Heavily T2-weighted imaging findings of spinal cord swelling in dogs with intervertebral disc extrusion.

ABSTRACT: This study investigated causes of attenuation of cerebrospinal fluid (CSF) signal on heavily T2-weighted (T2W) images in dogs with thoracolumbar disc extrusion. Medical records and magnetic resonance images were retrospectively reviewed. Dogs were classified into the following grades; grade 1, non-ambulatory paraparesis; grade 2, paraplegia with deep pain perception and grade 3, paraplegia without deep pain perception. The length of intramedullary T2W hyperintensity of the spinal cord, cranial/ caudal expansion of extradural compressive materials (ECM), and the CSF signal attenuation were measured. Ratios to the second lumbar vertebra (L2) were calculated for the length of intramedullary T2W hyperintensity (T2W:L2), cranial/caudal expansion of ECM (ECML:L2), and CSF signal attenuation (CSF:L2). The dogs were classified into focal or extended T2W hyperintensity groups according to the length [focal, shorter than length of L2; extended, longer than L2]. The area of EMC and the spinal canal were measured on transverse images at the lesion deriving occupancy ratio. The correlation between CSF:L2 and other data were analysed, and CSF:L2 was compared between the grades. In dogs with intramedullary T2W hyperintensity, the locations of CSF attenuation and the hyperintensity were compared if those locations were matched. Fifty-five dogs were included, 36 of which showed intramedullary T2W hyperintensity. Twenty-two of 36 dogs were considered as match of the location of the CSF attenuation and hyperintensity. CSF:L2 was significantly correlated with T2W:L2 in dogs with extended T2W hyperintensity (p = 0.0002), while CSF:L2 was significantly correlated with ECML:L2 in dogs with focal or no T2W hyperintensity (p = 0.0103 and p = 0.0364, respectively). CSF:L2 in grade 3 was significantly greater than those in patients who were grade 1 or 2 (both p < 0.001). In conclusion, higher CSF:L2, which was frequently seen in grade 3, would be most consistent with a higher T2W:L2 which might indicate spinal cord swelling.

A novel nonsecosteroidal VDR agonist (CH5036249) exhibits efficacy in a spontaneous benign prostatic hyperplasia beagle model.

To date, there have been no reports showing the efficacy of nonsecosteroidal vitamin D receptor (VDR) agonists in a benign prostatic hyperplasia (BPH) animal model. To examine the efficacy of CH5036249, a novel nonsecosteroidal VDR agonist, we orally administered the compound at 0.03 microg/kg to a beagle model with spontaneous BPH. Prostate volume was checked by rectal ultrasonic probe periodically during 11 months of administration and the prostate tissues histologically examined. CH5036249 inhibited prostate growth in two out of three dogs compared with vehicle-treated dogs. In the prostate specimens, substantial atrophy of the epithelium was observed in all dogs administered CH5036249. At the dose given, serum calcium levels slightly increased in the CH5036249-treated dogs but stayed within a normal range. We next examined the cell growth inhibition of CH5036249 using human prostate stromal cells and found the cell growth inhibitory activity of CH5036249 to be comparable to that of 1alpha,25(OH)2D3. The bioavailability from oral administration in rats was 95.1% with a t1/2 of 17.6 h. Both micro-AMES and micronucleus tests were negative. Although the results are still preliminary, we consider the novel nonsecosteroidal VDR agonist CH5036249 to be a possible new drug candidate for the treatment of BPH in humans.

In vitro model for the prediction of clinical CYP3A4 induction using HepaRG cells.

It is important to predict CYP3A enzyme induction in the drug-discovery process to avoid adverse effects in clinical. In the present study, we constructed a method to correct the variability of in vitro CYP3A induction assays and thereby a method for the prediction of CYP3A induction in the clinical setting. Induction assays were performed in vitro using HepaRG cells and seven typical inducers. An index value was determined for enzyme induction, termed the relative factor (RF), from the ratio of the concentration of the inducers to the reference standards. Using RF as an index, variation among the assays was reduced. A good relationship was obtained between the ratio of the free plasma concentration at steady-state (C(ss,u)) to RF (expressed as C(ss,u)/RF) and the in vivo induction response. Using rifampicin as a reference standard, compounds with a C(ss,u)/RF value greater than 7.31 nmol l(-1) may induce CYP3A in vivo in humans.

