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Biomedical subjects

G Milanesi

Publications and source records attributed to G Milanesi.

At least 37 records · Page 2Linked to original sources

Trans-membrane translocation of a myristylated protein amino terminus.

Hepatitis B virus surface protein variants are described, capable of translocating to the lumenal side of the endoplasmic reticulum membrane their myristylated N-termini, as revealed by the contestual modification of N-terminal, N-linked glycosylation sites. To our knowledge, this is the first example of transmembrane translocation of a preformed protein acyl adduct. The possible significance of this event for hepatitis B virus biology is discussed.

Amino Acid Sequence↗

Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction.

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.

Acquired Immunodeficiency Syndrome↗

Identification of a human cytomegalovirus mutant in the pp150 matrix phosphoprotein gene with a growth-defective phenotype.

Following amplification by PCR of a portion of the matrix phosphoprotein pp150 gene, electrophoretic analysis revealed the simultaneous presence of two viral variants of human cytomegalovirus in the blood of a heart transplant recipient. Repeated denaturation-annealing cycles during the amplification reaction led to the formation of heteroduplex molecules with altered electrophoretic mobility. Sequence analysis of the amplification products showed the presence of a viral variant carrying an in-frame three nucleotide deletion, which caused the absence of an aspartic acid in the corresponding protein. Attempts to plaque-purify the deletion mutant were unsuccessful, suggesting that the variant was growth-defective.

Base Sequence↗

Indobufen in the prevention of thromboembolic complications in patients with heart disease. A randomized, placebo-controlled, double-blind study.

BACKGROUND: The purpose of this randomized, double-blind study was to evaluate the efficacy of indobufen, a reversible inhibitor of platelet cyclooxygenase, in the prevention of embolic events of cardiac origin. METHODS AND RESULTS: One hundred ninety-six patients with heart disease and at risk for cardiogenic embolism (90 with atrial fibrillation and 106 in sinus rhythm) were randomly assigned to receive indobufen (100 mg b.i.d.) or placebo. All patients were reexamined every 3 months for the duration of the study. The primary study end points were cerebral ischemic attack (stroke and transient ischemic attack), systemic embolism, pulmonary embolism, and fatal myocardial infarction. The median duration of treatment was 854 days in the indobufen group and 865 days in the placebo group. The frequencies of primary end points (fatal and nonfatal) were 6.1% and 17.3%, respectively, in the indobufen and placebo groups (p < 0.05) for a reduction of 65% in the risk of a primary event (indobufen/placebo relative risk, 0.35; 95% confidence limits, 0.14-0.89). Adverse drug reactions, mostly gastrointestinal or hemostasis disorders, occurred in 9.2% of indobufen-treated patients. CONCLUSIONS: The results of the study indicate that indobufen may reduce the risk of ischemic events in patients with heart disease associated with an increased risk of embolism.

Adolescent↗

Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction.

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackie-virus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5' noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothiocyanate extraction from tissue stored frozen at -80 degrees C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrylamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 10(5) to 10(6) cells starting from 1 microgram total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1.

Adult↗

Human cytomegalovirus infection of the major leukocyte subpopulations and evidence for initial viral replication in polymorphonuclear leukocytes from viremic patients.

Fourteen immunocompromised patients were examined for viremia, pp65 and p72 antigenemia, and presence of viral DNA in leukocyte fractions of polymorphonuclear leukocytes (PMNL), monocytes/macrophages (M/M), and B and T lymphocytes after purification by fluorescence-activated cell sorting. Nearly all PMNL and M/M fractions were positive for DNA and pp65 antigenemia, while p72 antigenemia was detected in 73% and 62%, respectively. The virus isolation rate was 45% from PMNL and 17% from M/M. T lymphocytes were positive for DNA in 50% of cases and for pp65 and p72 antigenemia in only 11%, while B lymphocytes were DNA-positive in 43% of samples and consistently negative for antigenemia; neither T nor B lymphocytes had virus isolated. Immediate-early (IE)1 RNA was present in 23 (85.2%) of 27 dextran-enriched DNA-positive p72-positive PMNL samples and, in sequential PMNL samples from two heart-transplanted patients, was detected during peak infection in association with p72. Thus, PMNL and M/M are the subpopulations primarily involved in HCMV infection; PMNL may undergo IE replicative events and are not merely passive carriers of phagocytized viral material.

