[Clinicopathological characteristics and prognosis of diffuse midline gliomas with histone H3K27M mutation: an analysis of 30 cases].

Objective: To analyze the clinicopathological characteristics and prognosis of diffuse midline glioma (DMG) with H3K27M mutation. Methods: Thirty cases of DMG were collected in Guangdong Sanjiu Brain Hospital from October 2016 to May 2018. The patients' clinicopathological data including age, tumor site and histological grade, treatment and follow-up data were collected and analyzed. Results: There were 21 males and 9 females, with a mean age of 26 years (range 5-53 years). Fourteen tumors were located in thalamus, 12 in brainstem (one involved both thalamus and brainstem), and one each in hypothalamus, fourth ventricle, and sellar region, respectively. Two cases presented as diffuse intracranial lesions. Three cases (10.0%) were of WHO grade Ⅰ, 10 cases (33.3%) were grade Ⅱ, eight cases (26.7%) were grade Ⅲ, and nine cases (30.0%) were grade Ⅳ.All patients with gradeⅠ tumors were older than 20 years. Histologically, all were pilocytic astrocytoma-like. Immunohistochemical staining demonstrated that all tumors were IDH1 negative. Twenty-eight tumors showed diffuse expression of H3K27M, and two showed focal expression. Twenty-one tumors(100.0%, 21/21) showed absent expression of H3K27me3. Sixteen tumors (57.1%, 16/28) showed strongly positive expression of p53, and ATRX was negative in eight tumors (38.1%, 8/21). The Ki-67 proliferation index ranged from 5% to 40%. Eight cases (including two cases of H3K27M expression of individual cells) showed K27M mutation in H3F3A gene. Intracranial and spinal cord dissemination occurred in six cases (20.0%, 6/30). Median progression-free survival (PFS) was 9.5 months and median overall survival (OS) was 34 months. Mean PFS was 11.2 months and mean OS was 24.3 months. Compared with adults (>20 years old), children/adolescents (no more than 20 years old) had significantly shorter median OS (8 months vs. 34 months, P=0.013). There was no significant difference in PFS and OS between DMGs located in the brain stem/thalamus and other sites within midline (P>0.05). There was no significant difference in PFS and OS between WHO grade ⅠDMGs and WHO grade Ⅱ-Ⅳ DMGs (P>0.05). Conclusions: DMGs occur more commonly in children and adolescents with male predominance. DMGs present with WHO Ⅰ-Ⅳ tumors morphologically, and pilocytic astrocytoma-like lesions with WHO Ⅰ are more common in adults. Expression of H3K27M but not H3K27me3 is helpful for diagnosis of DMG. The prognosis of children/adolescents is significantly worse than that of adults, whereas histological grade and tumor location do not affect prognosis.

Association between COL9A2 Gln326Arg mutations and the development of intervertebral disc disease in a Chinese population.

Intervertebral disc disease is a multifactorial condition, yet disease pathogenesis that can be promoted by a single dominant mutation affecting the expression of susceptibility genes. We performed a case-control study to assess the influence of the COL9A2 Gln326Arg polymorphism on risk of intervertebral disc disease in a Chinese population. Between March 2014 and March 2015, a total of 215 patients and 230 healthy controls were recruited from Binzhou Medical University Hospital. Genotyping of COL9A2 Gln326Arg was carried out using polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate logistic regression analyses revealed that the Arg/Arg genotype of COL9A2 Gln326Arg was associated with increased risk of intervertebral disc disease in comparison to the Gln/Gln genotype [crude odds ratio (OR) = 2.25, 95% confidence interval (CI) = 1.12-4.62; adjusted OR = 2.46, 95%CI = 1.20-5.29]. Moreover, the Arg/Arg genotype correlated with an elevated risk of this disease compared to the Gln/Gln + Gln/Arg genotypes (crude OR = 2.21, 95%CI = 1.17-4.30; adjusted OR = 2.42, 95%CI = 1.28-5.51). In conclusion, our results suggest that the COL9A2 Gln326Arg polymorphism contributes to the development of intervertebral disc disease in the Chinese population.

Serological comparison of antibodies to avian influenza viruses, subtypes H5N2, H6N1, H7N3 and H7N9 between poultry workers and non-poultry workers in Taiwan in 2012.

