Inhibitory effect of low-dose pentazocine on the development of antinociceptive tolerance to morphine

Purpose  The development of antinociceptive tolerance to morphine is one of the major problems in its clinical use. Therefore, exploring effective measures to prevent morphine tolerance is of great clinical relevance. We evaluated whether pentazocine could prevent morphine tolerance in mice. Methods  Five groups of male ICR mice received repeated subcutaneous (s.c.) injections of morphine at a high dose (10 mg·kg⁻¹) or saline, concomitantly with s.c. injections of pentazocine at low, subanalgesic doses (0.1, 0.3, or 1.0 mg·kg⁻¹) or saline, respectively, once daily for 14 days. On day 15, mice received co-injections of morphine and pentazocine 120 min after pretreatment with nor-binaltorphimine (5 mg·kg⁻¹), a selective κ-opioid receptor antagonist. The tail pressure threshold was measured before and 60 min after the daily drug co-injections. Results  Repeated s.c. co-injections of morphine and saline resulted in a progressive decrease in morphine-induced antinociception, due to the development of morphine tolerance. Co-injections of pentazocine (0.1, 0.3, and 1.0 mg·kg⁻¹) with morphine potentiated the morphine-induced antinociception dose-dependently by preventing the development of morphine tolerance. Nor-binaltorphimine completely inhibited the chronic antinociception maintained by co-injections of morphine and pentazocine. Conclusion  When chronically co-administered with morphine, pentazocine at low, subanalgesic doses dose-dependently potentiated morphine-induced antinociception in morphine-tolerant mice, through its κ-opioid-receptor-mediated tolerance-preventing activity. Because pentazocine is the only agonist-antagonist analgesic that has an effective oral formulation suitable for chronic administration, the results of the present study warrant clinical trials of pentazocine to assess its tolerance-preventing activity in patients with cancer pain.     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.     

Removal of blood group A/B antigen in organs by ex vivo and in vivo administration of endo-beta-galactosidase (ABase) for ABO-incompatible transplantation

BACKGROUND: ABO incompatibility in organ transplantation is still a high risk factor for antibody-mediated rejection, despite the progress in effective treatments. We have explored the possibility of using the enzyme to remove the blood type A/B antigen in organs. METHODS: Recombinant endo-beta-galactosidase (ABase), which releases A/B antigen, was produced in E. coli BL-21. Human A/B red blood cells (RBC) were digested with ABase, and subjected to flow cytometric analysis after incubation with human sera. Purified recombinant ABase was intravenously administered to a baboon. Biopsies were taken from kidney and liver before and 1, 4 and 24 h after in vivo administration. Excised baboon kidneys were perfused with cold UW solution+/-purified recombinant ABase and preserved at 4 degrees C. Biopsies were taken before and 1 and 4 h after ex vivo perfusion. The change in A/B antigen expression was analyzed by immunohistochemical study. RESULTS: ABase removed 82% of A antigen and 95% of B antigen in human A/B red blood cells, and suppressed anti-A/B antibody binding and complement activation effectively. ABase was also found to remain active at 4 degrees C. In vivo infusion of ABase into a blood type A baboon demonstrated a marked reduction of A antigen expression in the glomeruli of kidney (85% at 1 h, 9% at 4 h and 13% at 24 h) and the sinusoids of liver (47% at 1 h, 1% at 4 h and 3% at 24 h) without serious adverse effects. After ex vivo perfusion and cold storage of excised baboon kidney (blood type B) with ABase, the expression levels of B antigen in glomeruli were reduced to 49% at 1 h and 6% at 4 h. CONCLUSIONS: This alternative approach might be useful for minimizing antibody removal and anti-B cell immunosuppression as an adjuvant therapy in ABO-incompatible kidney, liver and possibly heart transplantation.     

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but γ-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells.     

Molecular signaling mediated by angiotensin II type 1A receptor blockade leading to attenuation of renal dysfunction-associated heart failure

BackgroundIn patients with end-stage renal disease, angiotensin II type 1A receptor (AT1) blockade attenuates the associated cardiac dysfunction. We investigated the molecular signaling mediating that effect.Methods and ResultsWe used 5/6 nephrectomy to induce significant renal dysfunction in AT1 knockout (AT1KO) and wild-type mice (WT). Twelve weeks after nephrectomy, WT showed significant left ventricular dilation and dysfunction that were accompanied by cardiomyocyte hypertrophy, fibrosis, and reduced capillary density. All of these effects were significantly mitigated in AT1KO. Nephrectomy led to upregulation of myocardial expression of AT1, transforming growth factor-β1 (TGF-β1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and phosphorylated Akt (p-Akt), and also led to increased oxidative damage in cardiomyocytes. In AT1KO, TGF-β1, TIMP-1, oxidative damage levels were lower, whereas MMPs and p-Akt levels were higher. Treating nephrectomized WT mice with valsartan (an AT1 blocker), but not hydralazine, improved cardiac function and altered molecular signaling in a manner similar to that seen in AT1KO mice. Notably, AT1 expression was downregulated in valsartan-treated but not hydralazine-treated hearts.ConclusionsThese findings provide novel insight into the mechanism underlying the beneficial effects of AT1 blockade on cardiac function in a model of renal dysfunction–associated heart failure.     

