Giovanni Mantero

Gene expression of cardiac beta1-adrenergic receptors during the development of hypertension in spontaneously hypertensive rats

Objective: To study adrenergic receptors in the heart tissues of genetically hypertensive rats by evaluating the gene expression and the membrane protein density of ß1-adrenergic receptors using steady-state messenger RNA (mRNA) levels and a radioligand binding assay, respectively. Design: We compared prehypertensive (5-week-old) and early-hypertensive (13-week-old) spontaneously hypertensive rats (SHR) with age-matched Wistar-Kyoto (WKY) normotensive control rats Methods: Polyadenylated RNA was extracted from individual hearts and analysed by the slot-blot technique using a ß1-adrenergic receptor complementary DNA probe.ß1 -Adrenergic receptors in myocardial membranes were studied by radioligand binding assay using [125l]-cyanopindolol and the ß1- and ß2-selective antagonists CGP207.12A and IC1118.551, respectively Results: ß1-Adrenergic receptor mRNA levels were slightly higher, and membrane protein density was similar in prehypertensive SHR and age-matched WKY rats. However, both ß1-adrenergic receptor mRNA levels andß1-adrenergic receptor density were lower in the hypertensive SHR than in the control rats. ß1-Adrenergic receptor mRNA was significantly reduced in older rats of both strains, and this reduction was most evident in the SHR Conclusions: The absence of downregulation of ß1-adrenergic receptors in young SHR, despite published data indicating a higher cardiac noradrenaline turnover than in WKY rats, may suggest that the cardiac hyperadrenergic activity observed in prehypertensive SHR is maintained, at least in part, by the participation of peripheral, postsynaptic component(s) involving ß1-adrenergic receptor dysregulation. In addition, the present data suggest that the previously reported evidence of an age-related decrease in cardiac ß1-adrenergic receptors in rats may be determined at the transcriptional level

