Topologies of amyloidogenic proteins in Congo red-positive sliced sections of formalin-fixed paraffin embedded tissues by MALDI-MS imaging coupled with on-tissue tryptic digestion

Objectives

Matrix-assisted laser desorption time-of flight ionization (MALDI)-imaging MS (IMS) with MSMS analysis using on-tissue tryptic digests is a powerful tool for identification of disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections. We applied this novel IMS technique, not only to identify tryptic peptides of deposited amyloidogenic proteins but also to clarify topologies of these proteins in amyloidosis tissue sections.

Methods

Sequence determinations of tryptic peptides derived from amyloidogenic proteins were performed using MALDI-MSMS analysis directly from Congo red positive regions in tissue sections with/without procedure for retrieval of epitopes before on-tissue digestion.

Results

Tryptic peptides, m/z = 1073.5 and 1924.3 were identified with the sequences, from 48th to 56th and 1st to 19th positions of Ig lambda V-III region, respectively. Other peptides, m/z = 1365.5 and 1523.5 were with the sequences, from 22nd to 34th and 36th to 48th positions of TTR, respectively. Heat-map images of all four tryptic peptides were overlapped with Congo red positive regions. Immunohistochemistry of FFPE tissue sections was confirmed to only react with anti-λ chain antibody in a case of AL-type amyloidosis or anti-TTR antibody in two cases of TTR-type amyloidosis.

Conclusion

IMS with MSMS analysis using on-tissue tryptic digestion enables us not only to identify amyloidogenic molecule in a sliced tissue section but also to play a complementary role with the conventional pathological examination.     

Amlodipine treatment reduces stroke size in apolipoprotein E-deficient mice

BACKGROUND: This study investigated the effects of amlodipine, an L-type calcium channel blocker, on stroke size after focal brain ischemia in apolipoprotein E-deficient (ApoE KO) mice. METHODS: Mice were subjected to middle cerebral artery (MCA) occlusion after being given a high-cholesterol (HCD) or normal diet for 10 weeks with or without amlodipine at a nonhypotensive dose of 3 mg/kg/day. Ischemic brain area was measured by 2,3,5-triphenyltetrazolium chloride staining. Cerebral blood flow was analyzed by laser-Doppler flowmetry. Superoxide anion production in the brain was detected by dihydroethidium staining. RESULTS: The ApoE KO mice given HCD for 10 weeks showed a larger ischemic lesion size than mice with a normal diet. Amlodipine treatment in parallel with HCD feeding reduced the ischemic lesion size in ApoE KO mice. Interestingly, amlodipine treatment for only the last 2 weeks was also effective in reducing the ischemic lesion size in HCD-fed ApoE KO mice. The neurologic deficit after MCA occlusion was also improved by amlodipine treatment for either 10 weeks or 2 weeks. The decrease in surface cerebral blood flow after MCA occlusion was significantly attenuated in the peripheral region of the MCA territory in amlodipine-treated mice. Amlodipine treatment in HCD-fed ApoE KO mice also reduced superoxide production in the ischemic area of the brain. CONCLUSIONS: These results suggest that amlodipine treatment reduces stroke size and neurologic deficit after focal brain ischemia, possibly through an increase in cerebral blood flow and inhibition of superoxide production.     

Blood-brain barrier transport of opioid analgesics

Opioid analgesics exhibit cationic properties under physiological conditions, and the mechanism underlying permeation of the blood-brain barrier thus cannot be fully explained by simple diffusion alone. Various types of transporters that exhibit substrate specificity are localized on the blood-brain barrier, and play a role in transporting substances from circulating blood and from brain interstitial fluid. Progress is being made in explaining the mechanisms, functions, and physiological roles of polyspecific organic cation transporters, but little evidence has indicated that these previously identified organic cation transporters are involved in the transport of opioid analgesics across the blood-brain barrier. Consequently, clarifying the role of transporters in the distribution of opioid analgesics into the brain and determining their transport molecule will not only provide clues to effective drug delivery to the brain, but will also contribute to optimizing pain relief treatment, and by extension play a role in drug discovery for analgesics. Currently there are enthusiastic discussions in the literature regarding the existence of putative transporters involved in the transport of opioid analgesics across the blood-brain barrier. This review article introduces the results of our research as well as recent findings on the involvement of transporters in the blood-brain barrier transport of opioid analgesics such as morphine, morphine metabolites, oxycodone, fentanyl, codeine, and pentazocine.     