In Taiwan, avian influenza virus (AIV) subtypes H5N2, H6N1 and H7N3 have been identified in domestic poultry, and several strains of these subtypes have become endemic in poultry. To evaluate the potential of avian-to-human transmission due to occupational exposure, an exploratory analysis of AIV antibody status in poultry workers was conducted. We enrolled 670 poultry workers, including 335 live poultry vendors (LPVs), 335 poultry farmers (PFs), and 577 non-poultry workers (NPWs). Serum antibody titres against various subtypes of viruses were analysed and compared. The overall seropositivity rates in LPVs and PFs were 2·99% (10/335) and 1·79% (6/335), respectively, against H5N2; and 0·6% (2/335) and 1·19% (4/335), respectively, for H7N3 virus. Of NPWs, 0·35% (2/577) and 0·17% (1/577) were seropositive for H5N2 and H7N3, respectively. Geographical analysis revealed that poultry workers whose workplaces were near locations where H5N2 outbreaks in poultry have been reported face greater risks of being exposed to viruses that result in elevated H5N2 antibody titres. H6N1 antibodies were detected in only one PF, and no H7N9 antibodies were found in the study subjects. Subclinical infections caused by H5N2, H6N1 and H7N3 viruses were thus identified in poultry workers in Taiwan. Occupational exposure is associated with a high risk of AIV infection, and the seroprevalence of particular avian influenza strains in humans reflects the endemic strains in poultry in this region.

Reversal of ophthalmic artery flow as a predictor of intracranial hemodynamic compromise: implication for prognosis of severe carotid stenosis.

BACKGROUND AND PURPOSE: High-grade cervical carotid stenosis (70-99%) or occlusion often accompanies reversed ophthalmic artery flow (ROAF), but its potential clinical significances remain poor understood. This study assessed ROAF and the related variables caused by carotid hemodynamic compromise in patients with unilateral severe cervical carotid stenosis. METHODS: The study consisted of 200 patients diagnosed as unilateral high-grade cervical carotid stenosis/occlusion using ultrasonography. The hemodynamic parameters of 152 patients, excluding 48 with cervical carotid occlusion, were compared based on the presence of ROAF. Out of 200 patients, 159 underwent brain magnetic resonance imaging and were analysed for risk factors impacting functional outcomes including ROAF. RESULTS: The patients (n = 48) with internal carotid artery occlusion had significantly higher incidence (62.5%) of ROAF compared with that of 25.0% in those patients (n = 152) with unilateral high-grade carotid stenosis (P < 0.001). In ROAF patients (n = 38) with the unilateral high-grade stenosis, a significant retrobulbar arteries hemodynamic difference was observed between the stenotic and non-stenotic vessels. The patients (n = 159) with history of stroke (P = 0.035), ROAF (P = 0.023) and intracranial stenosis (P < 0.001) exhibited significantly higher incidence of poor functional outcome compared with the corresponding control groups. In the same patients (n = 159), those with both cervical and intracranial stenosis showed sevenfold higher risk (OR, 7.60; 95% CI, 3.44-16.81) for ROAF than those with only cervical stenosis. CONCLUSIONS: ROAF may result from intracranial hemodynamic compromise. Patients with unilateral high-grade cervical carotid stenosis/occlusion in combination with intracranial stenosis appear to be a significant risk factor for poor functional outcome and increased incidence of ROAF.

Surveillance of avian influenza A(H7N9) virus infection in humans and detection of the first imported human case in Taiwan, 3 April to 10 May 2013.

On 3 April 2013, suspected and confirmed cases of influenza A(H7N9) virus infection became notifiable in the primary care sector in Taiwan, and detection of the virus became part of the surveillance of severe community-acquired pneumonia. On 24 April, the first imported case, reported through both surveillance systems, was confirmed in a man returning from China by sequencing from endotracheal aspirates after two negative throat swabs. Three of 139 contacts were ill and tested influenza A(H7N9)-negative.

Correlation between magnetic properties and the electronic structures of soft magnetic ternary Fe(78-x)Y(x)B(22) (x = 4-9) bulk metallic glasses.

