Identification and differentiation of the human herpes virus group using the PCR method]

Six kinds of human herpes viruses have been identified and classified on the basis of structure and characteristics. We studied the identification and classification of these types using PCR to amplify the virus-specific DNA sequences. This method showed higher sensitivity than the conventional method of virus isolation and culture for HSV and CMV detection. For each positive control, the viral DNA was amplified only when the complementary primers themselves were used. PCR apparently detects only the activated virus, because normal controls were negative when this method was used. Therefore, the present method is thought to closely reflect viral activation and infectious diseases in patients with latent infections.     

Effects of vasopressin on water and NaCl transport across the in vitro perfused medullary thick ascending limb of Henle's loop of mouse,rat, and rabbit kidneys

Direct tubular effects of arginine vasopressin (AVP) on water and NaCl transport across the medullary thick ascending limb of Henle (MAL) were examined by the in vitro perfusion of isolated nephron fragments of mice, rats, and rabbits. Osmotic water permeability of the MAL of mice and rats was low and remained unchanged with 2 mU/ml AVP added to the bath. A dose-dependent increase in transepithelial electrical potential difference (PD) with AVP was observed in the mouse MAL when the ambient medium was isotonic. A similar result was also obtained when 2×10^–4 mol/l dibutyryl adenosine 3,5-cyclic-monophosphate was added to the bath. In this preparation, AVP also caused an increase in the unidirectional Cl efflux from 323±45 to 398±61 pmoles·mm^–1 ·min^–1 (n=6,P<0.05). In contrast, under similar condition, we could not demonstrate any effect of AVP on PD, Cl efflux, or net Na flux in the rat MAL and on PD and Cl efflux in the rabbit MAL. Both PD and Cl efflux in the rat MAL were unaffected by AVP when the perfusate was made hypotonic. However, when the ambient medium was made hypertonic by adding NaCl and urea, a significant increase in PD was observed. In addition, we confirmed that AVP stimulated adenylate cyclase activity in the MAL as well as in the collecting tubule of mice and rats. We conclude that AVP stimulates Cl transport across the MAL of mice and rats by activating adenylate cyclase-cyclic AMP system. However, this effect of AVP may quantitatively vary among species.     

Isolation, sequence and expression of a cDNA encoding the alpha-chain of the feline CD8.

We have cloned, sequenced and expressed a cDNA encoding the alpha-chain of feline CD8. This clone, named FT8-10, has an open reading frame with 720 nucleotides in length encoding a protein with 239 amino acid residues. Sequence analysis has revealed that the feline CD8 alpha-chain (CD8 alpha) shares significant homology with human (T8/Leu-2), bovine (BoCD8), rat (MRC OX8) and mouse (Lyt-2) CD8 alpha subunits. Cysteine residues as well as the tyrosine kinase p56lck binding site are well conserved. Besides, no putative N-linked glycosylation site was found. Interestingly, immunofluorescence analysis of COS-7 cells transfected with feline CD8 alpha expression plasmid driven by SR alpha promoter showed that the expressed feline CD8 alpha cross-reacted with an anti-human CD8 alpha monoclonal antibody OKT8, but did not react with an anti-feline monoclonal antibody, FT-2, which is thought to recognize the feline analogue of the human T8/Leu-2 and murine Lyt-2 molecules expressed on cytotoxic/suppressor T cells.     

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.     

Chronic hepatitis C infection: influence of the viral load, genotypes, and GBV-C/HGV coinfection on the severity of the disease in a Brazilian population

The distributions of the different genotypes of the hepatitis C virus (HCV) and GBV-C virus (GBV-C/HGV) vary geographically and information worldwide is still incomplete. In particular, there are few data on the distribution of genotypes (and their relationship to the severity of liver disease) in South America. Findings are described in 114 consecutive patients from Northeast Brazil (median age 52 years, range 18-72 years) who had abnormal levels of serum aminotransferases and seropositivity for HCV RNA. The patients were recruited from an outpatient clinic between November 1997 and April 1998. Quantitative HCV RNA and GBV-C/HGV RNA estimations were carried out by double-nested polymerase chain reaction (PCR) using primers from the 5'-untranslated regions (UTRs) of the genomes. HCV genotypes were determined by restriction fragment length polymorphism (RFLP) analysis with 5'-UTR primers and by PCR with type-specific 5'-UTR primers. GBV-C/HGV-RNA genotypes were determined by RFLP with specific 5'-UTR primers and phylogenetic trees were constructed using the Neighbour-Joining and Drawtree programs. Histological features were graded and staged according to international criteria. Of the 114 patients, 35 (30.7%) patients had cirrhosis and 22 (27.8%) had mild, 51 (64.6%) had moderate, and 6 (7.6%) had severe chronic hepatitis. Median HCV viral load was 10(6) genome equivalents per millilitre (range 10(4)-10(9)/ml). Frequencies of genotypes were 5.3% type 1a, 44.7% type 1b, 3.5% type 2, 41.2% type 3, and 5.3% mixed types. GBV-C/HGV-RNA was detected in the sera of 12 (10.5%) patients and was distributed among three phylogenetic groups. There were no significant differences between patients with the predominant HCV genotypes (1b and 3) with respect to gender, age group, viral load, severity of liver disease, or coinfection with GBV-C/HGV.     

Feline CD 4 molecules expressed on feline non-lymphoid cell lines are not enough for productive infection of highly lymphotropic feline immunodeficiency virus isolates

Summary To investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells andFelis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains.     

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.