GB virus C Infection in patients with type II mixed cryoglobulinemia

Type II mixed cryoglobulinemia is characterized by purpura, arthralgias, weakness, glomerulonephritis, peripheral neuropathy, and other manifestations of systemic vasculitis. Its pathogenesis probably depends on cryoprecipitable circulating immunocomplexes consisting of polyclonal IgG and monoclonal IgM, the latter having rheumatoid factor activity [1-3]. Although mixed cryoglobulinemia is commonly associated with hepatitis C virus (HCV) [4-8], a variable proportion of cryoglobulinemic patients does not have markers of infection with any known viral agent. We investigated the possible role of GB virus C (GBV-C), a newly discovered RNA virus that shares some sequence homology with HCV [9-11], in type II mixed cryoglobulinemia. Methods We studied 58 patients with type II mixed cryoglobulinemia that was not associated with hematologic malignant conditions, autoimmune disorders, or infectious diseases other than chronic viral hepatitis. One hundred forty-five volunteer blood donors served as healthy controls. We also studied a second control group that included 20 noncryoglobulinemic blood donors who were age-matched with cryoglobulinemic patients and were positive for antibodies to HCV. All patients were tested for liver and kidney function according to standard methods. The serum concentration of immunoglobulins, rheumatoid factor activity, and C3 and C4 components were measured by using routine nephelometric assays. Cryoglobulins were measured as the protein concentration of isolated cryoprecipitate and were characterized by using standard immunoelectrophoresis and immunofixation procedures. Antibodies to HCV were measured with a second-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, Illinois). To search for enrichment of antibodies to HCV in cryoglobulins, the isolated and washed cryoprecipitate was resuspended in a volume of phosphate-buffered saline equal to the volume of the original serum. The activity of the antibodies to HCV per mg of IgG was subsequently measured, as reported elsewhere [12], in serial fivefold dilutions of both cryoprecipitate and supernatant. Serum specimens were tested for HCV RNA with a reverse-transcription polymerase chain reaction (PCR) assay, as described elsewhere [5]. The HCV viral load was measured in both cryoprecipitate and supernatant by using a quantitative reverse-transcription PCR assay (Amplicor HCV Monitor, Roche Diagnostic Systems, Inc., Branchburg, New Jersey), according to the manufacturers instructions. The GBV-C genome was detected by using a reverse-transcription PCR method developed in our laboratory [13]. Total RNA was extracted from 100 L of serum and was reverse transcribed by using random esanucleotides as primers. Complementary DNA was amplified through 45 cycles by using a pair of primers spanning a 161-base pair sequence in the conserved GBV-C helicase region. An aliquot of the PCR product was hybridized to an internal probe with a DNA enzyme immunoassay [14]. The optical densities of this immunoassay were used to measure GBV-C RNA levels in serial tenfold dilutions of cryoprecipitate-supernatant pairs. Twenty-seven cryoglobulinemic patients received recombinant interferon-alpha2b, and 8 received human lymphoblastoid interferon-, 4.5 to 6 million U three times weekly for 6 months and then 3 million U three times weekly for 6 to 12 months. For our purposes, virologic response was defined as the disappearance from serum of GBV-C RNA, HCV RNA, or both as a result of interferon- therapy. A clear alleviation of the signs and symptoms of cryoglobulinemia, along with a reduction of 50% or more in serum cryoglobulin levels, was considered a clinical response. Statistical analyses were done by using the two-sample Wilcoxon test, the chi-square test, or the Wilcoxon matched-pairs signed-rank test, as appropriate. All P values are two tailed, and statistical significance was set at the 0.05 level. Results We detected GBV-C RNA in 23 of the 58 patients with type II mixed cryoglobulinemia (40%); 20 of the 23 were also infected with HCV. We found HCV RNA alone in 28 patients. We detected GBV-C RNA in 1 of 145 unselected blood donors; thus, the prevalence of this virus was much lower among unselected blood donors than among cryoglobulinemic patients (0.7% compared with 40%; P < 0.001). Similarly, only a small proportion of blood donors was positive for antibodies to HCV compared with cryoglobulinemic patients (2.7% compared with 81%; P < 0.001). In contrast, the prevalence of GBV-C infection was similar in cryoglobulinemic patients positive for antibodies to HCV and age-matched blood donors positive for these antibodies (43% compared with 35%; difference, 8% [95% CI, 18% to 33%]; P > 0.2). As indicated above, 20 of 58 cryoglobulinemic patients (34%) were co-infected with GBV-C and HCV. No obvious difference was found when the demographic, clinical, and laboratory features of these patients were compared with those of patients infected with HCV alone. However, the patients with GBV-C and HCV co-infection tended to have slightly higher levels of cryoglobulins (P = 0.18) and rheumatoid factor activity (P > 0.2) and lower concentrations of C4 (P = 0.05). We detected GBV-C RNA alone in the serum specimens of three cryoglobulinemic patients who seemed to be clinically similar in all respects to patients infected with HCV and patients without demonstrable HCV infection. We measured the concentration of viral markers in samples of cryoprecipitate and supernatant from 10 patients. Results in patients who were co-infected with HCV and GBV-C are shown in the (Table 1). The concentration of antibodies to HCV, as expressed by the antibody activity per mg of IgG, was clearly higher in the cryoprecipitate than in the supernatant (P = 0.022). Similarly, the HCV RNA level was consistently greater in the cryoprecipitates than in the supernatants obtained from the same patients (P = 0.036). In contrast, GBV-C RNA levels seemed to be lower in cryoprecipitate than in supernatant in 4 of the 5 patients studied in this group (P > 0.2). Finally, GBV-C RNA was undetectable in all three cryoprecipitate specimens obtained from patients infected with GBV-C alone. Table 1. Quantitative Evaluation of Antibodies to Hepatitis C Virus, Hepatitis C Virus RNA, and GB Virus C Supernatant from Cryoglobulinemic Patients Co-Infected with Hepatitis C Virus and GB Virus C* Twenty-eight of 35 patients receiving interferon- responded by clearing circulating GBV-C, HCV, or both. The clinical condition did not improve in either of the two patients in whom GBV-C RNA but not HCV RNA disappeared from serum. In contrast, a clinical response was evident in 5 of 6 patients who cleared HCV RNA alone and 6 of 7 patients who cleared both HCV RNA and GBV-C RNA. Of the 18 patients with isolated HCV infection, 13 had both a virologic and a clinical response. Discussion Infection with GBV-C or its homologue, hepatitis G virus, has been demonstrated in many patients with such conditions as acute and chronic hepatitis, intravenous drug use, hemophilia, aplastic anemia, and maintenance hemodialysis [9-11, 15-18]. However, the pathogenic role of these viruses has not been established. We show that GBV-C infection, usually associated with HCV, is frequently found in patients with type II mixed cryoglobulinemia. It is interesting that the prevalence of GBV-C infection is equally high in healthy blood donors and cryoglobulinemic patients who are positive for antibodies to HCV. This suggests a common route of transmission for both viruses and may be taken as evidence against a causal role for GBV-C in type II mixed cryoglobulinemia. Moreover, because risk factors for parenteral exposure were uncommon in our patients with cryoglobulinemia, it is likely that most of these patients had community-acquired infection with cotransmitted GBV-C and HCV. No clinical or biochemical characteristic clearly distinguished the cryoglobulinemic patients with GBV-C infection from those who were not infected. Nonetheless, the tendency of cryoglobulinemic patients with GBV-C and HCV co-infection to have more evident immunologic abnormalities than patients infected by HCV alone suggests a pathogenic role for GBV-C. This possibility is also supported by the fact that GBV-C was the only agent identified in three cryoglobulinemic patients. Because mixed cryoglobulinemia is commonly considered to be a vasculitic process mediated by cryoprecipitable circulating immunocomplexes, several authors have sought markers of HCV infection in cryoprecipitate; some have found increased concentrations of antibodies to HCV, HCV RNA, or both [5-8]. In our patients with GBV-C and HCV co-infection, we found that concentrations of both antibodies to HCV and HCV RNA were consistently higher in cryoprecipitates than in supernatants, whereas levels of GBV-C RNA were lower in most cryoprecipitates obtained from the same patients. Moreover, GBV-C RNA was not detected in the cryoprecipitates of any of the three patients with isolated GBV-C infection. Recent therapeutic trials [12, 19] indicate that the effectiveness of interferon- in HCV-associated mixed cryoglobulinemia is related to the drugs antiviral activity. We were able to study the effects of interferon- in patients co-infected with GBV-C and HCV. In these patients, we found that response with respect to GBV-C infection, expressed by the disappearance of viral genome from serum, was as frequent as response with respect to HCV infection. However, our most important finding was that clinical response was related to the virologic response with respect to HCV but not GBV-C infection. Thus, our results suggest that GBV-C does not have a primary role in the pathogenesis of type II mixed cryoglobulinemia in patients co-infected with GBV-C and HCV. Although it seems unlikely, the situation could be different in cryoglobulinemic patients with isolated GBV-C infection. These patients formed a small

