Knee osteoarthritis pain following medial meniscectomy in the nonhuman primate.

OBJECTIVE: A number of promising compounds developed for osteoarthritic pain have failed to demonstrate clinical efficacy. To enhance preclinical translational research for osteoarthritis, a model of knee osteoarthritis pain was developed in Macaca fascicularis and the effects of two distinct pharmacological classes of drugs were tested on pain-related behavior. DESIGN: Behavioral assessments were developed specifically for the macaque. Baseline knee pressure threshold and weight bearing were assessed prior to a unilateral medial meniscectomy (MMx). Fifteen days following MMx, macaques underwent a once daily exercise regimen for 36 days. Sixty-seven days following MMx, macaques were assigned to one of three treatment groups (n = 3/group), either non-steroidal anti-inflammatory drug (NSAID) diclofenac, NK1 receptor antagonist aprepitant or vehicle, and treated for 5 days. Animals were tested 3-4 h after p.o. dosing and testing was performed blinded. Treatment utilized a crossover design-each animal received all treatments-and a 9-day washout period was utilized between treatments. RESULTS: Vehicle-treated macaques consistently demonstrated decreased ipsilateral pressure threshold ("hyperalgesia") and decreased weight bearing. While diclofenac increased weight bearing and pressure threshold, full attenuation of pain was not obtained. No significant improvement of either knee pressure or weight bearing was observed with aprepitant. CONCLUSIONS: Unilateral MMx in the macaque evoked pain-related behaviors and knee joint pathology reminiscent of osteoarthritis. The behavioral endpoints were sensitive to NSAID treatment but not sensitive to NK1 receptor block, which parallel clinical findings. The current macaque osteoarthritis model could be used to test potential treatments for osteoarthritis pain.

Monitoring of cell layer coverage and differentiation with the organic electrochemical transistor.

Electrical, label-free monitoring of cells is a non-invasive method for dynamically assessing the integrity of cells for diagnostic purposes. The organic electrochemical transistor (OECT) is a device that has been demonstrated to be advantageous for interfacing with biological systems and had previously been shown to be capable of monitoring electrically tight, resistant, barrier type tissue. Herein, the OECT is demonstrated not only for monitoring of barrier tissue cells such as MDCK I, but also for other, non-barrier tissue adherent cells including HeLa cells and HEK epithelial cells. Transistor performance, expressed as transconductance (gm) is measured as a function of frequency; barrier tissue type cells are shown to have a more abrupt drop in transconductance compared to non-barrier tissue cells, however both tissue types are clearly distinguishable. Simple modelling of the cell layers on the transistor allows extraction of a resistance term (Rc). OECT monitoring shows that barrier tissue cells lose their barrier function in a standard calcium switch assay, but remain adhered to the surface. Re-addition of calcium results in recovery of barrier tissue function. The entire process is continuously followed both electronically and optically. Finally, high resolution fluorescence imaging of live cells labelled with a red fluorescent actin marker demonstrates the versatility of this method for tracking molecular events optically, with direct correlation to electronic readouts.

Comparison of matched-sibling donor BMT and unrelated donor BMT in children and adolescent with acquired severe aplastic anemia.

From January 1991 to March 2007, 61 children and adolescent with acquired severe aplastic anemia received BMT in our institutions. We retrospectively compared the outcome of 30 cases of matched-sibling donor BMT (MSD-BMT) and 31 cases of unrelated donor BMT (URD-BMT). We observed one graft failure among MSD-BMT recipients and three graft failures among URD-BMT recipients, respectively. No patients in the MSD-BMT group developed grades II-IV acute GVHD compared with 11 of 30 patients (37%) in the URD-BMT group (P<0.001). One of 30 MSD-BMT recipients (3%) developed chronic GVHD compared with 8 of 30 URD-BMT recipients (27%) (P=0.013). The incidence of EBV and CMV reactivation was 11 of 20 URD-BMT recipients and 23 of 30, respectively. One patient in the URD-BMT group died of a motor accident 5.5 years after BMT. Ten-year OS was 100% in MSD-BMT recipients and 93.8% (95% CI, 81.9-100%) in URD-BMT recipients, respectively (P=0.252). Ten-year failure-free survival was 96.7% (95% CI, 90.2-100%) in the MSD-BMT group and 84.7% (95% CI, 70.2-99.2%) in the URD-BMT group, respectively (P=0.161).

Late-onset non-infectious pulmonary complications following allogeneic hematopoietic stem cell transplantation in children.