Fe and Y K-edge extended x-ray absorption fine structure, Fe(Y) L(3,2)-edge (L(3)-edge) x-ray absorption near-edge structure (XANES) and valence-band photoemission spectroscopy (VB-PES) measurements have been carried out to study soft magnetic ternary Fe(78-x)Y(x)B(22) bulk metallic glasses (BMGs). The combined XANES and VB-PES results do not show broadening of the Fe 3d band to support the previous interpretation of the reduction of the magnetic moment in BMGs by Y-induced decrease of exchange splitting of Fe 3d orbitals. Instead, the density of delocalized/itinerant Fe 3d states in the vicinity of the Fermi level is found to be reduced by Y substitution, which reduces the strength of itinerant-states-mediated ferromagnetic coupling between local spins on the Fe ions and the total magnetic moment of the Fe-based BMGs.

Prognostic factors affecting the outcome of early cervical cancer treated with radical hysterectomy and post-operative adjuvant therapy.

The purpose of this study is to investigate the clinical and histological features that may affect the survival of the patients and to evaluate the impact of post-operative adjuvant therapy on the outcomes of patients with stage IB and IIA carcinoma of the cervix. From August 1998 to January 2005, 140 patients with International Federation of Gynecology and Obstetrics stage IB and IIA cervical cancer were treated with radical hysterectomy and post-operative pelvic radiation therapy with or without chemotherapy. The median age was 55 years (range, 29-86 years). Seventy-six patients had stage IB and 64 patients had stage IIA disease. Tumour size was <4 cm in 96 patients and > or = 4 cm in 44 patients. One hundred and eleven patients had histology of squamous cell carcinoma, 12 patients has adenocarcinoma and 17 patients had other histologic types. Depth of stromal invasion was <2/3 in 20 patients and > or = 2/3 in 120 patients. Twenty-three patients had parametrial invasion and 117 patients had no parametrial invasion. Thirteen patients had lymphovascular space invasion and 127 had no lymphovascular space invasion. Nine patients had positive surgical margin and 131 patients had negative margin. Twenty-seven patients had pelvic lymph node metastasis and 113 patients had no pelvic lymph node metastasis. Seventy-five patients received concurrent chemoradiotherapy and 65 patients received radiotherapy alone. The 5-year overall survival (OAS) and disease-free survival were 83% and 72% respectively. In the log rank test, tumour size (P = 0.0235), pararmetrial invasion (P = 0.0121), pelvic lymph node metastasis (P < 0.0001) and adjuvant chemotherapy + radiotherapy (P = 0.0119) were significant prognostic factors for OAS, favouring tumour size <4 cm, absence of parametrial invasion and pelvic lymph node metastasis, and those who received adjuvant chemoradiotherapy. The patients who received radiation with concomitant chemotherapy had a 5-year OAS rate of 90% versus those who received radiotherapy alone, with a rate of 76%. For patients with high-risk early stage cervical cancer who underwent a radical hysterectomy and pelvic lymphadenectomy, adjuvant chemoradiotherapy resulted in better survival than radiotherapy alone. The addition of weekly cisplatin to radiotherapy is recommended. The treatment-related morbidity is tolerable.

Clinical effectiveness and safety experience with efalizumab in the treatment of patients with moderate-to-severe plaque psoriasis in Taiwan: results of an open-label, single-arm pilot study.

BACKGROUND: No clinical trial of efalizumab has been conducted in Asia. OBJECTIVE: To determine the efficacy and safety of efalizumab in Taiwanese patients with psoriasis. METHODS: This is an open-label, single-arm pilot study conducted at two centres. Patients were given 1 mg/kg efalizumab subcutaneously once a week for 12 weeks and were then followed up for a further 12 weeks. RESULTS: A total of 49 patients participated in the study. The median improvement in Psoriasis Area and Severity Index (PASI) during the treatment period was 19.6%, and a >or= 50% improvement in PASI was seen in 20.4%. Rebound was seen in 17.8% of patients, and anti-efalizumab antibodies were detected in 41% of patients. The most frequent adverse events were headache (34.7%), arthralgia/arthritis (28.6%), psoriasis events (new form/exacerbation; 26.5%) and pruritus (22.4%). CONCLUSIONS: This small pilot study indicated that efalizumab was effective in improving psoriasis symptoms in Taiwanese patients, with no new safety issues identified.

Serotonin and neuroprotection in functional bowel disorders.