Messenger ribonucleic acid expression profile in peripheral blood cells from RA patients following treatment with an anti-TNF-alpha monoclonal antibody, infliximab.

OBJECTIVES: We monitored the mRNA expression profiles of peripheral blood cells during treatment with a TNF-alpha inhibitor, infliximab, in patients with RA. Using a DNA microarray analysis, we demonstrated a unique set of genes, with distinct baseline and post-treatment changes in expression between responders and non-responders to infliximab treatment. METHODS: Using a customized low-density cDNA microarray with 747 genes and a reliable data collection system, we monitored the mRNA expression profiles of whole blood cells from 18 RA patients before and after the infusion of infliximab for up to 22 weeks. The clinical response to treatment with infliximab was determined using the ACR response criteria, the disease activity score of 28 joints (DAS28), and individual clinical parameters. The patients were classified as responders or non-responders based on their ACR50% response at 22 weeks. RESULTS: Approximately 15% of the total genes were found to exhibit a >1.5-fold change, compared with their reference values, at one or more time points during the 22 weeks of infliximab therapy. The expression of inflammatory genes, such as IFN-related genes, was strongly correlated with the serum level of CRP and the DAS28. The increased expression of inflammatory genes in responders was normalized within 2 weeks and then remained at a normal level during the treatment period. In contrast, in the non-responders, the elevated expression at baseline, although it was significantly decreased at 2 weeks, returned to the baseline level after 14 weeks. In addition to inflammatory genes, we identified several groups of genes with distinct differences in expression between the responders and non-responders. CONCLUSIONS: Our results suggest that a customized low-density microarray is useful for monitoring mRNA expression profiles in peripheral blood cells, enabling us to identify a unique set of genes with differentially regulated expressions in responders and non-responders to a TNF inhibitor among patients with RA.

In vivo approach for the evaluation of mechanism-based inhibition of cytochrome P450 3A in rats.

1. There have been no reports showing that the area under the concentration-time curve (AUC) of a probe drug is elevated due to mechanism-based inhibition (MBI) of drug-metabolizing enzymes in animals. This study ascertained that mechanism-based inhibitors reported to induce drug-drug interactions (DDIs) in humans also caused MBI in rats. 2. Midazolam (MDZ), mainly metabolized by cytochrome P450 3A in rats, and mibefradil, which showed the most intense time-dependent inhibition among the inhibitors tested, were selected as the probe and the inhibitor, respectively. Following pretreatment of mibefradil at 24 h before MDZ administration in rats, the C(max) and AUC values of MDZ were significantly elevated in comparison with the control. The free plasma concentration of mibefradil was substantially lower than the IC(50) value observed in the in vitro inhibition study, suggesting that the DDI was due to MBI. 3. It is concluded that the evaluation of MBI in rats in vivo in combination with in vitro data using human enzymes could be useful to evaluate risk in clinical studies.

Long-term follow-up results of no initial therapy for ocular adnexal MALT lymphoma.

BACKGROUND: The majority of lymphomas in the ocular adnexa are low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma). Although radiotherapy is the most frequently applied management, cataract and dry eye are problematic complications. PATIENTS AND METHODS: Between 1973 and 2003, the clinical features of 36 patients with ocular adnexal MALT lymphoma with no symptoms who were managed with no initial therapy after biopsy or surgical resection were retrospectively analyzed. RESULTS: The median patient age was 63 years (range 22-84) and all patients had stage I disease, consisting of 31 unilateral cases and five bilateral cases. With a median follow-up of 7.1 years, 25 (69%) did not require treatment. The median time until the initiation of treatment in the remaining 11 patients (31%) was 4.8 years. Six patients (17%) died, and among them only two (6%) died due to progressive lymphoma. Seventeen patients (47%) progressed, but histologic transformation was recognized in only one (3%). The estimated overall survival rates of the 36 patients after 5, 10 and 15 years were 94%, 94% and 71%, respectively. CONCLUSIONS: In selected patients with ocular adnexal MALT lymphoma, no initial therapy might be an acceptable approach, because 70% of patients remained untreated at a median of 8.6 years, and their survival was comparable to that of reports on immediate therapy.

