The suppressive effect on CD4 T cell alloresponse against endothelial HLA-DR via PD-L1 induced by anti-A/B ligation.

While donor-specific human leukocyte antigen (HLA) antibodies are a frequent cause for chronic antibody-mediated rejection in organ transplantation, this is not the case for antibodies targeting blood group antigens, as ABO-incompatible (ABO-I) organ transplantation has been associated with a favorable graft outcome. Here, we explored the role of CD4 T cell-mediated alloresponses against endothelial HLA-D-related (DR) in the presence of anti-HLA class I or anti-A/B antibodies. CD4 T cells, notably CD45RA-memory CD4 T cells, undergo extensive proliferation in response to endothelial HLA-DR. The CD4 T cell proliferative response was enhanced in the presence of anti-HLA class I, but attenuated in the presence of anti-A/B antibodies. Microarray analysis and molecular profiling demonstrated that the expression of CD274 programmed cell death ligand 1 (PD-L1) increased in response to anti-A/B ligation-mediated extracellular signal-regulated kinase (ERK) inactivation in endothelial cells that were detected even in the presence of interferon-γ stimulation. Anti-PD-1 antibody enhanced CD4 T cell proliferation, and blocked the suppressive effect of the anti-A/B antibodies. Educated CD25+ CD127- regulatory T cells (edu.Tregs ) were more effective at preventing CD4 T cell alloresponses to endothelial cells compared with naive Treg ; anti-A/B antibodies were not involved in the Treg -mediated events. Finally, amplified expression of transcript encoding PD-L1 was observed in biopsy samples from ABO-I renal transplants when compared with those from ABO-identical/compatible transplants. Taken together, our findings identified a possible factor that might prevent graft rejection and thus contribute to a favorable outcome in ABO-I renal transplantation.

Encephalitis caused by human herpesvirus-6B in pancreas-after-kidney transplantation.

Human herpesvirus-6 (HHV-6) is a common pathogen among children, classically presenting with fever and rash that resolves without specific therapy. HHV-6 can be reactivated in the immunosuppressed patient. After bone marrow and solid organ transplantation, HHV-6 has been linked to various clinical syndromes, including undifferentiated febrile illness, encephalitis, myelitis, hepatitis, pneumonitis, and bone marrow suppression. However, HHV-6 encephalitis after pancreatic transplant has rarely been reported. Early diagnosis and treatment of HHV-6 encephalitis may be important for affected patients. We report the case of a 53-year-old pancreas-after-kidney transplant recipient who initially presented with high fever and confusion 3 weeks after operation. We managed to save the patient's life and preserve the pancreas graft function. We also review previously reported cases of HHV-6B encephalitis in solid organ transplant recipients.

Acceleration of diabetes development in CXC chemokine receptor 3 (CXCR3)-deficient NOD mice.

AIMS/HYPOTHESIS: The aim of this study was to understand the role of CXC chemokine receptor 3 (CXCR3), a T-helper 1(Th1) type chemokine receptor, in the pathogenesis of type 1 diabetes. METHODS: We observed the incidence of diabetes in Cxcr3 homozygous knockout mice. We compared the expression pattern of various cytokines and chemokines and the frequency of FOXP3(+) cells in the pancreas and pancreatic lymph nodes from Cxcr3 ( -/- ) NOD mice and wild-type NOD mice. In addition, we observed the migration ability of CXCR3(+)CD4(+) cells to pancreatic islets upon adoptive transfer. Finally, we examined whether Cxcr3 (+) regulatory T cells (Tregs) actually suppressed the onset of diabetes in vivo. RESULTS: Cxcr3 ( -/- ) NOD mice developed spontaneous diabetes earlier than did wild-type NOD mice. In Cxcr3 ( -/- ) NOD mice, Tregs were more frequent in pancreatic lymph nodes and less frequent in pancreatic islets than in wild-type NOD mice. While transferred CXCR3(-)CD4(+) cells from wild-type NOD mice did not infiltrate pancreatic islets of NOD-severe combined immunodeficiency (SCID) mice, CXCR3(+)CD4(+) cells from the same mice migrated into the recipient islets and contained Forkhead box P3 (FOXP3) upon adoptive transfer. Moreover, CD4(+)CD25(+) cells from wild-type NOD mice suppressed and delayed the onset of diabetes compared with those from Cxcr3 ( -/- ) NOD mice in a cyclophosphamide-induced diabetes model system. CONCLUSIONS/INTERPRETATION: The mechanism of accelerated diabetes onset in Cxcr3 ( -/- ) NOD mice was considered to be due to the lack of hybrid Tregs (CXCR3(+)FOXP3(+)CD4(+) cells), which could effectively migrate into and regulate Th1 inflammation in local lesions under Cxcr3 knockout conditions.

Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. METHODS: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. RESULTS: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). CONCLUSION: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.

Timing for orthotopic liver transplantation in children with biliary atresia: a single-center experience.

INTRODUCTION: Biliary atresia is the most common indication for orthotopic liver transplantation (OLT) in childhood. The purpose of this study was to determine predictive prognostic factors for children with biliary atresia related to the timing for OLT within 15 months after hepatoportoenterostomy (HPE). PATIENTS AND METHODS: We retrospectively analyzed the medical records of 25 children (7 boys and 18 girls) who underwent HPE because of biliary atresia between January 1990 and December 2005 at our center. Data examined included age and pathologic findings at HPE, Pediatric End-Stage Liver Disease score at first admission, whether phototherapy was given, liver function test results and total bilirubin level before and 30 days after HPE, and number of cholangitis events. RESULTS: Twelve children were alive with their native liver, 8 had undergone living donor OLT (all children alive), and 5 had died without OLT. Five- and 10-year survival rates without OLT after HPE were 47.4% and 26.3%, respectively. At univariate analysis, the predictive prognostic factors for children with biliary atresia were total bilirubin level at 30 days after HPE and Pediatric End-Stage Liver Disease score before HPE. At multivariate analysis, the only prognostic factor was total bilirubin level at 30 days after HPE. CONCLUSIONS: In this study, the predictive prognostic factor was total bilirubin level at 30 days after HPE. Orthotopic liver transplantation within 15 months after HPE is needed in children with biliary atresia with a high total bilirubin level at 30 days after HPE.

Living donor liver transplantation for a child with recurrent pediatric adult-type hepatocellular carcinoma.

INTRODUCTION: Pediatric hepatocellular carcinoma (HCC) is an uncommon disease with a poor prognosis. There are few reports about liver transplantation for pediatric adult-type HCC. We experienced a case of living donor liver transplantation (LDLT) for a child with recurrent pediatric adult-type HCC. CASE REPORT: A 12-year-old boy was admitted to the Department of Pediatrics in our institution due to HCC in May 2005. He underwent hepatectomy after 3 courses of chemotherapy in July 2005. After the operation, he had 2 more courses of the same chemotherapy. His posttheraputic course was uneventful for 1 year. However, his alpha-fetoprotein level increased and a computed tomography (CT) scan showed recurrent tumor in his remnant liver in October 2006. He underwent another chemotherapy session immediately. However, CT revealed multiple liver tumors after chemotherapy in December 2006. His mother requested to be an LDLT donor, which was performed on January 23, 2007. The donor operation was a right hepatic lobectomy. The postoperative course of the donor was unremarkable and she has now returned to work. The recipient's posttransplantation course was uneventful and he was discharged at postoperative day 53 and is currently doing well. CONCLUSION: Liver transplantation in conjunction with chemotherapy may have an increasing role in the management of pediatric HCC.

Slow Progression of Aortic Calcification Is a Potential Benefit of Pre-emptive Kidney Transplantation.

