dependence of tau n on temperature was gradual and the Arrhenius plot of tau n revealed no obvious break-points. In addition to its quantitative effect on activation kinetics, temperature also had a qualitative effect. Near physiological temperatures (above approximately 25 degrees C), the current was well described by n4 kinetics. At intermediate temperatures (approximately 15-25 degrees C), the current was well described by n4 kinetics, but only if the n4 curve was translated rightward along the time axis (i.e., the current had a greater delay than could be accounted for by simple n4 kinetics). At low temperatures (below approximately 15 degrees C), n4 kinetics provided only an approximate fit whether or not the theoretical curve was translated along the time axis. In particular, currents in the cold displayed an initial rapid phase of activation followed by a much slower one. Thus, low temperatures appear to reveal steps in the gating process which are kinetically "hidden" at higher temperatures. Taken together, the effects of temperature on potassium currents in rat skeletal muscle demonstrate that the behavior of potassium channels at physiological temperatures cannot be extrapolated, either quantitatively or qualitatively, from experiments carried out in the cold.

Slow components of potassium tail currents in rat skeletal muscle.

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.

Calcium currents in a fast-twitch skeletal muscle of the rat.

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.

Spatial differences in gap junction gating in the lens are a consequence of connexin cleavage.

Gap junctions in the vertebrate lens exhibit spatial differences in pH gating: those in the cortical fibre cells close upon tissue acidification while those in the core region do not. It has been speculated that this difference in channel gating is a consequence of the cleavage of the connexins (Cx) that form the gap junction channels. We report the construction of a truncation mutant of ovine Cx50 which mimicks the cleavage in the intact lens. The construct when expressed in Xenopus oocytes results in the formation of functional channels. Comparison with full-length Cx50 revealed a significant reduction in the pH-sensitivity of the truncated form. This is the first evidence linking the non-uniform gating of gap junction channels in the lens with connexin cleavage. It also reveals how fibre cells in the core region remain connected despite the acidic environment caused by elevated lactate levels.

Differential expression of two gap junction proteins in corneal epithelium.

Two distinct gap junction proteins (connexins) are expressed in rat corneal epithelium in a way which parallels cellular differentiation processes in this tissue. Connexin43 is restricted predominantly to the basal cells of the corneal epithelium and is present in significantly reduced amounts compared to the situation in the adjacent conjunctival epithelium. In contrast, a gap junction protein recognized by antibodies against MP70 which is the ovine homolog of mouse connexin50, is strongly expressed in the corneal epithelium and is present in the basal cells, wing cells and surface cells. While the functional significance of this differential expression of corneal epithelial connexins has yet to be established, the corneal epithelium is the third avascular tissue besides lens and heart valves which expresses a gap junction protein recognized by anti-MP70 antibodies.

Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain.

Gap junction channel forming connexins share a common membrane topology which has four transmembrane spanning segments with the amino- and carboxy termini both located on the cytoplasmic side. Both, mutation and truncation of the carboxyl tail of some connexins have been shown previously to have profound effects on channel function. Truncation of the carboxyl tail of connexin50 (Cx50) and connexin46 (Cx46) occurs naturally during the maturation of fiber cells in the mammalian lens. This system therefore offers the unique opportunity to study not only the cleavage process but also the functional role played by the cleaved domain, in a physiologically relevant context. As a first step, we now report on the cleavage of the 70 kDa ovine isoform of Cx50. The calcium-activated neutral protease calpain (EC 3.4.22.17) was identified as the enzyme which removed a 32 kDa carboxyl portion from the Cx50 molecule in mature lens fiber cells. The amino-terminal 38 kDa portion remained embedded in the plasma membrane and was isolated and visualized as channel structures. The amino-terminal sequence of the cleaved 32 kDa portion matched an interior portion of the published amino acid sequence of the ovine Cx50 isoform. Thus, two closely spaced calpain cleavage sites were identified in the Cx50 molecule which were located carboxy-terminal from the predicted exit of the fourth transmembrane spanning segment by 62 or 72 amino acid residues, respectively. These data provide the basic information required for the future construction of Cx50 mutants to explore the functional consequences of this cleavage.