Late-onset non-infectious pulmonary complications (LONIPCs) that arise beyond 3 months after allogeneic hematopoietic SCT include bronchiolitis obliterans (BO), bronchiolitis obliterans with organizing pneumonia (BOOP) and idiopathic pneumonia syndrome (IPS). We retrospectively analyzed the incidence and outcome of LONIPCs among pediatric hematopoietic SCT recipients. We included 97 patients who survived for more than 3 months among the 114 who underwent allogeneic hematopoietic SCT between April 1997 and May 2007. Of the 97 enrolled patients, 10 (10.3%) developed LONIPCs at a median of 187 days after hematopoietic SCT (range, 123-826 days). Of the 10 patients with LONIPCs, eight had BO and two had IPS. Multivariate analysis showed that the onset of LONIPCs was associated with high-risk underlying disease and extensive chronic GVHD (hazard ratio, 5.42 (95% confidence interval, 1.36-21.7) and hazard ratio, 11.7 (95% confidence interval, 2.40-57.1), respectively). Only two patients responded to therapy with steroids and six of the 10 patients died. The 5-year OS rate was significantly lower among patients with, than without LONIPCs (28.0 vs 87.2%, P=0.000). Considering that we are lacking optimal therapies for LONIPCs, strategies aimed at the prevention of LONIPCs should be attempted.

Hypercalcemia in childhood acute lymphoblastic leukemia: frequent implication of parathyroid hormone-related peptide and E2A-HLF from translocation 17;19.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (>12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (<20 x 10(9)/l), 15 showed hemoglobin> or =8 g/dl and 14 showed platelet count > or =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes.

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.

Role of metabotropic glutamate receptor subtype 5 (mGluR5) in the maintenance of cold hypersensitivity following a peripheral mononeuropathy in the rat.

The present series of experiments were designed to examine the contribution of metabotropic glutamate receptor subtype 5 (mGluR5) to neuropathic pain by determining the effects of the selective mGluR5 antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine) on neuropathy-induced cold hypersensitivity. Unilateral chronic constriction injury (CCI) to the sciatic nerve in rats produced an increase in the number of hind paw withdrawals from a cold surface (4 +/- 2 degrees C) which was dose-dependently inhibited by systemic (i.p.) injection of MPEP (ID(50) = 11.3 mg/kg). In vivo brain mGluR5 receptor occupancy following systemic (i.p.) MPEP revealed that >90% occupancy is required for behavioral efficacy. Intracerebroventricular (i.c.v.) injection of MPEP dose-dependently inhibited CCI-induced cold hypersensitivity (ID(50) = 123.5 nmol), while microinjection of MPEP directly into the rostral ventromedial medulla (RVM) potently inhibited this hypersensitivity (ID(50) = 1.3 pmol). A role for mGluR5 in the RVM was further supported by the observation that intra-RVM injection of the mGluR5 agonist CHPG (10 nmol; 2-chloro-5-hydroxyphenylglycine) produced cold hypersensitivity in naïve rats that was blocked by pretreatment with intra-RVM MPEP (3 nmol). Intrathecal (500 nmol; i.t.) or intraplantar (300 nmol; i.pl.) injection of MPEP was ineffective in reversing CCI-induced cold hypersensitivity. These results demonstrate that mGluR5 contributes to cold hypersensitivity following peripheral neuropathy exclusively at supraspinal sites in the CNS. Additionally, mGluR5 in the RVM significantly contributes to the maintenance of cold hypersensitivity, likely via activation of descending nociceptive facilitatory systems.

[A case of unilateral moyamoya disease presenting with hemichorea].

We reported a 12-year-old boy with unilateral moyamoya disease whose initial and predominant manifestation was hemichorea. Neurological examinations revealed chorea in his left upper extremity and muscle hypotonia in his left upper and lower extremities. Cranial MRI showed moyamoya vessels only in the right basal ganglia and infarction in the white matter of the right frontal lobe. Right carotid angiography revealed stenosis in the distal part of internal carotid artery, and in the proximal part of anterior and middle cerebral arteries with moyamoya vessels. Left carotid angiography showed normal findings. He was diagnosed as a suspected case of moyamoya disease (unilateral moyamoya disease) according to the diagnostic criteria proposed by the Research Committee on Moyamoya Disease of the Ministry of Health and Welfare of Japan. His chorea responded to haloperidol but encephalo-duro-arterio-synangiosis on the right side improved all symptoms. Chorea occurs in some patients with moyamoya disease. Hypofunction of the striatal indirect pathway is suggested as the cause of chorea. In this case an ischemic lesion in the right striatum may have caused hypofunction of the pathway and developed chorea and hypotonia.

The antinociceptive effect of intrathecal administration of epibatidine with clonidine or neostigmine in the formalin test in rats.

