Heterotopic transplantation of the failing rat heart as a model of left ventricular mechanical unloading toward recovery

The left ventricular assist device (LVAD) is usually used in patients with end-stage heart failure as a bridge to transplantation. Recently, some studies have reported functional recovery with the use of an LVAD, although the mechanisms responsible for recovery are not fully understood. We investigated the functional recovery of the infarcted, failing rat heart in response to mechanical unloading after heterotopic transplantation. Heart failure was induced in Lewis rats by ligating the left anterior descending artery. After 4 weeks, the infarcted hearts were harvested and heterotopically transplanted. The transplanted infarcted heart was removed after 2 weeks of unloading and examined for hypertrophy and fibrosis, as well as for mRNA levels encoding for brain natriuretic peptide, sarco(endo)plasmic reticulum Ca(2+)-ATPase2a (SERCA2a), and beta1- and beta2-adrenergic receptors. Normal and infarcted rats without transplantation served as control animals. The infarcted heart was hypertrophied as evidenced by an increase in heart weight and myocyte diameter. After unloading the infarcted heart for 2 weeks, there was a decrease in heart weight and myocyte diameter. However, the percentage of myocardial fibrosis increased after unloading. The mRNA expression of brain natriuretic peptide and the beta2-adrenergic receptor significantly improved after mechanical unloading. The levels of SERCA2a mRNA tended to increase after unloading. In conclusion, unloading the failing, infarcted heart can help normalize left ventricular hypertrophy and cardiac gene expression. This unloading model appears to partially mimic the conditions of hemodynamic support with an LVAD in heart failure patients and potentially offers insights into the mechanisms of functional recovery.     

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

Expression profiles and functional analyses of Wnt-related genes in human joint disorders

Rheumatoid arthritis (RA) and osteoarthritis (OA) are joint disorders that cause major public health problems. Previous studies of the etiology of RA and OA have implicated Wnt genes, although the exact nature of their involvement remains unclear. To further clarify the relationship between RA, OA, and the Wnt gene family, gene expression analyses were performed on articular cartilage, bone, and synovial tissues in knee joints taken from RA, OA, and nor-mal/control patients. Cytokine assays were also performed in cells transfected with Wnt-7b, a member of the gene family most closely linked to RA and OA. Of the human Wnt genes, real-time PCR analysis revealed significant up-regulation of Wnt-7b in OA cartilage and RA synovium. In situ hybridization and immunohistochemistry also revealed that Wnt-7b was present in articular cartilage, bone, and synovium of RA samples and in osteophytes, articular cartilage, bone marrow, and synovium of OA samples. The levels of the cytokines tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were significantly increased in RA synovium and Wnt-7b-transfected normal synovial cells when compared with normal samples. These results point to the potential involvement of Wnt signaling in the pathobiology of both RA and OA.     

Membrane Fusion In Mitochondria Ii. An Ultrastructural Interpretation Of Cristae-Enriched Megamitochondria Induced By Cuprizone

Ultrastructure of megamitochondria in mouse hepatocytes induced by cuprizone was analyzed in the light of the fusion of inner mitochondrial membranes. Hepatic parenchymal cells of mice fed with a 0.5% cuprizone-diet for 7–8 days were characterized by megamitochondria with numerous cristae including disoriented, shorter ones than those of the control and those in vesicular configuration. After feeding the animals with cuprizone for 15–16 days, megamitochondria increased their sizes further and the number of cristae decreased drastically. Numbers of cristae based on a unit-mitochondrial area were 41.3$pL10.8 for the former type of cristae-enriched megamitochondria and 3.8 $pL 1.8 for the latter type of megamitochondria with the value of 23.9 $pL 6.2 for the control. Considering the content of cytochromes in megamitochondria previously reported, these results might suggest that the fusion and defusion of inner mitochondrial membrane take place along with the fusion of outer membranes during the megamitochondrial formation. ACTA PATHOL. JPN. 34: 481–488, 1984.     

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide

⁵Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle ceils and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.     

Open food challenge with milk, egg white and wheat

Background: A method for open food challenge test to determine food allergy has not been established in an evidence-based manner. Methods: We conducted an analysis of 438 open food challenges of raw milk (n=133, mean age 2.7+/-1.9 years), boiled egg white (n=216, 2.8+/-2.1 years) or udon noodles (n=89, 2.7+/-1.7 years) for the patients aged 1 year or more. Doses were increased (trace amounts, 1 g, 2 g, 5 g, 10 g, 20-30 g) every 20 minutes. Results: In total, 151 (38.5%) of food challenges were positive. The positive rates of milk, egg and wheat challenges were 35.8%, 42.4% and 33.3%, respectively. Of these, 76.2%, 32.5%, 27.8% and 0.7% had, respectively, skin, respiratory, gastrointestinal and cardiovascular symptoms. Although the prevalence of positive challenge increased with level of specific IgE, it did not correlate with the threshold amount of positive food challenge or the severity of symptoms. Among the challnege positive patients, 10.6% required injection of antihistamines, corticosteroids or adrenalines for the treatment of the symptoms. Conclusions: This challenge protocol seemed to be appropriate and safe.     

Evaluation of the drug lymphocyte stimulation test (DLST) with shosaikoto]

BACKGROUND: Our aim is to evaluate the significance of DLST by Shosaikoto. METHODS: We clinically evaluated 3 cases of drug-induced pneumonia assumed to be caused by Shosaikoto, and we performed DLST of Shosaikoto for healthy controls, and compared the data with drug-induced pneumonia cases of Shosaikoto. RESULTS: As clinical characteristics of 3 cases, 2 cases were positive for hepatitis C virus antibody, and 1 case was positive for DLST of Shosaikoto. The observed chest high-resolution CT (HRCT) findings showed hypersensitivity pneumonia (HP) pattern in all 3 cases. Prognosis was good in all 3 cases. DLST of Shosaikoto was positive in 27.5% of healthy controls. Stimulation index (S.I.) of DLST in drug-induced pneumonia cases increased depending on drug dilution density, compared to that of healthy controls. CONCLUSION: DLST of Shosaikoto showed high false-positive rate. However, we may be able to distinguish the true-positive cases with the false-positive cases by comparing the S.I. of DLST according to drug dilution density.     

Redox-linked signal transduction pathways for protein tyrosine kinase activation

The signaling for activation of protein tyrosine kinases (PTKs) is usually started by binding of ligands to cell-surface receptors. However, recent evidence suggests the presence of ligand binding-independent signaling pathways that are mediated by oxidative stress. Oxidation and reduction of protein cysteine sulfhydryl (SH) groups may work as a molecular switch to start or to stop the signaling. It is known that oxidation of cysteine SH groups on protein tyrosine phosphatases switches off the action of protein tyrosine phosphatases. This event may not, however, signal for initial autophosphorylation of previously unphosphorylated PTKs, whereas it certainly prevents dephosphorylation of once-phosphorylated PTKs. We have suggested new mechanisms for oxidative stress-mediated PTK activation. First, cell-surface glycosylphosphatidylinositol-anchoring proteins and a phosphoglycolipid/cholesterol-enriched membrane microdomain termed a "raft" can be the direct targets of oxidative stress for inducing their clustering through an S-S-bonded or S-X-S-bonded crosslinking of cell-surface proteins and subsequent activation of raft-associating Src family PTKs. Second, intracellular specific cysteine SH groups on PTK proteins can be another target of oxidative stress for inducing a conformational change necessary for initial activation of PTKs. A possible relationship between cell-surface and intracellular events is that the former frequently induces superoxide production as the second messenger for the latter.