CD2+ acute promyelocytic leukemia is associated with leukocytosis, variant morphology and poorer prognosis.

The T cell-lineage marker CD2 is sometimes expressed in acute promyelocytic leukemia (APL), and CD2 expression is reported to correlate with some clinical characteristics. However, the significance of CD2 expression in APL has not been fully elucidated. We evaluated CD2 expression in APL treated by the same treatment strategy in a single institute, and whether it had any special characteristics. Among 29 APL, 6 were positive for CD2. Patients with CD2+ APL tended to have a higher leukocyte count than CD2- APL (34.5 +/- 13.1/l vs. 6.8 +/- 2.1/l), morphological characteristics as variant-APL (50 vs. 0%). They also showed poor clinical prognosis. The CR rate of CD2- APL was 87.0% while that of CD2+ APL was 50 %. The mortality was 13.0 and 66.7%, respectively, and the survival rate was significantly lower in CD2+ APL. CD2 expression was proven to be a risk factor associated with death in addition to the morphological characteristics of variant-APL and leukocytosis. These results indicated that CD2 expression might have a significant impact on the prognosis of APL. Whether CD2+ APL should be characterized as a special clinical entity should be discussed in a larger patient population.

A possible target of antioxidative therapy for diabetic vascular complications-vascular NAD(P)H oxidase.

A growing body of evidence has shown that oxidative stress may be involved in the development of vascular complications associated with diabetes. However, the molecular mechanism for increased reactive oxygen species (ROS) production in diabetes remains uncertain. Among various possible mechanisms, attention have increasingly been paid to NAD(P)H oxidase as the most important source of ROS production in vascular cells. High glucose level stimulates ROS production through protein kinase C (PKC)-dependent activation of vascular NAD(P)H oxidase. Furthermore, the expression of NAD(P)H oxidase components is increased in micro- and macrovascular tissues of diabetic animals in association with various functional disorders and histochemical abnormalities. These results suggest that vascular NAD(P)H oxidase-driven ROS production may contribute to the onset or development of diabetic micro- or macrovascular complications. In this point of view, the possible new strategy of antioxidative therapy for diabetic vascular complications is discussed in this review.

[Significance of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) activity in breast cancer tissue].

TS, DPD, uridine phosphorylase and thymidine phosphorylase are enzymes involved in the metabolism of the anticancer drug pyrimidine fluoride. In this study, levels of these enzymes were measured in 47 women with primary breast cancer. These enzyme levels were then compared to levels determined from breast cancer patients who received either preoperative chemotherapy or nothing, in order to determine whether they might predict clinical outcome. The TS inhibition rate was significantly higher (p < 0.05) in patients receiving preoperative chemotherapy (20.4 +/- 13.3%) than in the untreated group (11.4 +/- 9.8%). No other significant differences in activity were noted between the treated and untreated groups for any of the other enzymes studied. The activity of each enzyme at the tumor site and the tumor/normal (T/N) ratio were also compared between patients with and without recurrence. The TS inhibition rate at the tumor site was lower in recurring cases than in non-recurring cases, and the T/N ratio tended to be higher for DPD in patients with recurrences. These findings indicate that the TS inhibition rate and DPD activity may be useful predictors for early recurrence of breast cancer following surgery.

Reduced voltage sensitivity of activation of P/Q-type Ca2+ channels is associated with the ataxic mouse mutation rolling Nagoya (tg(rol)).