The 5-HT(4) partial agonist tegaserod is effective in the treatment of chronic constipation and constipation predominant irritable bowel syndrome. 5-HT(4) receptors are located on presynaptic terminals in the enteric nervous system. Stimulation of 5-HT(4) receptors enhances the release of acetylcholine and calcitonin gene related peptide from stimulated nerve terminals. This action strengthens neurotransmission in prokinetic pathways, enhancing gastrointestinal motility. The knockout of 5-HT(4) receptors in mice not only slows gastrointestinal activity but also, after 1 month of age, increases the age-related loss of enteric neurons and decreases the size of neurons that survive. 5-HT(4) receptor agonists, tegaserod and RS67506, increase numbers of enteric neurons developing from precursor cells and/or surviving in culture; they also increase neurite outgrowth and decrease apoptosis. The 5-HT(4) receptor antagonist, GR113808, blocks all of these effects, which are thus specific and 5-HT(4)-mediated. 5-HT(4) receptor agonists, therefore, are neuroprotective and neurotrophic for enteric neurons. Because the age-related decline in numbers of enteric neurons may contribute to the dysmotilities of the elderly, the possibility that the neuroprotective actions of 5-HT agonists can be utilized to prevent the occurrence or worsening of these conditions should be investigated.

Retrospective analysis of early steroid-induced adverse reactions in kidney and kidney-pancreas transplant recipients.

Corticosteroids (steroids) are associated with numerous adverse drug reactions (ADRs). Long-term ADRs are well characterized, but there are limited data on the incidence and likelihood of short-term ADRs. We sought to determine the incidence of ADRs potentially related to early administration of steroids in kidney and kidney-pancreas transplant recipients and to determine the probability that the ADR was due to the steroid. We retrospectively evaluated the records of all eligible kidney or pancreas-kidney transplants during 2003. ADRs were rated by two reviewers according to the Naranjo algorithm, and identified as "definite," "probable," "possible," or "doubtful." ADRs were identified in 100% of patients (n = 103) by 8.2 +/- 4.9 days. The mean ADRs per patient were 3.26 +/- 1.04. Weight gain occurred in 79.6%, hypertension in 71.8%, diabetes mellitus in 52.4%, hyperglycemia in 47.6%, leukocytosis in 31.1%, insomnia in 27.2%, anxiety in 10.7%, and psychosis in 1.9%. Based on mean interinvestigator score, leukocytosis was judged as "probable" and weight gain and psychosis were "possible to probable." Diabetes, hyperglycemia, hypertension, and insomnia were "possible" and anxiety was "possible to doubtful." These results provide evidence of the incidence and likelihood of early steroid-related ADRs.

Insights into molybdenum cofactor deficiency provided by the crystal structure of the molybdenum cofactor biosynthesis protein MoaC.

BACKGROUND: The molybdenum cofactor (Moco) is an essential component of a large family of enzymes involved in important transformations in carbon, nitrogen and sulfur metabolism. The Moco biosynthetic pathway is evolutionarily conserved and found in archaea, eubacteria and eukaryotes. In humans, genetic deficiencies of enzymes involved in this pathway trigger an autosomal recessive and usually deadly disease with severe neurological symptoms. The MoaC protein, together with the MoaA protein, is involved in the first step of Moco biosynthesis. RESULTS: MoaC from Escherichia coli has been expressed and purified to homogeneity and its crystal structure determined at 2 A resolution. The enzyme is organized into a tightly packed hexamer with 32 symmetry. The monomer consists of an antiparallel, four-stranded beta sheet packed against two long alpha helices, and its fold belongs to the ferredoxin-like family. Analysis of structural and biochemical data strongly suggests that the active site is located at the interface of two monomers in a pocket that contains several strictly conserved residues. CONCLUSIONS: Asp128 in the putative active site appears to be important for catalysis as its replacement with alanine almost completely abolishes protein activity. The structure of the Asp128-->Ala variant reveals substantial conformational changes in an adjacent loop. In the human MoaC ortholog, substitution of Thr182 with proline causes Moco deficiency, and the corresponding substitution in MoaC severely compromises activity. This residue is located near the N-terminal end of helix alpha4 at an interface between two monomers. The MoaC structure provides a framework for the analysis of additional dysfunctional mutations in the corresponding human gene.

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas.