Adult↗

Translational modulation in hepatitis B virus preS-S open reading frame expression.

A series of hepatitis B virus open reading frame (ORF) preS-S variants, including mutants in which the relative order of the in-frame start codons (AUG1, AUG2 and AUG3) and nearby sequences had been altered, was expressed both in vivo (in HepG2 hepatoblastoma cells) and in vitro (by T7 promoter-driven transcription followed by translation in a rabbit reticulocyte lysate). The ratio of the synthesis of the large, middle (M) and major (S) proteins or their chimeric counterparts was analysed to study the translational regulation of ORF expression. As expected on the basis of the ribosome scanning model, the AUG sequence context was found to be a prominent factor in determining the different translational behaviour of the two preS-S-specific mRNAs of 2.4 kb (predominantly translated from AUG1) and 2.1 kb (which includes AUG2 and/or AUG3 and can be translated from either). Results from both experimental systems suggested that initiation at internal AUGs in the 2.4 kb RNA is possible. In experiments in vitro, preS-S mutants bearing lesions in a region 5' to AUG2, which has been implicated in AUG2/AUG3 cis repression, showed no increase in the utilization of internal AUGs. In addition, the chimeric envelope polypeptides produced in transfected HepG2 cells in this study were informative with respect to preS-mediated endoplasmic retention: replacement of the preS2 N terminus with that from preS1 generated a chimeric M protein that was glycosylated within the putative preS1 retention sequence ad was not secreted. Thus, the preS1 retention sequence most likely acts inside the lumen of endoplasmic reticulum and its function is insensitive to glycosylation. A similar element might be active at the N terminus of M protein.

Base Sequence↗

Development and clinical significance of a diagnostic assay based on the polymerase chain reaction for detection of human cytomegalovirus DNA in blood samples from immunocompromised patients.

The presence of human cytomegalovirus (HCMV) DNA in blood was investigated by the polymerase chain reaction (PCR) in 293 blood samples from 86 immunocompromised patients. Of the 86 patients, 23 underwent clinical and virologic follow-up for HCMV infection. In parallel, blood samples were examined for viremia and antigenemia. Concordant results between PCR and assays for viremia and antigenemia were obtained on 124 positive and 110 negative samples, with an overall concordance of 79.8%, while 59 samples (most from patients with HCMV infection) were positive by PCR alone. PCR is a new powerful tool for detection of HCMV infections in blood samples from immunocompromised patients. However, its clinical significance appears to be restricted to the indication of a risk of reactivation of HCMV infection.

Antigens, Viral↗

Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leukocytes from AIDS patients with human cytomegalovirus viraemia.

Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

Acquired Immunodeficiency Syndrome↗

Monitoring of human cytomegalovirus infections and ganciclovir treatment in heart transplant recipients by determination of viremia, antigenemia, and DNAemia.

Fourteen heart transplant recipients were monitored for human cytomegalovirus (HCMV) infection based on determination of antigenemia, viremia, and DNAemia (by polymerase chain reaction [PCR]) in peripheral blood polymorphonuclear leukocytes (PMNL). Three patients had symptomatic primary, 10 had recurrent (3 asymptomatic), and 1 (seronegative) had no HCMV infection. Severe clinical symptoms appeared when levels of viremia/antigenemia were greater than 50 infected PMNL/2 x 10(5) cells examined. Of 200 blood samples examined, 93 (46.5%) were positive for viremia/antigenemia and DNAemia, whereas 48 (24.0%) were positive for DNAemia only; 59 (29.5%) were negative in all assays. Follow-up of HCMV infections in heart transplant recipients showed that PCR can detect viral appearance in blood 7-10 days earlier than assays for antigenemia/viremia. On the other hand, viral disappearance from blood, as assessed by PCR, occurred weeks or months later than revealed by other assays. Detection of virus by PCR only was never associated with overt HCMV-related clinical symptoms. Of the 8 symptomatic patients treated with ganiclovir, 2 became PCR-negative at the end of treatment and 1 cleared virus from blood in the following weeks, whereas 5 showed persistent or recurrent infection.

Antigens, Viral↗

Thromboxane production by platelets during tumor cell-induced platelet activation.