PURPOSE: Pre-emptive kidney transplantation (PKT) is expected to improve graft and cardiovascular event-free survival compared with standard kidney transplantation. Aortic calcification is reported to be closely associated with renal dysfunction and cardiovascular events; however, its implication in PKT recipients remains incompletely explored. This aim of this study was to evaluate whether PKT confers a protective effect on aortic calcification, renal function, graft survival, and cardiovascular event-free survival. METHODS: One hundred adult patients who underwent renal transplantation between January 1996 and March 2016 at Hirosaki University Hospital and Oyokyo Kidney Research Institute were included. Among them, 19 underwent PKT and 81 patients underwent pretransplant dialysis. We retrospectively compared pretransplant and post-transplant aortic calcification index (ACI), renal function (estimated glomerular filtration rate [eGFR]), and graft and cardiovascular event-free survivals between the 2 groups. RESULTS: The median age of this cohort was 45 years. Preoperative ACI was significantly lower in PKT recipients. There were no significant differences between the 2 groups regarding postoperative eGFR, graft survival, and cardiovascular event-free survival. However, the ACI progression rate (ΔACI/y) was significantly lower in PKT recipients than in those who underwent pretransplant dialysis. Higher ACI was significantly associated with poor cardiovascular event-free survival. CONCLUSIONS: PKT is beneficial in that it contributes to the slow progression of after transplantation. Although we could not observe significant differences in graft and cardiovascular event-free survivals between the 2 groups, slow progression of aortic calcification showed a potential to decrease cardiovascular events in PKT recipients during long-term follow-up.

Partial Cystectomy of Paraganglioma of the Urinary Bladder Before Living Kidney Transplantation: Case Report.

BACKGROUND: Paraganglioma (extra-adrenal pheochromocytoma) of the bladder is a very rare disease, accounting for 0.06% of all bladder tumors. Optimal management of bladder paraganglioma before kidney transplantation is unknown. We report a case of partial cystectomy for urinary bladder paraganglioma before living kidney transplantation. CASE PRESENTATION: A 59-year-old man with a 27-year history of hemodialysis was referred to our department for further examination of a bladder tumor detected during pre-transplantation testing. Cystoscopy revealed a submucosal tumor on the right side of the bladder. The patient experienced a hypertensive crisis during transurethral resection of the bladder tumor. Endocrinologic and pathologic examinations confirmed the diagnosis of paraganglioma in the urinary bladder. A partial cystectomy was performed before kidney transplantation. Nine months after partial cystectomy, the patient underwent AB0-incompatible living kidney transplantation from his spouse. No disease recurrence or graft rejection was observed 12 months after the transplantation. CONCLUSIONS: To our knowledge, this is the 1st report on the management of paraganglioma in the urinary bladder before living kidney transplantation. Kidney transplantation after partial cystectomy is an option that may be considered in patients with paraganglioma of the urinary bladder, with careful observations of bladder function and vesicoureteral reflux to the grafts.

Condyloma Acuminata of the Urethra in a Male Renal Transplant Recipient: A Case Report.

BACKGROUND: Condyloma acuminatum (CA) is a common sexually transmitted disease associated with human papilloma virus (HPV). CA occurring in the urethra is rare and has not been reported in male renal transplant recipients. In addition, despite immunosuppressive conditions and increased risk of HPV-related malignant neoplasms in transplant recipients, HPV testing in male transplant recipients has been uncommon. Here we report a case of urethral CA in a male deceased donor renal transplantation recipient and discuss the importance of HPV testing in male transplant recipients. CASE PRESENTATION: A 33-year-old male deceased donor renal transplant recipient presented with miction pain 5 years after the transplantation. He reported repeated urinary tract infections with no sexual contact since the renal transplantation. Multiple papillary tumors in his penile urethra were detected by cystoscopy, and a biopsy sample was pathologically diagnosed with CA. Transurethral tumor resection was performed, and the tumors were completely resected. Additional HPV risk type screening with a urethral smear sample showed the prevalence of low-risk HPV. Although tacrolimus was switched to everolimus and imiquimod cream was administered, the tumors recurred 6 months after the resection, and a second resection was performed. No further recurrence has been observed for 1 year to date. CONCLUSION: As the urethral CA was possibly related to immunosuppressive conditions and a risk for HPV-related malignant neoplasm, the case required careful diagnosis, including HPV risk type. The methodology of sampling for HPV testing in men has not been established. This case suggests the necessity for further discussion about HPV testing in male transplant recipients.

Efficacy of Eculizumab Therapy for Atypical Hemolytic Uremic Syndrome Recurrence and Antibody-Mediated Rejection Progress After Renal Transplantation With Preformed Donor-Specific Antibodies: Case Report.

