Subthalamic nucleus activity in the processing of body and mental action verbs in people with Parkinson's disease.

Local field potentials evoked by body action and mental action verbs were recorded in the subthalamic nucleus (STN) of 18 patients with Parkinson's disease through the electrodes implanted for deep brain stimulation. Compared with the medication on-condition, the medication off-condition showed a difference in activity in the early time segments, mainly in the right STN, with larger amplitudes for body action verbs. In the on-condition a similar pattern was detected in the left STN. These patterns of early differences in activity evoked by different types of verbs might indicate the potential of the STN to rapidly detect relevant behavioural clues in verbal content and to integrate these in subsequent cortico-subcortical interactions. In addition, these lateralizations allow speculations about shifts in processing activity correlating with dopaminergic denervation. Whether this detection relies on phonological, semantic or grammatical clues remains an open question.

Assessment of plasma anti-elastin antibodies for use as a diagnostic aid for chronic progressive lymphoedema in Belgian Draught Horses.

Diagnosis of chronic progressive lymphoedema (CPL) in draught horses, including the Belgian Draught Horse, is mainly based on clinical evaluation of typical lower limb lesions. A deficient perilymphatic elastic support, caused by a pathological elastin degradation in skin and subcutis, has been suggested as a contributing factor for CPL. Elastin degradation products induce the generation of anti-elastin Ab (AEAb), detectable in horse serum by ELISA. For a clinically healthy group of draught horses, a significantly lower average AEAb-level than 3 clinically affected groups (mild, moderate and severe symptoms) was demonstrated previously. To improve CPL-diagnosis, we evaluated the AEAb-ELISA as an in vitro diagnostic aid in individual horses. Test reproducibility was assessed, performing assays independently in 2 laboratories on a total of 345 horses. Possible factors associated with AEAb-levels (age, gender, pregnancy, test lab and date of blood collection) were analyzed using a mixed statistical model. Results were reproducible in both laboratories. AEAb-levels in moderately and severely affected horses were significantly higher than in healthy horses. Nevertheless, this was only demonstrated in barren mares, and, there was a very large overlap between the clinical groups. Consequently, even when a high AEAb cut-off was handled to obtain a reasonable specificity of 90%, a very low sensitivity (21%) of AEAb for CPL-diagnosis was obtained. Results on the present sample demonstrate that the described ELISA procedure is of no use as a diagnostic test for CPL in individual horses.

Chronic progressive lymphoedema in draught horses.

The objective of this review was to summarise and evaluate the current state of knowledge about chronic progressive lymphoedema in draught horses. Clinical signs of this multifactorial disorder are mainly restricted to the lower limbs, comprising progressively deteriorating skin, swelling and deformation. Although typical lesions were first reported at the beginning of the 20th century, chronic progressive lymphoedema was recognised as a specific syndrome only in 2003, and since then research has driven forward. Despite the high prevalence in some breeds and the serious economic impact, the pathogenesis is not fully understood, and the available treatment options remain symptomatic and noncurative. There is a need to improve diagnostic techniques and to develop selection tools.

Mck2-dependent infection of alveolar macrophages promotes replication of MCMV in nodular inflammatory foci of the neonatal lung.

Infection with cytomegalovirus (CMV) shows a worldwide high prevalence with only immunocompromised individuals or newborns to become symptomatic. The host's constitution and the pathogen's virulence determine whether disease occurs after infection. Mouse CMV (MCMV) is an appreciated pathogen for in vivo investigation of host-pathogen interactions. It has recently been reported that a single base pair deletion can spontaneously occur in the open reading frame of MCMV-encoded chemokine 2 (MCK2), preventing the expression of the full-length gene product. To study the consequences of this mutation, we compared the Mck2-defective reporter virus MCMV-3D with the newly generated repaired Mck2(+) mutant MCMV-3DR. Compared with MCMV-3D, neonatal mice infected with MCMV-3DR showed severe viral disease after lung infection. Viral disease coincided with high viral activity in multiple organs and increased virus replication in previously described nodular inflammatory foci (NIF) in the lung. Notably, MCMV-3DR showed tropism for alveolar macrophages in vitro and in vivo, whereas MCMV-3D did not infect this cell type. Moreover, in vivo depletion of alveolar macrophages reduced MCMV-3DR replication in the lung. We proposed an Mck2-mediated mechanism by which MCMV exploits alveolar macrophages to increase replication upon first encounter with the host's lung mucosa.

Genetic parameters for chronic progressive lymphedema in Belgian Draught Horses.