We have evaluated in a homologous system the mechanisms of platelet activation by cells isolated from fresh human tumor tissues and the role of thromboxane B2 (TxB2) generation in this process. Thirty-eight of the 46 tumor tissues considered showed a high platelet-aggregating activity, with no particular distribution in any specific tumor type. Apyrase caused a nonsignificant reduction in the aggregation response, hirudin did not change it, while iodoacetic acid or p-hydroxymercuriphenylsulfonate, specific cysteine proteinase inhibitors, significantly reduced the platelet-aggregating capacity of these tumor cells. In 9 colon carcinomas and in 8 breast carcinomas the levels of TxB2 produced by platelets after addition of tumor cells were measured: tumor cell-induced platelet aggregation was accompanied by a significant production of the metabolite; indobufen, a cyclooxygenase inhibitor, significantly reduced aggregation and particularly TxB2 production, while the drug had no effect on both parameters if preincubated with tumor cells only. These data suggest that cells isolated from different human tumor tissues activate platelets through the activity of tumor-associated cysteine proteinase(s); platelet aggregation by tumor cells is largely dependent on arachidonic acid metabolism in platelets, while such metabolism in tumor cells does not play a significant role.

Adult↗

Fine specificity of human antibody response to the PreS1 domain of hepatitis B virus.

The preS1 domain of hepatitis B virus envelope proteins contains a site of attachment to the hepatocyte membrane that has been shown to evoke virus-neutralizing antibodies. Using synthetic peptides, we have examined kinetics and specificity of the anti-body response to preS1 during acute and chronic HBV infection. Antibodies against two continuous B cell epitopes, p (21-32) and p (32-47), which overlap with the virus receptor for hepatocytes, were detected in 17 (28%) and 28 (47%) patients, respectively, of 60 patients who were tested during acute hepatitis B. Serial testing demonstrated these anti-preS1 specificities in more than 50% of patients who became virus free. By contrast, five patients with chronic evolution of hepatitis B and 61 of 66 patients with an established chronic HBV infection were negative, independent of serological profile and liver disease activity. Fifteen (22.7%) patients with chronic hepatitis B were positive for antibody to the C-terminus p (94-117) preS1 sequence that, unlike the acute-phase anti-(21-32) and anti-(32-47) reactivities, did not behave as a virus-precipitating antibody. Acute-phase sera were found to also contain virus-precipitating antibodies directed against conformational preS1 epitopes. These results indicate that the preS1 site, which contains the binding activity for the hepatocyte membrane, elicits an early antibody response during acute hepatitis B. A defect in such antibody repertoire may participate in the chronicity process as a result of continuing reinfection of hepatocytes by circulating virions.

Acute Disease↗

Major antigenic domain recognized by monoclonal antibodies maps within the carboxy-terminal moiety of a recombinant human immunodeficiency virus-1 p24 protein.

Antigenicity in mice of a recombinant polypeptide including the complete amino acid sequence of mature human immunodeficiency virus type 1 p24 protein was studied by induction of monoclonal antibodies (MAbs). A panel of nine recloned hybridomas secreting MAbs with anti-p24 reactivity was isolated and further characterized. Competitive inhibition experiments suggested that the MAbs could be grouped into four epitopic classes corresponding to at least two distinct determinants. Analysis of reactivity to recombinant p24 deletion variants indicated that all the recognized epitopes are localized within a carboxy-terminal domain (amino acids 168-208) which should be largely exposed in recombinant as well as authentic antigen. Lack of response to N-terminal and central portions of p24 suggests that the antigenicity of those regions in the natural polypeptide is strongly conformation-dependent.

Antibodies, Monoclonal↗

Isolation in Europe of 69 M-like (serotype 8) human rotavirus strains with either subgroup I or II specificity and a long RNA electropherotype.

During an epidemiological study on the prevalence of human rotavirus (HRV) serotypes 1-4 in Europe, we found that some strains could not be typed. However, when a monoclonal antibody directed to serotype 8 HRV was included in the typing assay, we detected seven 69 M-like (serotype 8) strains, six from Finland and one from Italy. The previously reported serotype 8 HRV strains, 69 M, B 37, and B 38 isolated in Indonesia, were of subgroup I specificity and presented a peculiar "super short" RNA electropherotype. In contrast, all the seven European strains possessed a long RNA pattern, and one of them had subgroup II specificity. Three of these strains were adapted to growth in cell cultures and were further characterized by neutralization and by Northern blot hybridization. They appeared to be closely related to serotype 8 HRV strain 69 M by neutralization, but showed partial homology with several human and animal strains by hybridization. The epidemiological importance of these serotype 8 strains circulating in Europe should be investigated, in view of their possible inclusion in a rotavirus vaccine.