HLA-DQB1 typing of north east Italian IDDM patients using amplified DNA, oligonucleotide probes and a rapid DNA-enzyme immunoassay (DEIA)

We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.

Detection of human chromosomes in somatic cell hybrids by PCR analysis

The detection of human chromosomes in somatic cell hybrids is usually made by chromosomal analysis, Southern blot analysis with human probes, and starch-gel electrophoresis of isoenzymes. We describe here a new, quick, and very efficient method to detect human chromosomes in somatic cell hybrids between human and rodent (rat and mouse) cells. The method is based on the polymerase chain reaction to promote amplification of human DNA, using primers derived from localized genes or DNA fragments from each human chromosome.

Detection of BCR/ABL transcripts in chronic myeloid leukaemia by polymerase chain reaction and DNA enzyme immunoassay: a DNA probe assay without DNA labelling

The detection of the t(9;22) translocation, typical of chronic myeloid leukaemia (CML), can be accomplished by cytogenetical detection of Philadelphia (Ph1) chromosome or by molecular analysis of the bcr/abl fusion gene with nucleic acid probes after amplification by polymerase chain reaction (PCR). PCR‐based approaches are now widely used for follow up of CML patients during therapy or after bone marrow transplantation (BMT). We describe here a microtitre, colorimetric assay (DNA Enzyme Immunoassay, DEIA) for analysis of t(9;22) translocation after enzymatical amplification of RNA from CML patients. This assay is based on the use of a monoclonal antibody specifically reacting with double stranded DNA, i.e. with hybridized DNA. The assay represents a nonisotopic alternative to other current hybridization assays and requires no modifications of primers, probe or target DNA.