Recent genetic analyses have revealed an important association of the gene encoding the P/Q-type voltage-dependent Ca(2+) channel alpha(1A) subunit with hereditary neurological disorders. We have identified the ataxic mouse mutation, rolling Nagoya (tg(rol)), in the alpha(1A) gene that leads to a charge-neutralizing arginine-to-glycine substitution at position 1262 in the voltage sensor-forming segment S4 in repeat III. Ca(2+) channel currents in acutely dissociated Purkinje cells, where P-type is the dominant type, showed a marked decrease in slope and a depolarizing shift by 8 mV of the conductance-voltage curve and reduction in current density in tg(rol) mouse cerebella, compared with those in wild-type. Compatible functional change was induced by the tg(rol) mutation in the recombinant alpha(1A) channel, indicating that a defect in voltage sensor of P/Q-type Ca(2+) channels is the direct consequence of the tg(rol) mutation. Furthermore, somatic whole-cell recording of mutant Purkinje cells displayed only abortive Na(+) burst activity and hardly exhibited Ca(2+) spike activity in cerebellar slices. Thus, in tg(rol) mice, reduced voltage sensitivity, which may derive from a gating charge defect, and diminished activity of the P-type alpha(1A) Ca(2+) channel significantly impair integrative properties of Purkinje neurons, presumably resulting in locomotor deficits.

Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.

Reduced expression of a novel peptide, prostacyclin-stimulating factor, in the kidneys of streptozotocin-induced diabetic rats.

Prostacyclin (PGI2) produced by vascular endothelial cells (ECs) is a potent vasoactive prostanoid involved in maintenance of vessel wall homeostasis. Reduced PGI2 synthesis by vascular ECs could be a mechanism of pathogenesis in the development of vascular lesions such as diabetic angiopathy. Recently, we purified and cloned a novel bioactive peptide, PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular ECs. PSF may act on vascular ECs in a paracrine and/or autocrine fashion to regulate PGI2 synthesis. Decreased PSF production in the vessel wall may result in an imbalance of prostanoid synthesis, leading to the development of vascular lesions such as diabetic angiopathy. Our immunohistochemical study demonstrated that PSF is located in vascular resident cells such as vascular smooth muscle cells (SMCs) and ECs, as well as in bronchial SMCs. Moreover, PSF mRNA was found to be expressed in various tissues in Wistar rats, particularly in the kidneys and lungs. The present study demonstrated that streptozotocin (STZ)-induced diabetic rats showed less PSF mRNA expression in the kidneys (PSF mRNA/28S rRNA ratio; STZ versus control; 1.7+/-0.2 versus 2.5+/-0.2, p < 0.05) and reduced immunohistochemical staining for PSF in arteries in the kidney. However, in the lungs, there were no changes in tissue PSF mRNA expression (STZ versus control; 10.9+/-0.9 versus 11.5+/-1.0, NS) or in the extent of PSF staining in bronchial SMCs of STZ-induced diabetic rats. These findings suggest that decreased expression of PSF in renal vessels of STZ-induced diabetic rats may cause an imbalance of prostanoid synthesis, leading to the development and progression of vascular damage in the kidney.

Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels.

Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as L-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature 325, 717-720). In addition to "receiving" the EC coupling signal from the DHPR, RyR-1 also "transmits" a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72-76). A similar kind of retrograde signaling (from RyRs to L-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719-722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635-2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+ channel function, whereas a chimera containing adjacent RyR-1 residues (2, 659-3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Increased mRNA expression of a novel prostacyclin-stimulating factor in human colon cancer.

We recently cloned a prostacyclin (PGl2)-stimulating factor (PSF), which stimulates PGl2 production by cultured vascular endothelial cells. Immunohistochemistry and Northern blot analysis demonstrated that PSF was highly expressed in colon cancer sites compared with normal colon mucosa obtained from the same patient, as well as in cultured adenocarcinoma cell lines compared with cultured normal colon mucosal cell lines. Increased levels of the PSF protein were detected in the culture media of these adenocarcinoma cells compared with levels in the culture media of normal mucosal cells. These results suggest that PSF is closely associated with carcinogenesis of colon mucosa.