Spinal glial TLR4-mediated nociception and production of prostaglandin E2 and TNF

Background and purpose:

Toll-like receptor 4 (TLR4) expressed on spinal microglia and astrocytes has been suggested to play an important role in the regulation of pain signalling. The purpose of the present work was to examine the links between TLR4, glial activation and spinal release of prostaglandin E₂ (PGE₂) and tumour necrosis factor (TNF), and the role these factors play in TLR4-induced tactile allodynia.

Experimental approach:

Toll-like receptor 4 was activated by intrathecal (i.t.) injection of lipopolysaccharide (LPS) and KDO₂-Lipid A (KDO₂) to rats. Tactile allodynia was assessed using von Frey filaments and cerebrospinal fluid collected through spinal dialysis and lumbar puncture. PGE₂ and TNF levels were measured by mass spectometry and elisa. Minocycline and pentoxifylline (glia inhibitors), etanercept (TNF-blocker) and ketorolac (COX-inhibitor) were given i.t. prior to injection of the TLR4-agonists, in order to determine if these agents alter TLR4-mediated nociception and the spinal release of PGE₂ and TNF.

Key results:

Spinal administration of LPS and KDO₂ produced a dose-dependent tactile allodynia, which was attenuated by pentoxifylline, minocycline and etanercept but not ketorolac. Both TLR4 agonists induced the spinal release of PGE₂ and TNF. Intrathecal pentoxifylline blunted PGE₂ and TNF release, while i.t. minocycline only prevented the spinal release of TNF. The release of PGE₂ induced by LPS and KDO₂ was attenuated by i.t. administration of ketorolac.

Conclusions and implications:

Activation of TLR4 induces tactile allodynia, which is probably mediated by TNF released by activated spinal glia.     

mdx muscle pathology is independent of nNOS perturbation

In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS- dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.      

Inhibition of inositol monophosphatase (IMPase) at the calbindin-D28k binding site: Molecular and behavioral aspects

Bipolar-disorder (manic-depressive illness) is a severe chronic illness affecting ～1% of the adult population. It is treated with mood-stabilizers, the prototypic one being lithium-salts (lithium), but it has life threatening side-effects and a significant number of patients fail to respond. The lithium-inhibitable enzyme inositol-monophosphatase (IMPase) is one of the viable targets for lithium's mechanism of action. Calbindin-D28k (calbindin) up-regulates IMPase activity. The IMPase-calbindincomplex was modeled using the program MolFit. The in-silico model indicated that the 55–66 amino-acid segment of IMPase anchors calbindin via Lys59 and Lys61 with a glutamate in between (Lys–Glu–Lys motif) and that the motif interacts with residues Asp24 and Asp26 of calbindin. We found that differently from wildtype calbindin, IMPase was not activated by mutated calbindin in which Asp24 and Asp26 were replaced by alanine. Calbindin's effect was significantly reduced by a linear peptide with the sequence of amino acids 58–63 of IMPase (peptide 1) and by six amino-acid linear peptides including at least part of the Lys–Glu–Lys motif. The three amino-acid peptide Lys–Glu–Lys or five amino-acid linear peptides containing this motif were ineffective. Mice administered peptide 1 intracerebroventricularly exhibited a significant anti-depressant-like reduced immobility in the forced-swim test. Based on the sequence of peptide 1, and to potentially increase the peptide's stability, cyclic and linear pre-cyclic analog peptides were synthesized. One cyclic peptide and one linear pre-cyclic analog peptide inhibited calbindin-activated brain IMPase activity in-vitro. Our findings may lead to the development of molecules capable of inhibiting IMPase activity at an alternative site than that of lithium.     

Synthesis and anticonvulsant activity of 3-(6-substituted-benzothiazol-2-yl)-6-phenyl-[1, 3]-xazinane-2-thiones

A new series of 3-(6-substituted-benzothiazol-2-yl)-6-phenyl-[1, 3]-oxazinane-2-thiones (4a-j) has been synthesised using an appropriate synthetic route (Scheme 1) and characterised by elemental analyses and spectral (IR, (1)HNMR, (13)C NMR, and EI MS) data. The anticonvulsant activity of all the title compounds (4a-j) was evaluated against Maximal Electroshock (MES) induced seizures and furthermore the most potent compounds were evaluated against subcutaneous pentylenetetrazole (sc PTZ) induced seizures model in mice. The neurotoxicity was assessed using the rotorod procedure. All the test compounds were administered intraperitoneally at various dose levels ranging from 30-200 mg/kg body wt and the median effective dose (ED(50)), median toxic dose (TD(50)), and protection index (PI) values were determined (Table 2). Among the compounds tested, the 3-(6-dimethylaminobenzothiazol-2-yl)-6-phenyl-[1, 3]-oxazinane-2-thiones (4j) was found to be the most potent (ED(50): 9.85 and 14.8 in MES model and 12 and 17 in scPTZ model at t = 0.5 h and 4 h, respectively, and TD(50) 42.8 and 44 at t = 0.5 h and 4 h, respectively, which has been found to be significant at p < 0.01 with respect to reference standard phenytoin) with protection index (PI) 4.85.     