Blotting, Northern↗

Human cytomegalovirus viraemia in HIV-1-seropositive patients at various clinical stages of infection.

Eighty-two HIV-1-seropositive subjects were examined for the presence and quantification of human cytomegalovirus (HCMV) in peripheral blood polymorphonuclear leukocytes (PMNL) by polymerase chain reaction, culture and immunofluorescence in order to investigate the relationship between viraemia and immunosuppression. Patients were divided into three groups: (1) asymptomatic subjects with greater than 400 x 10(6)/l CD4 lymphocytes (n = 30); (2) asymptomatic subjects with less than 400 x 10(6)/l of CD4 lymphocytes and zidovudine (n = 20), and (3) AIDS-related complex (ARC)/AIDS patients on zidovudine (n = 32). Evidence of HCMV infection in circulating PMNL was found in 15 out of 29 ARC/AIDS patients examined (51.7%), whereas no infection was detected among the 50 asymptomatic HIV-1-seropositive subjects. HCMV-related symptoms were found only where the number of infected PMNL was greater than 50 per 2 x 10(5) cells.

AIDS-Related Complex↗

Serotype 3 human rotavirus strains with subgroup I specificity.

During an epidemiological study on human rotavirus (HRV) infections in Italy, three subgroup I strains not associated with serotype 2 reactivity were detected. All three strains were serotype 3, each with a distinct RNA pattern showing fast-moving tenth and eleventh segments (long electropherotype). Following successful adaptation to growth in cell cultures, the serotype 3 strains (MZ58, PCP5, and PA710) were further characterized by neutralization and by RNA-RNA (Northern blot) hybridization. Antiserum to reference HRV strain YO (subgroup II, serotype 3), as well as a monoclonal antibody to VP7 of YO neutralized, at comparable titers, the homologous virus, the three unusual HRV strains, and two reference simian strains (SA11 and RRV-2, both subgroup I, serotype 3), whereas SA11 antiserum and a monoclonal antibody to VP7 of SA11 neutralized simian strains more efficiently. However, antiserum to PCP5 neutralized the three unusual isolates and the simian strains at significantly higher titers than it did with reference strain YO. With 32P-labeled RNA from MZ58 as a probe, a high degree of homology was detected by Northern blot hybridization with strains PCP5, PA710, SA11, and UK (bovine rotavirus) at the level of several segments and with strain YO only at the level of genes 7 to 9. Conversely, labeled RNA of strain YO hybridized extensively with Wa (subgroup II, serotype 1 HRV strain) but only at the level of genes 7 to 9 with MZ58, PCP5, PA710, SA11, and UK. Finally, the labeled SA11 probe hybridized at the level of RNA segments 1 to 3 and 6 to 11 to the three unusual strains. These findings suggest that the unusual subgroup I, serotype 3, strains isolated from humans are more likely to be animal rotaviruses rather than natural reassortants between different HRV strains.

Blotting, Northern↗

Identification of human cytomegalovirus isolates by the polymerase chain reaction.

Fifty human cytomegalovirus (HCMV) isolates were recovered from different clinical specimens (buffy coat, throat washing and urine) obtained from fifty patients (23 AIDS patients, 20 heart transplant recipients, 1 bone marrow transplant recipient, 2 newborns with congenital HCMV infection and 4 immunocompetent individuals with acute HCMV infection). The isolates were previously identified by immunological methods and then examined for identification by the polymerase chain reaction. In parallel, reference HCMV strains as well as other human members of the Herpesviridae family (reference and wild strains) were examined as controls. Two pairs of primers relevant to the immediate-early and late regions of HCMV DNA, respectively, were used. The DNA amplification product was highly specific; in addition, all fifty HCMV isolates were amplified by both pairs of primers and thus identified as HCMV. These preliminary results show that selected pairs of primers are able to amplify DNA regions conserved enough to allow virus identification among a large number of HCMV strains. In addition, they are highly promising in view of the use of PCR for direct detection of HCMV DNA in clinical samples.

Cytomegalovirus↗