Genetic parameters for chronic progressive lymphedema (CPL)-associated traits in Belgian Draught Horses were estimated, using a multitrait animal model. Clinical scores of CPL in the four limbs/horse (CPLclin ), skinfold thickness and hair samples (hair diameter) were studied. Due to CPLclin uncertainty in younger horses (progressive CPL character), a restricted data set (D_3+) was formed, excluding records from horses under 3 years from the complete data set (D_full). Age, gender, coat colour and limb hair pigmentation were included as fixed, permanent environment and date of recording as random effects. Higher CPLclin certainty (D_3+) increased heritability coefficients of, and genetic correlations between traits, with CPLclin heritabilities (SE) for the respective data sets: 0.11 (0.06) and 0.26 (0.05). A large proportion of the CPLclin variance was attributed to the permanent environmental effect in D_full, but less in D_3+. Date of recording explained a proportion of variance from 0.09 ± 0.03 to 0.61 ± 0.08. Additive genetic correlations between CPLclin and both skinfold thickness and hair diameter showed the latter two traits cannot be used as a direct diagnostic aid for CPL. Due to the relatively low heritability of CPLclin , selection should focus on estimated breeding values (from repeated clinical examinations) to reduce CPL occurrence in the Belgian Draught Horse.

Evaluation of an IgE ELISA with Culicoides spp. extracts and recombinant salivary antigens for diagnosis of insect bite hypersensitivity in Warmblood horses.

Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n=176) according to the owners' descriptions. IBH-affected horses were subdivided based on the severity of their clinical signs at sampling and whether or not their IBH history was considered to be classical. The accuracies of the tests increased when clinical signs at sampling were more pronounced or when the IBH history could be considered as classical. A combination of IgE levels against the three best performing Culicoides spp. recombinant proteins (Culn4, Culo1 and Culo2) and ObsWBE resulted in the best performing test. When IBH-affected horses showing a classical history of the disease and severe clinical signs were compared with IBH-unaffected horses, the Youden's index at the optimal cut-off for the three tests in combination was 0.67. This optimal cut-off had a sensitivity of 70%, a specificity of 97% and a total accuracy of 92%. The performance of the IgE ELISA was affected by the severity of IBH clinical signs at sampling and was improved when IgE levels against several recombinant proteins were combined.

Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells.

Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

Genetically modified CD34+ cells do not contribute to the mesenchymal compartment after autologous transplantation in the baboon.

BACKGROUND: There is ongoing controversy about the transdifferentiation of hematopoietic stem cells (HSC) into different tissues such as mesenchymal cells. This transdifferentiation or 'plasticity' would be an appealing concept for many therapeutic strategies. While studies in the murine model show encouraging results, reports from clinical allogeneic stem cell transplantations do not support the concept of HSC plasticity. Our aim was to determine whether transplantation of transduced autologous marrow CD34+ cells leads to long-term engraftment of gene-marked cells with mesenchymal characteristics in the baboon. METHODS: We analyzed marrow of two baboons that had received green fluorescence protein (GFP)-marked CD34+ autologous marrow cells after myeloablative conditioning. Marrow was obtained 1 and 2.5 years after transplantation and adherent CD11a- (pan-leukocyte Ab) cells were cultured for 3 weeks. Cultures were then analyzed by flow cytometry and fluorescence microscopy for the presence of GFP+ cells. For further analysis fresh and cultured cells were also labeled with multiple Ab and functional analysis was performed. RESULTS: Both animals showed persistent and stable GFP marking by flow cytometry in peripheral blood leukocytes as well as in CD34+ marrow cells at 1 and 2.5 years after transplantation. There was no evidence of GFP+ mesenchymal cells by either flow cytometry or fluorescence microscopy, while functional and phenotypical analysis identified mesenchymal stem cells in these cultures. DISCUSSION: We conclude that genetically modified CD34+ cells do not contribute to the adherent marrow-derived mesenchymal cell population after autologous transplantation.

MLA-sensitive cholinergic receptors involved in the detection of complex moving stimuli in retina.

Acetylcholine, acting through nicotinic acetylcholine receptors, mediates the response properties of many ganglion cells in the rabbit retina, including those that are directionally selective (DS; Ariel & Daw, 1982a, b). For example, Grzywacz et al. (1998) showed that cholinergic input is necessary for DS responses to drifting gratings, a form of textured stimulus. However, the identities and locations of the neuronal acetylcholine receptor (nAChR) subtypes that mediate this input are not clear (Keyser et al., 2000). We investigated the role of methyllycaconitine-sensitive, alpha7-like nAChRs in mediating DS responses to textured stimuli and apparent motion. We recorded extracellularly from On-Off DS ganglion cells in rabbit retina using everted eyecup preparations. Our data provide evidence that MLA-sensitive nAChRs are involved in mediating directionally selective responses to apparent motion and to a variety of complex, textured stimuli such as drifting square-wave gratings, transparent motion, and second-order motion.

The value of loop electrosurgical conization in the treatment of stage IA1 microinvasive carcinoma of the uterine cervix.

