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M P Landini

Publications and source records attributed to M P Landini.

At least 19 recordsLinked to original sources
PubMed2003

Human cytomegalovirus glycoprotein N (gpUL73-gN) genomic variants: identification of a novel subgroup, geographical distribution and evidence of positive selective pressure.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

Amino Acid Sequence↗
PubMed2002

Immunoelectron microscopy analysis of HCMV gpUL73 (gN) localization.

Human Cytomegalovirus (HCMV) UL73 encodes for a polymorphic structural glycoprotein, gpUL73(gN), conserved among herpesviruses. This study analyzed the intracellular and intraviral localization of gpUL73 by immunoelectron-microscopy comparing the reactivity of two different antibodies. We found that gN is an envelope component of the mature viral particle with at least a portion exposed at the virus surface and another at the internal side of the envelope. Furthermore, gpUL73 is also present in the matrix of dense bodies and "black holes". These results, as well as immunoblotting analysis, suggest that the two antibodies recognize different forms, fully processed or unprocessed, of gpUL73-gN.

Antibodies, Viral↗
PubMed2002

Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein.

Human cytomegalovirus (HCMV) UL53 belongs to a family of conserved herpesvirus genes. In this work, the expression and localization of the UL53 gene product was analysed. Results obtained showed that pUL53 is a new structural protein. In infected human fibroblasts, pUL53 localizes in cytoplasmic perinuclear granular formations together with other structural viral proteins. In the nucleus, pUL53 forms patches at the nuclear periphery and co-localizes with lamin B at the internal nuclear membrane level. Immunoelectron microscopy studies have disclosed that nuclear pseudo-inclusions are labelled, whereas nucleocapsid formations within the intranuclear skein are negative. Furthermore, the mature virus particle maintains pUL53 at its tegumental level. These data suggest that pUL53 could be involved either in nucleocapsid maturation or in the egress of nucleocapsids from the nucleus to the cytoplasm through the nuclear membrane, a role compatible with the function hypothesized for UL31, its positional homologue in herpes simplex virus type 1.

Cytomegalovirus↗
PubMed2001

Clinical evaluation of a new recombinant antigen-based cytomegalovirus immunoglobulin M immunoassay in liver transplant recipients.

BACKGROUND: Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality among transplant recipients. Monitoring transplant recipients by CMV IgM serology has been questioned by several studies due to the reported insensitivity of serologic tests relative to antigen detection methods. METHODS: In this retrospective study, we have evaluated the performance of the new recombinant antigen-based Abbott AxSYM CMV IgM assay and compared it with CMV culture technique in a cohort of 40 liver transplant recipients who did not receive antiviral prophylaxis. RESULTS: The sensitivity, specificity, and positive and negative predictive values for detection of CMV disease by the AxSYM CMV IgM assay were 90.0%, 60.0%, 69.2%, and 85.7%, respectively, and by culture the values were 100%, 55.0%, 69.0%, and 100%, respectively. Detection of CMV IgM occurred before or at the time of CMV disease in only R+ recipients. CONCLUSION: Although this assay is a sensitive test for CMV-specific IgM, detection of CMV IgM preceded detection of virus by culture in patients only when the liver transplant recipient was CMV immune before transplantation (R+).

Antibodies, Viral↗
PubMed2001

Complete replication of human cytomegalovirus in explants of first trimester human placenta.

Tissue integrity and viability of first trimester placenta explants were obtained in culture for 3 weeks. Explants were infected with human cytomegalovirus (HCMV), several cycles of HCMV replication were obtained and the progression of the infection was observed within a tissue that maintains its normal cellular organization. In agreement with recent clinical data, 3 weeks were necessary for the virus to colonize the placenta fully. Complete HCMV replication was observed in trophoblasts, followed by subsequent transmission of the infection to the stromal fibroblasts and fetal endothelial capillary cells. Viral DNA replication was monitored and the production of infectious viral progeny documented.

Antigens, Viral↗
PubMed2001

Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1.

Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1-positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD.