Atypical hemolytic uremic syndrome (aHUS) develops as the result of unregulated complement progression and precipitates de novo thrombotic microangiopathy. Plasma therapy is used to control the progression of the complement cascade, but that therapy is not effective in all patients and is accompanied by risk of infection and/or allergy. Eculizumab has been reported as an efficient therapy for aHUS. We report the case of a 35-year old woman who underwent effective eculizumab therapy for aHUS recurrence and antibody-mediated rejection (AMR) progress after renal transplantation with preformed donor-specific antibodies (DSA). She developed end-stage renal disease due to suspicious IgA nephropathy at age 33 years. Kidney transplantation was performed at age 35 years, and aHUS recurred 2 weeks later, leading to the progressive hemolytic anemia and renal dysfunction. Therefore, she underwent plasma therapy several times. Because it was difficult to continue to plasma therapy for severe allergy, eculizumab was proposed as an alternate therapy. Treatment with eculizumab was initiated 36 days after renal transplantation. After 3 years of eculizumab treatment, and without plasma therapy, schistocytes decreased, haptoglobin increased to within normal limits, creatinine levels stabilized, and no further episodes of diarrhea were reported. At protocol biopsy 1 year after transplantation, she was diagnosed with C4d-negative subclinical AMR. However, her pathologic findings at follow-up biopsy 3 years after transplantation were recovered. We conclude that eculizumab alone, without plasma therapy, is sufficient to treat recurrence of aHUS and AMR due to DSA after renal transplantation and to maintain long-term graft function.

Optimal MR Plaque Imaging for Cervical Carotid Artery Stenosis in Predicting the Development of Microembolic Signals during Exposure of Carotid Arteries in Endarterectomy: Comparison of 4 T1-Weighted Imaging Techniques.

BACKGROUND AND PURPOSE: Preoperative identification of plaque vulnerability may allow improved risk stratification for patients considered for carotid endarterectomy. The present study aimed to determine which plaque imaging technique, cardiac-gated black-blood fast spin-echo, magnetization-prepared rapid acquisition of gradient echo, source image of 3D time-of-flight MR angiography, or noncardiac-gated spin-echo, most accurately predicts development of microembolic signals during exposure of carotid arteries in carotid endarterectomy. MATERIALS AND METHODS: Eighty patients with ICA stenosis (≥70%) underwent the 4 sequences of preoperative MR plaque imaging of the affected carotid bifurcation and then carotid endarterectomy under transcranial Doppler monitoring of microembolic signals in the ipsilateral middle cerebral artery. The contrast ratio of the carotid plaque was calculated by dividing plaque signal intensity by sternocleidomastoid muscle signal intensity. RESULTS: Microembolic signals during exposure of carotid arteries were detected in 23 patients (29%), 3 of whom developed new neurologic deficits postoperatively. Those deficits remained at 24 hours after surgery in only 1 patient. The area under the receiver operating characteristic curve to discriminate between the presence and absence of microembolic signals during exposure of the carotid arteries was significantly greater with nongated spin-echo than with black-blood fast spin-echo (difference between areas, 0.258; P < .0001), MPRAGE (difference between areas, 0.106; P = .0023), or source image of 3D time-of-flight MR angiography (difference between areas, 0.128; P = .0010). Negative binomial regression showed that in the 23 patients with microembolic signals, the contrast ratio was associated with the number of microembolic signals only in nongated spin-echo (risk ratio, 1.36; 95% confidence interval, 1.01-1.97; P < .001). CONCLUSIONS: Nongated spin-echo may predict the development of microembolic signals during exposure of the carotid arteries in carotid endarterectomy more accurately than other MR plaque imaging techniques.

Safety and Effectiveness of Marginal Donor in Living Kidney Transplantation.

BACKGROUND: We evaluated the safety and feasibility of living kidney transplantation from marginal donors. PATIENTS AND METHODS: Between June 2006 and March 2015, we performed 61 living related renal transplantations at two renal transplantation centers. Marginal donors were defined as those who were older than 70 years or who had hypertension, reduced renal function, body mass index greater than 30 kg/m(2), or mildly impaired glucose tolerance. We retrospectively compared renal function and graft survival between marginal and standard living donor kidney transplantations. To evaluate renal function, creatinine clearance (CCr) was preoperatively used for donors, and estimated glomerular filtration rate (eGFR) was postoperatively used for donors and recipients. RESULTS: Among 61 donors, 14 (23%) met the marginal criteria, the major reason being hypertension (91%). The mean age tended to be higher in the marginal group. Preoperative eGFR was significantly lower in the marginal group, whereas postoperative renal function decline ratio at two years was not significantly different between the groups (67% vs 67%, P = .960). Five-year graft survival rates were not significantly different between the two groups. However, recipient eGFR 1 year after kidney transplantation was lower in the marginal group than in the standard group (44 ± 8 vs 55 ± 9 in eGFR, P = .003). CONCLUSIONS: No significant differences were observed between the groups regarding donor renal function. Careful marginal donor selection can be safe and feasible for donors and recipients of living kidney transplantation; however, it may have a negative impact on recipient renal function.