Reference values for pulse oximetry at high altitude

OBJECTIVE: To determine reference values for oxygen saturation (Sao2) in healthy children younger than 5 years living at high altitude. DESIGN: One hundred and sixty eight children were examined for Sao2 at 4018 m during well child visits. Physiological state was also noted during the examination. RESULTS: The mean Sao2 was 87.3% (95% confidence intervals (CI) 86.7%, 87.9%) with a median value of 87.7%. A significant difference was observed in Sao2 between children younger than 1 year compared with older children, although the difference was no longer demonstrable when sleeping children were excluded. CONCLUSIONS: This study has provided a reference range of Sao2 values for healthy children under 5 years old so that pulse oximetry may be used as an adjunct in diagnosing acute respiratory infections. Younger children were also shown to have a lower mean Sao2 than older children living at high altitude, which suggests physiological adaptation to high altitude over time. In addition, sleep had a lowering effect on Sao2, although the clinical importance of this remains undetermined.     

MAG3 renal scintigraphy: Improved ability to make anatomical diagnoses in neonates

Technetium-99m mercaptoacetyltriglycine(MAG3) is the most recently introduced renal radiopharmaceutical in Australia and is established as the agent of choice for use in diuresis renography, particularly in neonates and infants. It provides superior anatomical information compared to previously used agents. Three cases are reported in which MAG3 diuresis renography was performed in neonates, who were found to have hydronephrosis detected antenatally. In two neonates, a previously unrecognized horseshoe kidney was demonstrated and in case 3 there were scan features characteristic of a ureterocele. It is highly unlikely that these abnormalities would have been delineated with ^99mTc dimethyltriamine penta-acetic acid (DTPA) study, as confirmed in case 1, because of the relatively poor uptake of DTPA when compared to MAG3.     

Gastroesophageal reflux in children with recurrent abdominal pain

In this study we investigated the presence of gastroesophageal reflux in children with recurrent abdominal pain and its possible relationship to food intolerance-associated duodenal inflammation. Twenty-four-hour intra-esophageal pH monitoring, an endoscopic duodenal biopsy and a small bowel 51Cr-EDTA permeability test were performed in 25 children with recurrent abdominal pain. In 14 cases (56%) the pH monitoring was abnormal, pointing to the presence of pathological gastroesophageal reflux. Treatment of gastroesophageal reflux in the latter patients resulted in resolution or improvement of abdominal pain in 10 cases (71%). Gastroesophageal reflux did not appear to be associated with either intestinal permeability to 51Cr-EDTA or duodenal biopsy findings. We conclude that pathological gastroesophageal reflex is a frequent finding in children with recurrent abdominal pain, that it is unrelated to duodenal inflammation and that there might be a causal relationship between pathological gastroesophageal reflux and recurrent abdominal pain in children.     

Pineal tumours in the north of England 1968-93

The records of 38 patients under 25 years of age presenting with pineal tumours between 1968 and 1993, identified from the Northern Region Children's and Young Adults' Malignant Disease Registry, were analysed retrospectively with regards to clinical presentation, diagnostic approach, treatment strategy, and outcome. The overall five year survival was 45%. Fifteen patients had a histological diagnosis: six with germinomas, three with teratomas, three with astrocytomas, and three with pinealoblastomas. One patient had a definitive diagnosis of teratoma made on the basis of raised tumour markers (alpha fetoprotein). Treatment consisted of surgery (87%) (ventriculoperitoneal or atrial shunt and/or biopsy), and/or radiotherapy (82%), and/or chemotherapy (26%). Those patients with a tissue diagnosis appeared to have a more favourable outcome, especially after 1976 when treatment was determined by tumour type (five year survival for those with a tissue diagnosis was 91% v 51% for those without, 95% confidence intervals 74 to 100% and 26 to 75%). This study suggests that tissue diagnosis allows more appropriate treatment to be delivered for children with pineal tumours resulting in improved survival. Referral to a centre with neurosurgery, radiotherapy, neuropathology, and paediatric oncology collaboration is essential.     

Influence of HLA-A2 on the effectiveness of platelet transfusions in alloimmunized thrombocytopenic patients

Platelet transfusions from donors selectively mismatched for cross- reactive and certain non-cross-reactive HLA antigens were found to be more effective in HLA-A2 negative than in HLA-A2 positive, alloimmunized thrombocytopenic patients. The two groups of patients responded equally well to platelets matched for antigens of the HLA-A and B loci. Certain alloimmunized patients negative for HLA-A2 continued to respond satisfactorily to platelets selectively mismatched for non-cross-reactive HLA antigens as long as platelets containing HLA- A2 were avoided. The data indicate that platelet transfusion support can be provided within a broader range of donor-recipient HLA antigenic disparity to HLA-A2 negative alloimmunized patients than to those who are positive for this antigen.