Amino Acid Sequence↗
PubMed2001

gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes.

Clinical isolates of human cytomegalovirus (HCMV) show differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent infections, characteristics that are thought to be related to genomic variation among strains. This work analysed the genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73. This ORF encodes the envelope glycoprotein gpUL73 (gN), which associates in a high molecular mass complex with its counterpart, gM, and induces a neutralizing antibody response in the host. Detailed sequence analysis of ORF UL73 and its gene product from clinical isolates and laboratory-adapted strains shows that this glycoprotein is highly polymorphic, in the N-terminal region in particular. gpUL73 hypervariability is not randomly distributed, but the identified genomic variants are clearly clustered into four distinct genotypes (gN-1, gN-2, gN-3 and gN-4), which are not associated with the gB subtype.

Amino Acid Sequence↗
PubMed2001

Evaluation of the Abbott AxSYM cytomegalovirus (CMV) immunoglobulin M (IgM) assay in conjunction with other CMV IgM tests and a CMV IgG avidity assay.

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.

Antibodies, Viral↗
PubMed2001

Evaluation of five commercial enzyme immunoassays for the detection of human cytomegalovirus-specific IgM antibodies in the absence of a commercially available gold standard.

In the recent years the number of commercially available immunoassays for the detection of human cytomegalovirus (HCMV)-specific immunoglobulin M (IgM) antibodies has rapidly increased. The aim of the present study was to evaluate five commercial immunoassays for the serological diagnosis of HCMV-infection. These methods, namely the IMx CMV IgM assay, the AxSYM CMV IgM assay (both Abbott), the Gull CMV IgM, the CMV-IgM-ELA test PCS Medac and the Biotest Anti-HCMV recombinant IgM ELISA, were compared for their diagnostic effectiveness and interference with substances eventually producing cross-reactions with HCMV-IgM (Epstein-Barr-virus (EBV)-IgM, rheumatoid factor (RF)). In addition, repeated measurements on samples from kidney and heart transplant recipients with active HCMV infection were examined to compare the temporal development of the HCMV-IgM measured with the five assay systems. Since there is no commercially available gold standard, it was assumed that the true classification, of whether the patient sample is HCMV-IgM positive or negative, was unknown. Hence sensitivity and specificity were assessed based on a maximum likelihood approach using a "latent class" model. The cross-reactions were quantified by a Bayesian statistical model using prior information for the expected prevalences in the EBV-IgM and rheumatoid factor sample groups. The results of the study demonstrated that there are great differences in sensitivity and specificity as well as in cross-reactions with EBV-IgM and RF between the tested ELISAs.

Adolescent↗
PubMed2001

Neonatal aortic arch thrombosis as a result of congenital cytomegalovirus infection.

Thrombotic disease is rare in neonates. The main risk factors at this age are perinatal asphyxia, maternal diabetes, sepsis, polycythemia, dehydration, a low cardiac output, and in primis the catheterization of central lines. Another important risk factor is inherited thrombophilia. Arterial thrombosis is even more rare than venous thrombosis and less related to most of the risk factors listed above; it occurs more frequently in the iliac, femoral, and cerebral arteries but very rarely in the aorta. Most of the described cases of aortic thrombosis are associated with the catheterization of an umbilical artery and involve the descending tract and the renal arteries; very few relate to the ascending tract and the aortic arch. The possible role of virus-induced primary vascular endothelium damage in the etiopathogenesis of neonatal arterial thrombosis has been previously hypothesized. Herpesviruses, particularly human cytomegalovirus (HCMV), can infect endothelial cells and directly damage intact vascular endothelium, altering its thromboresistant surface as a result of procoagulant activity mediated by specific viral surface phospholipids, necessary for the coagulation enzyme complex assembly that leads to thrombin generation. We describe a case of congenital aortic arch thrombosis. The clinical, laboratory, and virologic pictures; the anatomopathologic findings (fully compatible with viral infection); the detection of HCMV in various tissues (including the aorta); and the absence of other causes of aortic thrombosis make it possible to attribute the case to a severe congenital HCMV infection with multiple organ involvement, after the primary infection of the mother. The hemostatic system disorders and hemodynamic disturbances related to viral cardiac damage explain the clinical features of the case and indicate that congenital HCMV infection should be included among the causes of neonatal aortic thrombosis.