Tissue-specific expression of murine IP-10 mRNA following systemic treatment with interferon gamma.

We have examined the tissue distribution of 10-kd inflammatory protein (IP-10) mRNA expression in C57Bl/6 mice injected intravenously (i.v.) with various inflammatory stimuli. IP-10 mRNA was strongly induced by interferon-gamma (IFN-gamma) in liver and kidney but only poorly in skin, heart, and lung. IFN-gamma had nearly equivalent access to these tissues as indicated by the distribution of radiolabeled recombinant IFN-gamma 1 h after injection. The time course of IP-10 mRNA appearance was rapid and transient in both liver and kidney; maximal expression in the liver (2 h) preceded that in the kidney (3 h) and declined rapidly thereafter in both tissues. Expression of IP-10 mRNA in the liver and kidney was highly sensitive to IFN-gamma treatment; nearly maximal stimulation occurred with injection of 500 U of IFN-gamma per mouse. Comparable stimulation of IP-10 mRNA expression in splenic macrophages required 10,000 U of IFN-gamma administered i.v., indicating that liver and kidney responses are 10- to 20-fold more sensitive. IP-10 mRNA expression in both tissues was not restricted to stimulation by IFN-gamma but was also seen with injection of lipopolysaccharide (LPS) (25 micrograms/mouse) or IFN-beta (100,000 U/mouse). Two other members of the IP-10 gene family, KC (gro) and JE (MCP-1), were expressed at lower levels under similar treatment conditions. Analysis of IP-10 mRNA distribution in the liver and kidney by in situ hybridization indicated that expression in both tissues was most prominent in the reticuloendothelial cell system, particularly in the endothelial lining of the microvascular circulation. Although the function of the IP-10 gene product has not been defined, these results suggest that it may play an important role in the response of both the liver and kidney to systemic inflammation.

Elevated serum IP-10 levels observed in type 1 diabetes.

OBJECTIVE: Although most patients with type 1 diabetes are considered to have T-cell-mediated autoimmune disease, a method of measuring of pancreatic beta-cell-specific T-cell function in cases of type 1 diabetes has yet to be established. Here, we focused on interferon-inducible protein-10 (IP-10), a chemokine that promotes the migration of activated T-helper 1 (Th1) cells and measured serum IP-10 levels in patients with human type 1 diabetes, which is regarded as a Th1-mediated disease. RESEARCH DESIGN AND METHODS: Serum samples were obtained from diabetic patients, and the levels of autoantibodies (GAD and insulinoma-associated protein-2 [IA-2]) and IP-10 were measured. Diabetic patients positive for either or both of the autoantibodies were classified as Ab+ type 1, and those negative for both were classified as Ab type 1. To evaluate islet antigen-specific responses, peripheral blood from patients stimulated with or without GAD was used, and intracellular cytokine staining for flowcytometry was performed. RESULTS: The Ab+ and Ab- type 1 groups both showed a significantly higher serum IP-10 level than the healthy subjects (P < 0.001 and P < 0.05, respectively), and the IP-10 level in the recent-onset Ab+ subgroup was significantly higher than that in the established (longstanding) Ab+ subgroup (P < 0.002). Furthermore, there was a significant positive correlation between the serum IP-10 level and the number of GAD-reactive gamma-interferon-producing CD4+ cells in the Ab+ type 1 group (P < 0.007). CONCLUSIONS: Our findings demonstrate that measurement of serum IP-10 concentrations is useful in patients with type 1 diabetes.

Carotid plaque characterization using 3D T1-weighted MR imaging with histopathologic validation: a comparison with 2D technique.