Human leukocyte antigen-DQB1* genotypes encoding aspartate at position 57 are associated with 3beta-hydroxysteroid dehydrogenase autoimmunity in premature ovarian failure.

Premature ovarian failure (POF) has an autoimmune pathogenesis in a significant proportion of cases. Autoantibodies to the steroid cell enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD) are present in one fifth of patients and may identify an autoimmune subgroup. As autoimmune diseases are associated with alleles of the human leukocyte antigen (HLA) genes, we examined the distribution of HLA-DRB1 and -DQB1 genotypes in 118 women with POF, of whom 21% had 3betaHSD autoantibodies, and 134 racially matched control subjects. Two HLA-DQB1 alleles, 0301 and 0603, were associated with 3betaHSD autoantibody positivity (P = 0.04 and P = 0.006, respectively). As the DQB1*0301 and -0603 genes share an identical codon at position 57 (aspartate, Asp), we analyzed the frequency of DQbeta-Asp57 encoding DQB1 genes in our series. Eighteen of 21 POF patients with 3betaHSD autoantibodies had DQbeta-Asp57-encoding genotypes (haplotype frequency, 27 of 42; 64%) compared with 92 of 134 control subjects (haplotype frequency, 109 of 268; 41%; P = 0.004), and 9 of 21 (43%) cases were homozygous for codon 57 genotypes compared with 17 of 134 (13%) control subjects (P = 0.0006). These probability values were not significant after correction for multiple testing, and these trends will therefore require confirmation in larger cohorts. HLA class II molecules present antigenic peptides to CD4+ T lymphocytes. DQbeta57 occupies a key site at the boundary of the peptide binding groove, with a major impact on peptide binding. Our preliminary demonstration of an association between POF, 3betaHSD autoimmunity, and a distinctive HLA-DQ molecule supports the hypothesis that autoantibodies to this steroid cell enzyme may be markers of autoimmune ovarian failure and suggests that presentation of autoantigenic or external peptides to T lymphocytes by HLA-DQ molecules with Asp57-beta-chains is important in the pathogenesis of this disease.

The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves' disease.

The lymphoid tyrosine phosphatase (LYP), encoded by the protein tyrosine phosphatase-22 (PTPN22) gene, is a powerful inhibitor of T cell activation. Recently, a single nucleotide polymorphism (SNP), encoding a functional arginine to tryptophan residue change at LYP codon 620 has been shown to be associated with type 1 diabetes and other autoimmune disorders. We have used a PCR-restriction fragment (XcmI) assay to examine genotypes at the codon 620 polymorphism in 549 unrelated probands with Graves' disease, 104 unrelated subjects with autoimmune Addison's disease and 429 controls. The T nucleotide at the SNP, encoding the tryptophan 620 residue, was present in 151 of 1098 (13.8%) Graves' disease alleles compared to 67 of 858 (7.8%) control alleles (chi(2) = 17.2, p = 3.4 x 10(-5)' odds ratio = 1.88, 5-95% confidence intervals [CI] 1.39 to 2.55). Similarly, the T nucleotide at the codon 620 SNP was present in 26 of 208 (12.5%) Addison's disease alleles vs 7.8% of controls (chi(2) = 4.63, p = 0.031; odds ratio = 1.69, 5-95% CI 1.04 to 2.73). These data suggest that this LYP polymorphism is a susceptibility allele for Graves' disease with a major effect, and which is likely to have a role in many other autoimmune conditions.

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3.

BACKGROUND: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. RESULTS: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. CONCLUSIONS: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Genetic association of the HLA DRB1 gene locus on chromosome 6p21.3 with schizophrenia.