The analgesic effect of intrathecal injection of epibatidine, clonidine and neostigmine, compounds that elevate ACh, was examined in the formalin test, a model of post-injury central sensitization in the rat. The compounds were injected alone and in combination. Intrathecal injection of epibatidine alone did not alter pain behaviors, compared to vehicle-treated rats. Intrathecal injection of clonidine dose-dependently reduced tonic pain behaviors (ED(50)+/-95% confidence limits=6.7+/-4.8 microg). The combination of clonidine and epibatidine (C:E), in the ratio of 26:1, dose-dependently reduced tonic pain behaviors; and the ED(50) of C:E was 1.1+/-0.98 microg a significant 6-fold leftward shift of the dose response curve, compared with clonidine alone. The antinociceptive effect of C:E (26:1) was attenuated by pre-treatment with the nAChR antagonist mecamylamine. Neostigmine dose-dependently reduced tonic pain behaviors (ED(50)=1.5+/-1.3 microg). The combination of neostigmine and epibatidine, in a ratio of 8:1, significantly shifted the dose response curve 4-fold to the left (ED(50)=0.4+/-0.3 microg). The effect is mediated in part by the activation of the nAChR and possibly by the enhanced release of ACh. These data demonstrate significant enhancement of the antinociceptive effects of spinally delivered analgesics by a nAChR agonist, suggesting that this class of compounds may have utility as adjuvants when combined with conventional therapeutics.

Antagonist of nicotinic acetylcholine receptors (nAChR) enhances formalin-induced nociception in rats: tonic role of nAChRs in the control of pain following injury.

Following tissue injury, spinal neurons increase in spontaneous activity and in responsiveness to peripheral stimulation. These changes in spinal neurons may underlie abnormal pain behavior. Nicotinic acetylcholine receptor (nAChR) agonists are analgesic when evaluated in animal models of pain, but it is not known if the nAChRs differentially modulate acute and tonic pain. To test this, mecamylamine, a non-subtype selective nAChR antagonist, was systemically injected into rats prior or after hind paw injection of formalin. Formalin injection results in biphasic pain-related behaviors, characterized by a first phase (i.e. acute pain) immediately following formalin injection, then by a second phase (i.e. tonic pain) 15-60 min after formalin injection. Either pre- or post-formalin treatment with mecamylamine decreased phase 1 behaviors and significantly increased phase 2 pain behaviors in a dose-dependent manner. These results suggest that nAChRs may exert opposing effects on acute versus tonic pain and, as such, may have implications for the potential development of nAChR ligands for the treatment of pain.

NMDA-induced spinal hypersensitivity is reduced by naturally derived peptide analog [Ser1]histogranin.

N-methyl-D-aspartate (NMDA) receptor activation is thought to initiate a cellular cascade of events in the spinal cord that leads to neuronal hyperactivation and exaggerated persistent pain behaviors. Previous studies have demonstrated that implantation of adrenal medullary tissue into the spinal subarachnoid space reduces abnormal pain behaviors such as hyperalgesia and allodynia, possibly by intervening in the NMDA hyperexcitability cascade. Histogranin is a 15-amino acid peptide possessing NMDA receptor antagonist activity that has been isolated from adrenal medullary tissue. The present study examined the ability of stable analog [Ser1]histogranin to reduce abnormal pain-related behaviors induced in rats by direct activation of spinal NMDA receptors. The intrathecal injection of NMDA (5.0, 10.0, 20.0 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in a dose-related fashion. [Ser1]histogranin injected intrathecally prior to NMDA injections dose dependently attenuated or completely blocked hyperalgesia and allodynia. In addition, [Ser1]histogranin administration following NMDA-induction of abnormal pain behaviors reversed these effects. These results demonstrate that a naturally derived adrenal medullary neuropeptide can prevent and reverse NMDA-mediated spinal hyperexcitability. The distinct profile and robust activity of [Ser1]histogranin suggest novel alternative approaches in the management of pain and other CNS disorders involving abnormal excitatory neurotransmission.

Differential distribution of (GABA)A receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat.

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial gamma-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA(A) receptor alpha1 and alpha3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the alpha1 subunit, or to serotonin and the alpha3 subunit of the GABA(A) receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the alpha1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the alpha3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No alpha1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the alpha3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the alpha3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA(A) receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA(A) receptor antagonists in the VMM.

Suppression of neuropathic pain by a naturally-derived peptide with NMDA antagonist activity.

Chronic pain may result from hyperexcitability following activation of spinal NMDA receptors. A naturally-derived mammalian peptide, histogranin, may possess NMDA antagonist activity. This study explored the possibility that stable analog [Ser1]Histogranin (SHG) could reduce chronic pain. Neuropathic pain was induced using the chronic constriction injury model (CCI). Intrathecal injection of SHG markedly attenuated the hyperalgesia and allodynia resulting from CCI, nearly normalizing responses. These results suggest that the natural peptide histogranin may be a novel adjunct in neuropathic pain management.

Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts.

Abnormal pain-related behaviour that accompanies peripheral nerve injury may be the result of altered spinal neuronal function. The long-term loss of inhibitory function by GABA neurons in particular may be a mechanism by which abnormal neural hyperactivity occurs, leading to exaggerated sensory processing following nerve injury. In order to assess this, changes in spinal GABA immunoreactivity at several time points following constriction nerve injury were quantified in parallel with behavioural assessments of abnormal sensory responses to noxious and innocuous stimuli. In addition, the effects of spinal adrenal medullary transplants were determined since previous findings have demonstrated alleviation of behavioural pain symptoms by such transplants. In response to unilateral sciatic nerve injury, GABAergic profiles normally found in lumbar dorsal horn laminae I-III significantly decreased. The decrease was apparent three days following ligation, particularly on the side ipsilateral to the nerve injury. By two weeks, no GABAergic profiles could be seen, with the deficit appearing in the spinal dorsal horn both ipsilateral and contralateral to the unilateral peripheral nerve injury. Marked decreases in GABA-immunoreactive profiles persisted for at least up to five weeks post-injury, with partial restoration occurring by seven weeks. However, even at seven weeks, losses in GABA-immunoreactive profiles persisted in the dorsal horn ipsilateral to peripheral nerve injury. These findings were comparable in animals receiving control striated muscle transplants. In contrast, adrenal medullary transplants markedly reduced the loss in GABA-immunoreactive profiles at all time-points examined. In addition, GABA-immunoreactive profile levels were normalized near that of intact animals by five to seven weeks following nerve injury in animals with adrenal medullary transplants. Parallel improvements in sensory responses to innocuous and noxious stimuli were also observed in these animals. The results of this study indicate that peripheral nerve injury can result in severe losses in spinal inhibitory mechanisms, possibly leading to exaggerated sensory processes in persistent pain states. In addition, adrenal medullary transplants may provide a neuroprotective function in promoting recovery and improving long-term survival of GABAergic neurons in the spinal dorsal horn which have been damaged by excitotoxic injury.

Alterations in rat spinal cord cGMP by peripheral nerve injury and adrenal medullary transplantation.

Adrenal medullary chromaffin cells implanted into the spinal subarachnoid space can reduce abnormal pain-related responses in chronic pain models. Persistent pain is thought to involve the activation of N-methyl-D-aspartate (NMDA) receptors and subsequent production of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP). Changes in dorsal horn levels of cGMP in the rat were determined in conjunction with alterations in pain behaviors following peripheral nerve injury and adrenal medullary transplantation. Results indicated increased spinal cGMP levels in parallel with thermal and mechanical hyperalgesia and tactile allodynia consequent to chronic constriction injury of the sciatic nerve in rats. Adrenal medullary, but not control transplants, attenuated the hyperalgesia and allodynia and decreased spinal cGMP content. These results suggest that adrenal medullary transplants may reduce abnormal pain by intervention in the spinal NMDA-NO cascade.

Expression of class II MHC molecules in the rat pineal gland during development and effects of treatment with carbon tetrachloride.

Cells expressing major histocompatibility complex (MHC) class II (Ia) antigen have been examined during the development of rat pineals and in the pineal gland of adult rats treated with carbon tetrachloride. Cells positive for MHC class II are first detected in the pineal gland of the 7-day-old rat. These positive cells increase in number gradually during development, MHC class II immunoreactivity reaching adult levels at 4 weeks after birth. The MHC class II antigen is intensely labeled on the cell surface, and labeled cells are distributed throughout the organ, several positive cells being gathered into groups. The positive cells are small (7-12 microm in diameter), irregular in shape, and frequently exhibit one or more processes. At the electron-microscopic level, the cytoplasm of positive cells contains few organelles, variously sized empty vacuoles, and a few electron-dense lysosome-like structures. Pinealocytes with synaptic ribbons have been found adjacent to immunoreactive cells. Double-immunoperoxidase staining for MRC OX6, MRC OX42, and ED1 results in OX6(-)/ED1(+)/OX42(+), OX6(-)/ED1(-)/OX42(+), and OX6(+)/ED1(-)/OX42(- )cells. These findings suggest that OX6-positive cells in the pineal can be considered as peripheral dendritic cells. The number of cells expressing MHC class II (Ia) antigen significantly increases in the pineal gland of rats after treatment with carbon tetrachloride (P<0.005). Our results indicate that at least some of the OX6-positive cells migrate into the gland from the circulation under these conditions.