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes alpha-bungarotoxin (alpha-Bgt)-insensitive nAChRs, and a polyclonal antibody raised against the alpha-Bgt-sensitive receptor subunit, alpha7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35-receptor (mAb35-R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35-R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35-R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT-immunoreactive nerve fibers. In addition, the majority of ChAT-positive neurons expressed both cell surface and internal stores of mAb35-R. In the bowel, clusters of mAb35-R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin-immunoreactive myenteric neurons. In contrast, calbindin-immunoreactive submucosal neurons did not express cell surface mAb35-R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35-R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35-R immunoreactivity was only observed in ganglia. Alpha7-immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre- and postsynaptic nAChRs are present in the gut and pancreas.

Differential localization of Ca2+ channel alpha1 subunits in the enteric nervous system: presence of alpha1B channel-like immunoreactivity in intrinsic primary afferent neurons.

Immunocytochemistry was employed to locate calcium (Ca2+) channel proteins in the enteric nervous system (ENS) of the rat and guinea pig. Anti-peptide antibodies that specifically recognize the alpha1 subunits of class A (P/Q-type), B (N-type), C and D (L-type) Ca2+ channels were utilized. Alpha1B channel-like immunoreactivity was abundant in both enteric plexuses, the mucosa, and circular and longitudinal muscle layers. Immunoreactivity was predominantly found in cholinergic varicosities, supporting a role for Ca2+ channels, which contain the alpha1B subunit, in acetylcholine release. Immunoreactivity was also associated with the cell soma of calbindin-immunoreactive submucosal and myenteric neurons, cells that have been proposed to be intrinsic primary afferent neurons. Alpha1C channel-like immunoreactivity was distributed diffusely in the cell membrane of a large subset of neuronal cell bodies and processes, whereas alpha1D was found mainly in the cell soma and proximal dendrites ofvasoactive intestinal polypeptide-immunoreactive neurons in the guinea pig gut. Alpha1A channel-like immunoreactivity was found in a small subset of cell bodies and processes in the rat ENS. The differential localization of the alpha1 subunits of Ca2+ channels in the ENS implies that they serve distinct roles in neuronal excitation and signaling within the bowel. The presence of alpha1B channel-like immunoreactivity in putative intrinsic primary afferent neurons suggested that class B Ca2+ channels play a role in enteric sensory neurotransmission; therefore, we determined the effects of the N-type Ca2+ channel blocker, omega-conotoxin GVIA (omega-CTx GVIA), on the reflex-evoked activity of enteric neurons. Demonstrating the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (pCREB) identified neurons that became active in response to distension. Distension elicited hexamethonium-resistant pCREB immunoreactivity in calbindin-immunoreactive neurons in each plexus; however, in preparations stimulated in the presence of omega-CTx GVIA, pCREB immunoreactivity was found only in calbindin-immunoreactive neurons in the submucosal plexus and not in myenteric ganglia. These data confirm that intrinsic primary afferent neurons are located in the submucosal plexus and that N-type Ca2+ channels play a role in sensory neurotransmission.

In situ identification and visualization of neurons that mediate enteric and enteropancreatic reflexes.

To identify neurons participating in enteric and enteropancreatic reflexes, we validated the use of the activity-dependent markers FM1-43 and FM2-10 as "on-line" probes for the visualization of activated guinea pig enteric and pancreatic neurons. FM1-43 or FM2-10 labeling of neuronal perikarya and processes was induced by KCl (70 mM), veratridine (1.0 microM), intracellular injection of depolarizing current pulses, stimulation of afferent inputs, evoking reflexes (by inflating an intraluminal balloon, blowing puffs of N2 at, or applying glucose to, the villous surface of the duodenum), or injury; labeling was prevented by tetrodotoxin (0.5 microM). Intracellular recording and injection of Neurobiotin confirmed that FM1-43 labeled neurons that spike, but not those that exhibit only fast excitatory postsynaptic potentials. Perikarya did not label if axonal transport was blocked by colchicine. When pulses of N2 or glucose were directed at duodenal villi in vitro, labeling by FM1-43 or FM2-10 was observed in myenteric and pancreatic neurons, as well as in subsets of cells in pancreatic islets and intestinal crypts. Hexamethonium blocked the spread of label via nicotinic synapses and thus enabled primary afferent neurons to be located. Balloon distension elicited hexamethonium-resistant labeling of epithelial cells, interstitial cells, and Dogiel type II neurons in each plexus; however, in preparations stimulated with pulses of N2 or glucose, hexamethonium-resistant labeling of neurons occurred only in the submucosal plexus and not in myenteric ganglia. These observations suggest that primary afferent neurons responsible for mucosal pressure- or glucose-induced enteric and enteropancreatic reflexes are submucosal, whereas myenteric afferent neurons become activated only when the wall of the bowel is distended. The data are compatible with the possibility that primary afferent neurons are activated by a signaling molecule released from intestinal epithelial cells.