OBJECTIVE: The authors investigated the human leukocyte antigen (HLA) DRB1*04 gene in schizophrenic patients because it is positively associated with rheumatoid arthritis, an autoimmune disease that exhibits a strong negative association with schizophrenia. The HLA DQB1*0602 allele was also studied because of previous reports of genetic association between it and schizophrenia. Maternal HLA was investigated because of the reported association between prenatal influenza and schizophrenia and the central role of HLA molecules in the immune response to viral infections. METHOD: Polymerase chain reactions and sequence-specific oligonucleotide probes were used to genotype 94 unrelated patients with DSM-III-R schizophrenia, 92 mothers of schizophrenic offspring who were not related either to each other or to the 94 patients, and 177 healthy comparison subjects. RESULTS: The frequency of DRB1*04 alleles was significantly lower in both the schizophrenic patients and the unrelated mothers of schizophrenic offspring than in the healthy comparison subjects. No significant differences were found for DQB1*0602. CONCLUSIONS: DRB1*04 alleles may partially account for the genetic predisposition to schizophrenia. The association reported here may be explained by genetic linkage or by an autoimmune pathophysiology for a proportion of schizophrenia cases. Alternatively, it may be that maternal B lymphocytes that do not express the DR4 antigen encoded by DRB1*04 respond to influenza virus by producing antibodies that perturb neurodevelopment, thus underpinning a proportion of schizophrenia cases.

Collateral sprouting of insect motorneurons.

The metathoracic extensor tibiae muscle of the cricket, Teleogryllus oceanicus, is innervated by two excitatory axons: a fast axon, which initiates large twitches to single stimuli, and a slow axon, which evokes minute twitches to single stimuli, but which, through facilitation and summation, evokes readily measurable tension to repetitive stimulation. The fast axon and the slow axon leave the metathoracic ganglia in different nerve roots, the fast axon through nerve 5 and the slow axon through nerve 3. The fast axon innervates muscle fibers in the middle of the extensor tibiae, and the slow axon innervates muscle fibers at the proximal and distal ends of the muscle. A central region of muscle fibers is innervated by only the fast axon. This region is flanked on either side by dually innervated fibers, fibers that receive both the fast and the slow axons. Fibers with only slow axon innervation are restricted to a wedge-shaped patch in the proximal extensor tibiae and a larger region in the most distal portion of the muscle. Sectioning nerve 5 containing the fast axon, or nerve 3 containing the slow axon, partially denervates the extensor tibiae. Functional transmission by the fast axon fails 7-10 days after nerve section. The innervation field of the intact motorneuron expands in a partially denervated muscle. The linear expansion rate of the slow axon field is about 20-40 micrometer per day. The enlarged slow field does not regress when axons regenerate to the muscle through nerve 5. The progressive expansion of the slow innervation field suggests that the expansion is due to collateral sprouting of slow axon terminals.

Repeat HLA-B and -DR loci mismatching at second liver transplantation improves patient survival.

The role of HLA matching in liver transplantation remains uncertain, and the effect of HLA mismatches on the outcome of second transplants has not been studied. In renal transplantation, if HLA mismatches are repeated in the second graft there is a greatly increased risk of immunological graft failure. In the present study, 78 patients who had received second liver transplants were studied. HLA typing was performed using standard complement-dependent microcytotoxicity for class I (A and B) antigens, and a combination of restriction fragment length polymorphism and serology for HLA-DR. Patient survival at follow-up for those with a repeat B locus mismatch was improved (79% compared with 43%, P < 0.02), and a similar effect was noted for repeat DR mismatches (67% vs. 47%, P = 0.06). In the subgroup of patients who received a second transplant for graft rejection, 90% of patients with a repeat B mismatch were alive at follow-up compared with 46% without B mismatches (P = 0.02). This improvement in patient survival was evident during the first 2 months after the second transplant. In this study, repeat HLA-B and -DR mismatching improves survival after second transplantation.

Role of HLA compatibility in pediatric liver transplantation.