Aorta, Thoracic↗
PubMed2001

New developments in the diagnosis of maternal and congenital CMV infection.

Recent advances in the screening of pregnant women with Cytomegalovirus (CMV) IgM, CMV IgG and CMV IgG avidity serologic tests, has led to a more accurate diagnosis of CMV infection. When serologic screening is performed early in gestation, it is possible to identify those women at risk of intrauterine transmission of the virus, i.e., those women with a primary CMV infection, who should be enrolled in prenatal diagnosis. The use of quantitative PCR on amniotic fluid from pregnant women at 21-22 weeks of gestation in prenatal diagnosis is an effective diagnostic tool to distinguish between CMV infection and CMV disease in the fetus and newborn. Quantitative PCR on peripheral blood leukocytes from CMV infected newborns can be used to monitor viral load, especially during treatment with ganciclovir. These advances in serology and quantitative virology should lead to more accurate diagnosis of maternal and congenital CMV infection.

Antibody Affinity↗
PubMed2001

[Amino acid sequence analysis of peptides that can react to the nuclear localization signals of human cytomegalovirus PPUL44].

OBJECTIVE: To find the peptides that have strong binding ability to the nuclear localization signals (NLS) of human Cytomegalovirus (HCMV) PPUL44 protein, and to analyze their amino acid sequences. METHODS: Peptide clones that have binding ability to the NLSs were selected from a random peptide display library by using synthesized HCMV PPUL44 NLSA and NLSB respectively. The DNA sequences of these clones were detected by using "ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit". The amino acid sequences of the clones were analyzed for their homology, and the homologous sequences were compared with known sequences in protein bank. RESULTS: The peptides that can bind to the HCMV PPUL44 NLSA(named as bNLSApep) have higher homologous amino acid sequence to importin alpha subunit, the bNLSApep48 sequence AVVTPVLTEILK is more similar to the sequence of the importin alpha subunit Arm 7 from amino acid 18 to 29, ANIFPVLTEILQ; The bNLSBpep 39 is similar to the sequence of all 8 Arm regions of importin alpha subunit from amino acid 10 to 21. CONCLUSION: It is possible that the HCMV PPUL44 NLSA is a specific recognition site by importin alpha subunit, it can bind to the Arm 7 region of importin alpha subunit; HCMV PPUL44 NLSB is a non-specific recognition site for importin alpha subunit. This study provided an experimental data for the proposal that the Arm repeat region of importin alpha subunit is the binding region of NLS.

Amino Acid Sequence↗
PubMed2000

Abstension from treatment of low-level pp65 cytomegalovirus antigenemia after liver transplantation: a prospective study.

BACKGROUND: Ganciclovir is a highly effective and relatively safe drug to treat cytomegalovirus (CMV) infection in liver transplant patients; CMV resistance to ganciclovir is progressively emerging due to the extensive use of the drug in transplant and AIDS patients; CMV pp65 antigenemia allows early diagnosis of CMV infection and quantitation of the viral load; preemptive antigenemia-guided therapy of CMV infection can prevent CMV disease but the threshold of antigenemia value above which treatment has to be instituted is unclear. METHODS: To demonstrate the safety of abstention from preemptive treatment in the presence of low levels of antigenemia 77 consecutive liver transplant recipients were prospectively evaluated. Antigenemia was tested twice a week from transplantation until discharge, then once a week until the third postoperative month. In absence of risk factors for CMV disease, namely donor positive/recipient negative CMV serology, treatment with antibodies to lymphocytes and retransplantation, only patients with antigenemia of more than 50 or symptoms possibly related to CMV infection had preemptive treatment. RESULTS: A total of 32 patients had at least one positive antigenemia test with a value less than 50; 22 (68.7%) spontaneously cleared the virus, 3 were treated with i.v. ganciclovir for the presence of fever, and the other 7 (21,8%) progressed to values of antigenemia of more than 50 and were treated even if asymptomatic. No CMV disease was observed in these patients. CONCLUSION: CMV antigenemia less than 50 in liver transplant recipients with low and intermediate risk for CMV disease does not mandate preemptive ganciclovir treatment. Close surveillance with repeated determination of antigenemia until its negativization and careful clinical and laboratory monitoring is advisable.