The objective of this study is to assess the value of Loop Electrosurgical Conization (LEC) in the treatment of stage IA1 microinvasive squamous cell carcinoma (MIC) of the uterine cervix. Retrospectively, 82 patients with FIGO stage IA1 MIC, primarily treated with LEC on see and treat basis, were analyzed. After the initial LEC, 16 patients received cytologic and colposcopic follow-up only, 66 patients underwent a second procedure (repeat LEC, Cold Knife Conization (CKC), or hysterectomy), and four patients underwent a third procedure (hysterectomy). In 63 patients (77%) no residual CIN 3 or MIC was present after the initial LEC. Treatment of residual CIN 3 or MIC was equally effective with a repeat LEC as with CKC. One patient defaulted follow-up and developed a recurrence in the vaginal vault and was treated with a radical hysterectomy. LEC can be used as an alternative for CKC in treatment of patients with stage IA1 MIC. The advantage of LEC is that it can be performed as an outpatient procedure in addition to a diagnostic colposcopy and does not require a major anesthetic. Only a small number of patients will need a more extensive procedure.

Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits.

Some members of the epithelial Na+ channel/degenerin (ENaC/DEG) family of ion channels have been detected in mammalian brain. Therefore, we examined the RNA and protein expression of these channels in another part of the central nervous system, the rabbit retina. We next sought to demonstrate physiological evidence for an amiloride-sensitive current in Müller glia, which, on the basis of a previous study, are thought to express alpha-ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160-168, 2001). RT-PCR of retinal RNA revealed the presence of alpha-, beta-, gamma-, and delta-ENaC as well as acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemical localization with antibodies against alpha-ENaC and beta-ENaC showed labeling in Müller cells and neurons, respectively. The presence of alpha-ENaC, beta-ENaC, and ASIC1 was detected by Western blotting. Cultured Müller cells were whole cell patch clamped. These cells exhibited an inward Na+ current that was blocked by amiloride. These data demonstrate for the first time both the expression of a variety of ENaC and ASIC subunits in the rabbit retina as well as distinct cellular expression patterns of specific subunits in neurons and glia.

Rabbit retinal ganglion cell responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors.

The responses of many ganglion cells in the rabbit retina are mediated, at least in part, by acetylcholine (ACh) acting on neuronal nicotinic acetylcholine receptors (nAChRs). nAChRs are comprised of alpha and beta subunits; three beta subunits and nine alpha subunits of nAChRs have been identified and these subunits can combine to form a large number of functionally distinct nAChR subtypes. We examined the effects of cholinergic agents on the light-evoked responses of ganglion cells to determine which nAChR subtypes mediate the effects of ACh. Extracellular recordings of retinal ganglion cells were made in intact everted eyecup preparations and nicotinic agonists and antagonists were added to the superfusate. While several ganglion cell classes exhibited methyllycaconitine (MLA) sensitivity, the directionally selective (DS) ganglion cells were most sensitive; exposure to 30 nanomolar MLA, a concentration reportedly too low to affect alphaBgt-insensitive nAChRs, suppressed the stimulus-evoked responses of DS cells without eliminating directional selectivity. Epibatidine, which at low concentrations is an agonist selective for alphaBgt-insensitive nAChRs, stimulated firing of various cell types including DS ganglion cells at low nanomolar concentrations. The effects of the various agents tested persisted under cobalt-induced synaptic blockade. The low nanomolar MLA and epibatidine sensitivity of DS cells suggests that DS ganglion cells express both alphaBgt-sensitive and alphaBgt-insensitive nAChRs. Other ganglion cell types appear to express only alphaBgt-sensitive nAChRs but not alphaBgt-insensitive nAChRs.

Neuronal nitric oxide synthase and the autonomic innervation of the mouse lacrimal gland.

PURPOSE: To determine the expression patterns of the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) in the pterygopalatine ganglion (PPG) and the exorbital lacrimal gland of normal mice. METHODS: Mouse PPG and lacrimal glands were processed for single- and double-labeled indirect immunofluorescence studies. Slides were examined with conventional fluorescence microscopy and confocal laser scanning microscopy. RESULTS: All the somata in the PPG expressed both VAChT and nNOS immunoreactivity (IR). The postganglionic axons within the ganglion showed less VAChT-immunoreactive intensity than that seen in the somata, whereas nNOS IR was almost undetectable. In the lacrimal gland, nNOS-positive nerve bundles and fibers were observed to be associated with tear-collecting ducts, blood vessels, and acini. Some nNOS-positive punctate elements appeared to be distributed among acini. Many nerve fibers were VAChT immunoreactive and a small number of fibers were TH immunoreactive in the gland. Most of the VAChT-positive fibers and some of the TH-positive nerves displayed nNOS IR. CONCLUSIONS: The expression of nNOS in cells of the PPG and in lacrimal gland nerves suggests that NO may play a role in modulating tear production. The site of action may include the PPG, ducts, blood vessels, acini, nerve fibers, and myoepithelial cells within the gland. NO may modulate parasympathetic and/or sympathetic synaptic transmission or by acting directly on lacrimal gland components. The interaction between NO-ergic and the conventional autonomic input illustrates the complexity of the innervation pattern of the mouse lacrimal gland.