NADPH diaphorase (nitric oxide synthase)-containing nerves in the enteropancreatic innervation: sources, co-stored neuropeptides, and pancreatic function.

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. NADPH-diaphorase is present in a subset of enteric neurons. We investigated the presence of NADPH-diaphorase in the enteropancreatic innervation, the contribution of extrinsic nerves to the NADPH-diaphorase-containing fibers of the gut and pancreas, and the coincident expression of NADPH-diaphorase NADPH-diaphorase in intrinsic neurons of these organs with neuropeptides. The possible role of nitric oxide in the neural regulation of pancreatic secretion was studied in isolated pancreatic lobules. Neuronal perikarya with NADPH-diaphorase activity were found in both Dogiel type I and type II neurons of the myenteric plexus of the stomach and duodenum. All galanin (GAL)-immunoreactive neurons and a small subset of vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-immunoreactive neurons contained NADPH-diaphorase activity. NADPH-diaphorase activity was also found in a subset of VIP and NPY-immunoreactive pancreatic neurons. Retrograde tracing with FluoroGold established that NADPH-diaphorase-containing neurons in the bowel project to the pancreas. NADPH-diaphorase-containing fibers were also found to be provided to both organs by neurons in dorsal root ganglia. Secretion of amylase was evoked by L-arginine. This effect was prevented by N(G)-nitro-L-arginine (L-NNA), which also inhibited VIP-stimulated secretion of amylase; however, L-NNA had no effect on amylase secretion stimulated by carbachol. These results provide support for the hypothesis that nitric oxide plays a role in the neural regulation of pancreatic secretion.

Detection of the 5-HT1A receptor and 5-HT1A receptor mRNA in the rat bowel and pancreas: comparison with 5-HT1P receptors.

We tested the hypothesis that the rat bowel and pancreas contain 5-HT1A receptors. 3H-8-hydroxy-2-(di-n-propylamino)tetralin (3H-8-OH-DPAT) was used as a radioligand. Binding of 3H-8-OH-DPAT to membranes derived from the myenteric plexus and the pancreas was investigated by rapid filtration. Alternatively, radioautography was employed to locate 3H-8-OH-DPAT binding sites in frozen sections of unfixed bowel or pancreas. An excess of 5-HT (10 microM) was used to define nonspecific binding. Saturable, high affinity binding of 3H-8-OH-DPAT to enteric (Kd = 2.8 +/- 1.1 nM; Bmax = 83.8 +/- 4.3 fmol/mg protein) and pancreatic (Kd = 6.6 +/- 1.3 nM; Bmax = 44 +/- 2.2 fmol/mg protein) membranes was found. The binding of 3H-8-OH-DPAT to enteric and pancreatic membranes was inhibited by 8-OH-DPAT, NAN-190, and spiperone. In contrast, the binding of 3H-8-OH-DPAT to enteric and pancreatic membranes was not inhibited by 5-carboxyamidotryptamine, or by a variety of compounds known to bind to other subtypes of 5-HT receptor. Digoxigenin-labeled oligonucleotides were found to detect mRNA encoding the 5-HT1A receptor in a subset of neurons in myenteric and submucosal ganglia. In contrast, 5-HT1A mRNA was not found in the pancreas. Radioautography revealed that the highest density of 3H-8-OH-DPAT binding sites was found in the stomach. These sites were especially numerous in the lamina propria adjacent to gastric glands, and in myenteric ganglia. Pancreatic 5-HT1A receptors were located on nerves, lymphoid tissue (especially the capsule of nodes), and on cells scattered in the pancreatic parenchyma. The concentration of 3H-8-OH-DPAT binding sites in the rat bowel and pancreas was less than that of 3H-5-HT binding sites; however, the distribution of 3H-8-OH-DPAT binding sites was similar to that of sites that bind 3H-5-HT. It is concluded that the rat gut and its extension in the pancreas contains 5-HT1A receptors. Many, if not all, of the nerve cells and processes that express 5-HT1A receptors express 5-HT1P receptors as well. The function of these receptors in the physiology of the entero-pancreatic innervation remains to be determined.