BACKGROUND AND PURPOSE: 3D FSE T1WI has recently been used for carotid plaque imaging, given the potential advantages in contrast and spatial resolutions. However, its diagnostic performance remains unclear. Hence, we compared the ability of this technique to readily assess plaque characteristics with that of conventional images and validated the results with histologic classification. MATERIALS AND METHODS: We prospectively examined 34 patients with carotid stenosis who underwent carotid endarterectomy by using 1.5T scanners and obtained 3D-FSE T1WI and 2D spin-echo T1WI scans. After generating reformatted images obtained from the 3D-FSE T1-weighted images, we calculated the contrast ratios for the plaques and the adjacent muscles and compared these findings with the pathologic classifications. RESULTS: Carotid plaques were histologically classified as types VII, VIII, IV-V, or VI. With 3D-FSE T1WI, the range of contrast ratios for each classification was the following: 0.94-0.97 (median, 0.95), 0.95-1.29 (median, 1.10), 1.33-1.54 (median, 1.42), and 1.53-2.12 (median, 1.80), respectively. With 2D imaging, the range of contrast ratios for each classification was the following: 0.79-1.02 (median, 0.90), 0.88-1.19 (median, 1.01), 1.17-1.46 (median, 1.23), and 1.55-2.51 (median, 2.07), respectively. Results were significantly different among the 4 groups (P < .001). Sensitivity and specificity for discriminating vulnerable plaques (IV-VI) from stable plaques (VII, VIII) were both 100% for the 3D technique and 100% and 91%, respectively, for the 2D technique. CONCLUSIONS: 3D-FSE T1WI accurately characterizes intraplaque components of the carotid artery, with excellent sensitivity and specificity compared with those of 2D-T1WI.

A TRH analog (DN-1417): motor stimulation with rearing related to catecholaminergic mechanisms in rats.

The effect of an analog of TRH, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417) on motor activity was studied in rats. Peripheral administration of DN-1417 (0.2-20 mg/kg, i.p.) caused a significant, dose-dependent increase in total spontaneous motor activity, with a definite increase in rearing behaviour. Both increases in spontaneous motor activity and rearing behaviour were markedly inhibited by pretreatment with chlorpromazine (1, 5 mg/kg, i.p.), haloperidol (0.1, 0.5 mg/kg, i.p.), pimozide (1 mg/kg, i.p.) or alpha-methyltyrosine (250 mg/kg, i.p.). Only stimulation of rearing behaviour was selectively attenuated by phenoxybenzamine (5 mg/kg, i.p.) or FLA-63 (25 mg/kg, i.p.) at doses producing no significant effect on spontaneous motor activity. Although propranolol (10 mg/kg, i.p.) and methysergide (10 mg/kg, i.p.) had no effect, atropine (10 mg/kg, i.p.) and mecamylamine (10 mg/kg, i.p.) respectively potentiated and counteracted the effects of DN-1417. Concerning the stimulation of spontaneous motor activity, the nucleus accumbens and lateral hypothalamic area were most sensitive to DN-1417, and the lateral hypothalamic area was the most sensitive site for the stimulation of rearing. Furthermore, DN-1417 (5 X 10(-5) M) significantly enhanced the spontaneous release of [3H]dopamine from the rat nucleus accumbens slices in vitro. These findings indicate that the motor stimulatory action of DN-1417 appears to be mediated primarily via a dopaminergic mechanism by enhancing the release of dopamine from nerve terminals, including the nucleus accumbens in the mesolimbic dopamine system, and, in turn, the rearing may be mediated via noradrenergic mechanism.

Characteristics of memory impairment following lesioning of the basal forebrain and medial septal nucleus in rats.