BACKGROUND: The influence of HLA mismatching in liver transplantation remains controversial. To date, few studies have focused solely on the pediatric population, and none have investigated DR and DQ mismatches using molecular genotyping. We sought to investigate HLA-A, -B, -DR, and -DQ mismatches in a large series of primary pediatric liver transplant recipients. Living-related liver transplants were excluded. METHODS: A total of 138 consecutive first liver transplants performed between January 1991 and July 1996 were studied. Minimum follow-up was 1 year, and both patient and graft survival rates were assessed. The incidence of the most common complications was analyzed. HLA-A and -B phenotyping was performed by complement-dependent microcytotoxicity or polymerase chain reaction (PCR)-sequence-specific primer protocols in 133 of 138 patients. HLA-DR and -DQ genotyping was performed by standard PCR-sequence-specific oligonucleotide and/or PCR-sequence-specific primer protocols in 135 patients. RESULTS: Overall, there was no influence of HLA mismatching on either graft or patient survival rates. However, patients with two mismatches at the A locus showed a significantly lower incidence of acute rejection than those with one A mismatch (52% vs. 72%; P < 0.03) and patients with two B locus mismatches had a better graft survival rate at 5 years than those with one mismatch (76% vs. 62%), although this was of only borderline significance (P < 0.09). No differences were found in the severity of the episodes of rejection, incidence of chronic rejection, cytomegalovirus hepatitis, and other causes of graft loss. CONCLUSION: This study indicates that HLA-A, -B, -DR, and -DQ mismatches are not detrimental in primary pediatric liver transplantation.

Immunopathogenesis of hepatitis B virus recurrence after liver transplantation.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation is associated with inflammatory graft changes, despite immunosuppression and donor/recipient HLA mismatch. We investigated whether immune mechanisms are involved in the pathogenesis of hepatitis B after liver transplantation. METHODS: The virus-specific T helper (Th) cell response, activation of Th1/Th2 subpopulations, donor/recipient HLA, and expression of tumor necrosis factor (TNF)-alpha/TNF receptors were determined in 28 patients who underwent transplantation for HBV-related cirrhosis (17 with HBV recurrence and 11 without recurrence) in comparison to 30 nontransplant patients with chronic hepatitis B. RESULTS: Orthotopic liver transplantation recipients with HBV recurrence showed significant hepatitis B core antigen-specific T-cell proliferation, comparable to nontransplant patients, which was not present in transplant recipients without recurrence. In addition, hepatic and serum interleukin (IL)-2, interferon-gamma, and TNF-alpha were enhanced, without changes in IL-4 and IL-10. Phenotypically, hepatic infiltrates in allografts with HBV recurrence were comprised of CD4+ lymphocytes and macrophages with a correlation between interferon-gamma- and TNF-alpha-producing cells and the degree of necroinflammatory activity. There was a marked up-regulation of both TNF-alpha receptors, significantly greater than in nontransplant patients. CONCLUSIONS: These findings suggest that despite immunosuppression, HLA class I-independent immune mechanisms have a significant pathogenic role in liver damage associated with HBV recurrence after liver transplantation.

IgG donor-specific crossmatches are not associated with graft rejection or poor graft survival after liver transplantation. An assessment by cytotoxicity and flow cytometry.

Early studies in liver transplantation suggested that there was no association between graft outcome or rejection and the presence of alloantibodies before transplantation. More recent reports have suggested lower graft survival rates and a higher incidence of chronic rejection in patients with IgG warm-T crossmatches. In the present study, panel reactive antibody, direct crossmatch testing, and flow cytometry were used to detect preformed antibodies in sera from 158 consecutive adult recipients of first hepatic grafts. The relationship between preformed antidonor antibodies and liver allograft survival and rejection was determined. Twenty-six (17%) patients were panel reactive antibody (PRA)-positive before transplantation, 22 (15%) had positive donor-specific crossmatches, and 14 (9%) were positive by IgG-specific flow cytometry. Cumulative survival distribution and multivariate analysis failed to reveal any significant associations between overall graft survival and antibody status. Graft survival in patients with PRA-positive sera was 81% compared with 77% for those with PRA-negative sera, 68% for those with positive donor-specific crossmatches compared with 80% for those who were donor-specific crossmatch negative, and 79% for those who were antibody positive by flow cytometric analysis compared with 78% for those who were antibody negative. Subgroup analysis also failed to reveal any significant associations. In addition, Cox proportional hazards regression analysis failed to reveal a relationship between acute or chronic graft rejection with the presence or absence of preformed antibodies, irrespective of immunoglobulin class, cell type (T or non-T), specificity, or technique used for antibody detection. In conclusion, there appears to be no association between either donor-specific or "third-party" alloreactive IgG or IgM antibodies and liver transplant survival or rejection. These data do not indicate a need for prospective crossmatching of liver transplant recipients.