Identification of cells that express 5-hydroxytryptamine1A receptors in the nervous systems of the bowel and pancreas.

Although serotonin (5-HT)1A receptors are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Earlier investigations have suggested that 5-HT1A receptors are synthesized in enteric, but not pancreatic ganglia, and that they mediate pre-and postjunctional inhibition. Wholemount in situ hybridization was used to identify cells that contain mRNA encoding 5-HT1A receptors, and immunocytochemistry was employed to locate receptor protein. mRNA encoding 5-HT1A receptors was found in the majority of neurons in both submucosal and myenteric plexuses. 5-HT1A immunoreactivity, however, was abundant only on the surfaces of a limited subset of nerve cell bodies and processes. 5-HT-immunoreactive axons were found in close proximity to sites of 5-HT1A immunoreactivity. Myenteric, but not submucosal calbindin-immunoreactive neurons (with Dogiel type II morphology) were surrounded by rings of 5-HT1A immunoreactivity. The cytoplasm of the cell bodies and dendrites of a small subset of Dogiel type I neurons was also intensely 5-HT1A immunoreactive. Most of the Dogiel type I 5-HT1A-immunoreactive myenteric neurons, and some of the type II neurons that were ringed by 5-HT1A immunoreactivity became doubly labeled following injections of the retrograde tracer, FluoroGold (FG), into the submucosal plexus. 5-HT1A-immunoreactive neurons in distant submucosal ganglia also became labeled by retrograde transport of FG. None of the 5-HT1A-immunoreactive cells were labeled by the intraluminal administration of the beta-subunit of cholera toxin, a marker for vasoactive intestinal peptide-containing secretomotor neurons. These observations suggest that some of the myenteric 5-HT1A-immunoreactive neurons project to submucosal ganglia and that the submucosal 5-HT1A-immunoreactive cells are interneurons. In addition to neurons, a subset of 5-HT-containing enterochromaffin cells expressed 5-HT1A immunoreactivity, which was co-localized with 5-HT in secretory granules. In the pancreas, 5-HT1A immunoreactivity was observed in ganglia, acinar nerves, and glucagonimmunoreactive islet cells. Serotonergic enteropancreatic axons have been found to terminate in close proximity to each of these structures, which may thus be the targets of this innervation. The abundance of 5-HT1A receptor immunoreactivity on nerves of the gut and pancreas suggests that drugs designed to interact with these receptors may have unanticipated visceral actions.

Chemokine expression and viral infection of the central nervous system: regulation of host defense and neuropathology.

An effective host response against viral infection of the central nervous system (CNS) is the principal factor dictating the outcome of infection. It is the responsibility of the immune response to contain and control viral replication. Paradoxically, it is the immune response that may also contribute to the development of neuropathology. We have used mouse hepatitis virus (MHV), apositive-strand RNA virus, infection of the CNS to understand the dynamic interaction between viral replication, protection, and pathology with an emphasis on understanding how chemokines participate in these interrelated processes. Herein, we demonstrate the complexity of the chemokine response to MHV infection of the CNS and the delicate balance that exists between host defense and development of disease.

Collagen induces cytokine production by synovial fluid mononuclear cells in rheumatoid arthritis.

Synovial fluid (SF) mononuclear cells (MNC) from 13 patients with rheumatoid arthritis (RA) and 12 patients with other arthritic diseases (OD) including osteoarthritis (OA), gout and spondyloarthritis (SA) were cultured in the presence of collagen types I and II or lipopolysaccharide (LPS) for 24 h. Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) in the SF and culture supernatants were assayed using ELISA. The results showed that one-half of the RA patients with high SF monocyte count had high SF IL-6 levels that coincided with the high spontaneous release of IL-6 by SF MNC. In the other RA patients with lower SF monocyte count, type II collagen induced significantly higher IL-1 beta than the medium control levels by SF MNC (P < 0.01) or that of the other diseases (P < 0.01). Similarly, type II collagen-induced IL-6 and TNF-alpha production rose significantly (P < 0.01) from SF MNC of RA but less from OD (P < 0.05). In addition, type I collagen could also induce IL-1, IL-6 and TNF-alpha in these samples from RA and OD patients but was less potent than type II collagen. Our results indicate that collagen-induced cytokines may be important in the pathogenesis of the disease.