Myocardial fibrosis attenuates the effect of cibenzoline on left ventricular diastolic function in patients with hypertrophic cardiomyopathy

The aim of this study was to investigate the relationship between late gadolinium enhancement (LGE) and the effect of cibenzoline (CBZ) on left ventricular (LV) diastolic function in hypertrophic cardiomyopathy (HCM) patients. Echocardiography before and after intravenous CBZ (1.4 mg/kg over 5 minutes) and magnetic resonance imaging (MRI) were performed in 22 consecutive patients with HCM [mean age: 65 ± 14 years, obstructive HCM: 14, nonobstructive HCM (HNCM): 8]. The extent of LGE (%LGE = LGE volume/total LV volume) was obtained by contrast-enhanced MRI using custom software. LGE was observed in 19 patients (mean %LGE = 5.1% ± 3.9%). The propagation velocity of LV early filling flow (Vp) increased significantly in patients with obstructive HCM (26 ± 7 to 36 ± 14 cm/s, P = 0.001) and nonobstructive HCM (25 ± 9 to 36 ± 16 cm/s, P = 0.007). A significant negative correlation was observed between the change in Vp and %LGE in patients with HCM (r = 20.542, P = 0.009). Less extensive myocardial fibrosis, as demonstrated by LGE on contrast-enhanced MRI, may predict the ability of CBZ to improve LV diastolic function in HCM.     

Phase II trial of carboplatin in patients with advanced germ-cell testicular tumors and transitional cell carcinomas of the urinary tract

Summary Carboplatin, an analog of cisplatin, was evaluated in a phase II study involving 25 patients with advanced testicular tumor and 45 with transitional cell carcinoma (TCC) of the urinary tract; 21 and 38 cases, respectively, were evaluable for response. Prior treatment with cisplatin-based chemotherapy had occurred in 7 of the testicular cancer patients and in 11 with TCC. The response rate (complete + partial response) in testicular tumors was 47.6%. The best response rate was observed in seminomas (70.0%), whereas the response rate in nonseminomas was 27.3%. The seminoma patients had mainly stage IIIA or less than IIA disease, with metastatic lesions restricted to the lymph nodes. Three responses were seen in patients previously treated with cisplatin. In TCC, the response rate was 18.4%. Good-risk patients were treated with a dose of 400 mg/m² every 4 weeks, whereas poor-risk patients received a lower dose of 300 mg/m². The response rates for good-risk patients were 50.0% in testicular lesions and 26.1% in TCC. For poor-risk patients, the response rates were 40.0% and 6.7%, respectively. Carboplatin was well tolerated, with no significant renal impairment or ototoxicity detected. Nausea and vomiting were experienced by 51.7% of patients, but the severity was low; half of these patients demonstrated WHO grade I toxicity. However, myelosuppression was severe. In conclusion, carboplatin demonstrated activity in both testicular tumors and TCC and is worthy of further study, especially in combination with other active drugs.     

Ectopic Nesidioblastosis

We observed one case of ectopic nesidioblastosis. A solitary nesidioblastoma was observed in the interior wall of the duodenum. Ectopic nesidioblastosi has not been previously reported, but our experience suggests the necessity of examination for ectopic pancreas in cases of SIDS or its near-miss.     

Prognostic significance of electrocardiographic findings in patients with dilated cardiomyopathy

Summary The prognostic significance of electrocardiographic variables was retrospectively investigated in 88 patients with dilated cardiomyopathy and with normal coronary arteriograms. During an average follow-up of 3.7 ± 2.9 years, 43 patients died, 26 of progressive heart failure, 15 patients with sudden death, and one due to cerebral embolism. Excluding one patient, who died of esophageal cancer, the cumulative survival rate was 73% at 2 years and 60% at 5 years. By univariate life table analysis, abnormal Q-waves, a QRS duration 0.12s, a cardiothoracic ratio 60%, systolic blood pressure <110mmHg, and left ventricular end-diastolic pressure 15 mmHg were significantly associated with increased mortality within 5 years. Multivariate analysis using Cox's proportional hazards model determined the major independent risk factors in the following order: (1) for all patients; the presence of abnormal Q-waves, left bundle branch block or intraventricular conduction disturbances, left ventricular end-diastolic pressure, systolic blood pressure and the cardiothoracic ratio; (2) for patients without left bundle branch block or intraventricular conduction disturbances; abnormal Q-waves, left ventricular end-diastolic pressure and systolic blood pressure. The present study thus demonstrated that the electrocardiogram could provide independent prognostic predictors in patients with dilated cardiomyopathy, possibly reflecting the severity of myocardial damage.This study was supported in part by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan.     

Peritransplant VLA‐4 blockade inhibits endogenous memory CD8 T cell infiltration into high‐risk cardiac allografts and CTLA‐4Ig resistant rejection

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC‐mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen‐4 (VLA‐4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti‐VLA‐4 mAb given to C57BL6 (H‐2^b) recipients of AJ (H‐2^a) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra‐allograft IFN‐γ‐induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA‐4Ig treated recipients. Anti‐VLA‐4 mAb also inhibited priming of donor‐specific T cells producing IFN‐γ until at least day 7 posttransplant. Peritransplant anti‐VLA plus anti‐CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti‐VLA‐4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.