The relationship between GABA-containing cells and the cholinergic circuitry in the rabbit retina.

As a part of ongoing efforts to understand the cholinergic circuitry in the mammalian retina, we studied the coexpression of nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA), the GABA transporter 1 (GAT-1), or choline acetyltransferase (ChAT) immunoreactivity in the rabbit retina. Double-label experiments with monoclonal antibody 210 (mAb 210) against nAChRs and antibodies against GABA revealed that several populations of GABA-containing amacrine, displaced amacrine, and ganglion cells displayed nAChR immunoreactivity. Some of them also exhibited ChAT immunoreactivity and were identified as the cholinoceptive starburst cells. Other GABAergic amacrine cells positive for mAb 210 were not cholinergic. Simultaneous visualization of mAb 210 and GAT-1 immunoreactivity revealed that 10% of GAT-1 immunoreactive amacrine cells contained nAChRs. Ninety-nine percent of the GAT-1 labeled cells demonstrated GABA immunoreactivity, but only 75% of the GABAergic cells were outlined by GAT-1 staining. Neither population of starburst cells exhibited GAT-1 immunoreactivity. Thus, mAb 210 expressing, GAT-1 positive cells in the rabbit retina constitute a noncholinergic subset of GABAergic amacrine cells. Taken together, our results suggest that some GABAergic amacrine cells are cholinoceptive, raising the possibility that ACh, acting through nAChRs, can modulate the release of GABA in the rabbit retina.

[Treatment of hepatitis B virus-associated membranous glomerulonephritis with interferon alfa in a 7 year old boy]. / Therapie der membranösen Glomerulonephritis mit alpha-Interferon bei einem 7-jährigen Jungen mit chronisch persistierender Hepatitis B.

BACKGROUND: The association between Hepatitis B virus infection and membranous nephropathy has been confirmed by sources in several countries. Most commonly, the illness is seen as a nephrotic syndrome. Optimal treatment remains undefined. Antiviral therapies observed with recombinant human interferon alpha may be the best treatment option. CASE REPORT: We present a 7-year old boy with membranous glomerulonephritis and nephrotic syndrome. Twelve months after the initial hospitalization therapy was started with recombinant alpha-interferon s.c. three times weekly for six months. After the therapy the patient is stable, without proteinuria, edema or renal failure. He was seronegative for HBsAg, HBV-DNA and antibody to HBeAg. CONCLUSIONS: This case report suggests that alpha interferon is effective in the complete resolution of proteinuria in HBV membranous nephropathy.

Charged residues in the M2 region of alpha-hENaC play a role in channel conductance.

The epithelial Na(+) channel (ENaC) is a low-conductance channel that is highly selective for Na(+) and Li(+) over K(+) and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of alpha-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in alpha-subunit human ENaC (alpha-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na(+):K(+) permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 alpha-hENaC mutants along with wild-type (wt) beta- and gamma-hENaC was low (46-93 nA) compared with the wt channel (1-3 microA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant alpha-hENaC subunits with green fluorescent protein-tagged beta- and gamma-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of alpha-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

Amacrine, ganglion, and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors.

Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three beta and eight alpha subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain beta2 subunits, some of which are in combination with alpha3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 microm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 microm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.

Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells.

Human nicotinic acetylcholine receptor (AChR) subtypes alpha3 beta2, alpha3 beta2 alpha5, alpha3 beta4, and alpha3 beta4 alpha5 were stably expressed in cells derived from the human embryonic kidney cell line 293. alpha3 beta4 AChRs were found in prominent 2-micrometer patches on the cell surface, whereas most alpha3 beta2 AChRs were more diffusely distributed. The functional properties of the alpha3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on alpha3 beta4 AChRs and nearly a full agonist on alpha3 beta2 alpha5 AChRs. Chronic exposure of cells expressing alpha3 beta2 AChRs or alpha3 beta2 alpha5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of alpha3beta4 or alpha3 beta4 alpha5 AChRs, i.e. the up-regulation of alpha3 AChRs depended on the presence of beta2 but not beta4 subunits in the AChRs. This was also found to be true of alpha3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, alpha3 beta2 AChRs were expressed at much lower levels than alpha3 beta4 AChRs, but in the presence of nicotine, the amount of alpha3 beta2 AChRs exceeded that of alpha3 beta4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated alpha3beta2 AChRs were functional. The nicotinic antagonists curare and dihydro-beta-erythroidine also up-regulated alpha3 beta2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of alpha3 beta2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of alpha3 beta2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.