Macrophage-derived chemokine in malignant and tuberculous pleural effusions

BACKGROUND AND OBJECTIVES: Macrophage-derived chemokine (MDC/CCL22) is recognized as a T-helper (Th) 2-type chemokine. Both malignant and tuberculous pleural effusions are typically lymphocytic pleural effusions. Tuberculous pleural effusions have a more polarized Th1 reaction than malignant effusions, which are predominantly Th2 in nature. The aim of this study was to compare the levels of MDC in malignant pleural effusions with those in tuberculous pleural effusions to help delineate the role of MDC in Th2 versus Th1 effusions. METHODS: Forty-three patients with pleural effusions (32 malignant, 11 tuberculous) were studied. The concentration of MDC in the pleural effusion was measured by ELISA. RESULTS: The median concentration of MDC was lower in malignant pleural effusions than in tuberculous pleural effusions (P < 0.005). CONCLUSIONS: MDC has been reported to both promote and suppress antitumour immunity. The low concentration of MDC in malignant effusions is likely to minimise its antitumour activity but the precise role of MDC in malignant and tuberculous effusions needs to be investigated further.     

Establishment of external quality control program for hs-CRP and three-year follow-up of the performance for precision and accuracy

AIM: We established an external quality control (QC) program for high-sensitivity C-reactive protein (hs-CRP) for a collaborative epidemiological study.METHODS: External QC was performed 3 times in 3 years to follow hs-CRP performance for precision and accuracy.RESULTS: For precision, the mean coefficient of variation (CV) of the internal QC by 9 laboratories was 2.2% and 1.9% in the 1st and 2nd tests, respectively. The mean CV of the external QC by 4 laboratories was 2.7% in the three tests. The CV of both the internal and external QC satisfied the acceptable range specified by the AHA/CDC Scientific Statement, CV < 10%. For accuracy, the mean values of the 1st external QC by 9 laboratories were set as the consensus value and the acceptable range was set to within +/- 10% from it. The mean accuracy by 9 laboratories was 0.51% in the 2nd external QC. The mean accuracy by 4 laboratories was - 0.37% in the 3rd external QC. These findings demonstrated that the initial consensus value was valid in the continued external QC, and hs-CRP was stable for 3 years.CONCLUSION: We demonstrated both the precision and accuracy of hs-CRP by an external QC program applied for 3 years in a collaborative epidemiological study.     

Possible role of human cytomegalovirus in pouchitis after proctocolectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis

AIMTo detect the presence of human cytomegalovirus(HCMV)proteins and genes on the ileal pouch of patients with ulcerative colitis who have undergone proctocolectomy with ileal pouch-anal anastomosis(IPAA).METHODSImmunohistochemistry,polymerase chain reaction(PCR)and PCR sequencing methods were utilized to test the presence of HCMV in pouch specimens taken from 34 patients in 86 endoscopies.RESULTSHCMV genes and proteins were detected in samples from 12(35.2%)patients.The rate of detection was significant in the endoscopies from patients diagnosed with pouchitis(5 of 12,41.6%),according to the Japanese classification of pouchitis,in comparison to patients with normal pouch(7 of 62,11.2%;P = 0.021).In all patients with pouchitis in which the HCMV was detected,it was the first episode of pouchitis.The virus was not detected in previous biopsies taken in normal endoscopies of these patients.During the followup,HCMV was detected in one patient with recurrent pouchitis and in 3 patients whose pouchitis episodes improved but whose positive endoscopic findings persisted.CONCLUSIONHCMV can take part in the inflammatory process of the pouch in some patients with ulcerative colitis who have undergone proctocolectomy with IPAA.     

PGD(2) DP1 receptor protects brain from ischemia-reperfusion injury

Prostaglandin D(2) is the most abundant prostaglandin in the brain. It has long been described as a modulator of the neuroinflammatory process, but little is known regarding the role of its Galpha(s)-coupled receptor, DP1. Therefore, in this study, the effect of the DP1 receptor on the outcome of cerebral ischemia in wildtype (WT) and DP1 knockout (DP1(-/-)) C57Bl/6 mice was investigated. Ischemia-reperfusion injury was produced by a 90-min occlusion of the right middle cerebral artery followed by a 4-day reperfusion. Infarct size was 49.0 +/- 11.0% larger in DP1(-/-) mice (n = 11; P < 0.01) than in WT mice (n = 9 per group). However, no differences were detected in the relative cerebral blood flow (CBF) or any of the physiological parameters measured (n = 5 per group) or in the large blood vessel anatomy (n = 3 per group). To further address whether the DP1 protective role in the brain could be extended to neurons, mouse primary corticostriatal neuronal cultures were exposed to the DP1-selective agonist, BW245C, which provided dose-dependent protection against excitotoxicity induced by glutamate. Protection was significant at a dose as low as 0.05 microm. The results indicate that the DP1 receptor is neuroprotective in both in vivo and in vitro paradigms. Development of drugs to stimulate the DP1 receptor in brain could provide a new therapeutic strategy against cerebral ischemia and potentially other neurological conditions.     

Botanical origin of Indian celery seed (fruit)

In the course of our study on the traditional medicines and foodstuffs used in Pakistan, we investigated the origin of Indian celery by using the analysis of the internal transcribed spacer (ITS) sequence of nuclear rDNA and a phytochemical approach. We found that the source plant of the Indian celery containing coumarin derivatives such as seselin (1), bergapten (2) and isopimpinellin (3) was not common celery, Apium graveolens. Our results suggest the source plant is Seseli diffusum even though Indian workers reported that A. graveolens seeds contain the aforementioned compounds. In addition, a market survey of the Indian celery in Pakistan and related countries revealed that the Indian celery seeds in Pakistani markets are mainly composed of three species which have been confused in rural markets. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.