Memory impairment in rats with lesions of the basal forebrain (BF) and medial septal nucleus (MS) including cell bodies of the cortical and septohippocampal cholinergic systems, respectively, were compared in order to evaluate the functional contribution of the two cholinergic systems to memory. Biochemical assay revealed that lesioning of the BF and MS resulted in marked and selective decreases in both choline acetyltransferase and acetylcholinesterase activities in the cerebral cortex and hippocampus, respectively. Rats with BF lesions exhibited a severe deficit in a passive avoidance task; acquisition of passive avoidance by repeated training was sluggish, and the acquired response was rapidly eliminated in a subsequent extinction test. However, only slight impairment of passive avoidance was observed in rats with MS lesions. Memory impairment in rats with BF or MS lesions was also investigated using two spatial localization tasks, the Morris water task and the 8-arm radial maze task. Both BF and MS lesions elicited a significant impairment in the Morris water task that required reference memory, as demonstrated by an apparent increase in the latency to escape onto a hidden platform in a large water tank. The impairment was much more obvious in the BF-lesioned rats. In contrast, in the radial maze task primarily requiring working memory, rats with lesions of the MS showed severe disruption, exhibiting a marked increase in total errors, a decrease in the number of initial correct responses, and an apparent change in the strategy pattern. However, corresponding changes in the rats with BF lesions were slight. These results suggest that BF lesions may lead to substantial long-term memory impairment while MS lesions may primarily produce short-term or working memory impairment, indicating a qualitatively different contribution of the two cholinergic systems to memory. It is also suggested that these two experimental animal models may be useful for evaluation of therapeutic drugs for senile dementia of the Alzheimer type.

Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme development in vivo.

The development of (Na+ + K+) ATPase, carbonic anhydrase and HCO3--stimulated ATPase activity was studied in developing rat brain in vivo, and in primary astrocyte cultures from 1--3-day-old rat brain as a function of increasing cell growth. The primary cultures showed an increase in all the above enzyme activities during cell growth, with time courses which were qualitatively similar to their development in vivo. Cell cultures grown separately from the cerebellum plus brain stem regions showed greater carbonic anhydrase activity than cerebral cultures over the entire 4-week growth period, corresponding to development of this activity in these same regions in vivo, HCO3-stimulated ATPase activity was slightly greater in cerebellar cultures and (Na+ + K+) ATPase activity was greater in cerebral cultures up to the second week of growth, resembling development of the same enzyme activities in vivo. C6 glioma and neuroblastoma cells showed no and 10-fold lower carbonic anhydrase activities respectively, compared to the primary astrocyte cultures. Addition of 1 mM N6-2'-O-dibutyryladenosine-3',5'-monophosphate (DBcAMP) in the presence of serum caused marked formation of cellular processes and increased carbonic anhydrase and (Na+ + K+) ATPase activity. Maximum effects were found 2 h after addition of 1 mM DBcAMP and thereafter declined. In the absence of serum such effects persisted for at least 24 h. Electron microscope studies showed large numbers of microtubule (approximately 20 nm diameter) and filamentous structures (less than or equal to 10 nm diameter) in the cytoplasm, which showed changes in distribution in cells treated with DBcAMP. This study suggests that the increase in ATPase and carbonic anhydrase activities in rat brain with increasing age may be in part a reflection of proliferation and development of astroglia cells. Together with the morphological data, it also provides additional evidence that primary cultures derived from neonatal rats may closely resemble developing astroglia in vivo.

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in oxygen consumption and cyclic AMP formation of rat cerebral cortex slices.

Effects of TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L- prolinamide ) and pentobarbital, alone or in combination, on oxygen consumption and cyclic AMP formation in rat cerebral cortex slices were investigated. The oxygen consumption of rat cerebral cortex slices as measured with a Warburg apparatus, increased linearly over time (0 to 60-min incubation at 37C). Addition of pentobarbital (1 to 7 x 10-4M) inhibited oxygen consumption, in a concentration-dependent manner, up to 45% of control. A concomitant application of DN-1417 (10-5M) or TRH (10-4M) and pentobarbital (5 x 10-4M) led to a partial recovery of the pentobarbital effect. The similar anti-pentobarbital effects were observed with the addition of carbachol (10-4M) or dibutyryl cyclic AMP (10-3M), but not norepinephrine (10-4M) or dopamine (10-4M). DN-1417, TRH, carbachol, norepinephrine or dopamine at 10-4M stimulated cyclic AMP formation in the cerebral cortex slices. Addition of pentobarbital (1 to 7 x 10-4M) inhibited the cyclic AMP formation, in a concentration-dependent manner. DN-1417, TRH or carbachol at 10-4M but not norepinephrine or dopamine at 10-4M significantly reversed the reduction of cyclic AMP formation induced by pentobarbital (5 x 10-4M). Atropine (10-4M) almost completely abolished DN-1417-, TRH- and carbachol-induced cyclic AMP formation in the presence and absence of pentobarbital.