Adult↗
PubMed2000

Relationship between IgM antibody to human cytomegalovirus, virus load, donor and recipient serostatus, and administration of methylprednisolone as risk factors for cytomegalovirus disease after liver transplantation.

A retrospective study was performed on a selected cohort of 40 liver transplant recipients derived from the previous prospective follow-up of 162 liver transplant patients. The criterion for selection of this cohort was the presence of human cytomegalovirus (HCMV) DNAemia after transplantation, as determined by qualitative polymerase chain reaction (PCR). These 40 patients were followed longitudinally by quantitative PCR and by the new recombinant antigen-based AxSYM immunoassay for IgM to HCMV. The detection of IgM to CMV after transplantation was significantly associated with the development of HCMV disease in patients who had evidence of active HCMV replication in the blood by PCR (P=.01). On the basis of multivariate logistic regression analyses, the maximum titer of IgM detected after transplantation was a risk factor that was independent of augmented methylprednisolone and donor seropositivity. However, in multivariate analyses, elevated virus load continued to be the predominant risk factor for progression to HCMV disease.

Anti-Inflammatory Agents↗
PubMed2000

Laboratory signs of acute or recent cytomegalovirus infection are common in cirrhosis of the liver.

Human cytomegalovirus (CMV) is an ubiquitous pathogen that can cause severe and often fatal infections in immunocompromised patients. Patients with cirrhosis often show various degrees of impaired cellular immunity that could lead to acute CMV reactivation. The aim of the present study was to determine whether laboratory findings of active CMV infections are common in patients with cirrhosis. Fifty-five patients with cirrhosis were studied for acute CMV infection by virological (antigenemia and quantitative polymerase chain reaction in polymorphonuclear leukocytes) and serological (detection of anti-CMV IgM by immunoblot) methods. The same tests were carried out on 50 blood donors and on 20 chronic hepatitis patients, considered as control populations. Acute or recent CMV infection had occurred in 31 (56%) of 55 patients with cirrhosis, whereas only 1 out of 20 (5%) patients with chronic non-cirrhotic liver disease and none of the 50 blood donors had laboratory signs of active CMV infection. The difference between patients with cirrhosis and the control groups was significant (P < 0.001, chi(2) test). CMV in patients with cirrhosis was not related to age, gender, hepatitis C virus infection or hepatocellular carcinoma. There was no significant correlation between impairment of liver function and the presence of active CMV infection. Patients with cirrhosis should be considered at risk for CMV infection, that seems to be mild and asymptomatic.

Acute Disease↗
PubMed2000

pUL25 immunolocalization in human cytomegalovirus-infected and gene-transfected cells.

By means of confocal and electron microscope immunocytochemistry we have studied the localization of a recently described structural protein (pUL25) of human cytomegalovirus, in both infected cells and in cells transiently transfected with UL25. pUL25 localization in infected cells was observed in typical cytoplasmic structures characterized by a very electrondense texture previously reported to accumulate other tegument proteins. At the virion level pUL25 seems to localize at the interface between the tegument and the capsid of both intracytoplasmic and extracellular virions. In UL-25-transfected cells, pUL25 has been found in characteristic para-crystalline cytoplasmic aggregates, suggesting its intrinsic ability to aggregate in a regular subunit pattern.

Animals↗
PubMed2000

Humoral immune response to proteins of human cytomegalovirus latency-associated transcripts.

Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame [ORF] 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124). The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection. Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized. Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6. Patterns of antibody prevalence to these proteins in long-term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150. Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins. Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease. These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients.

Antibodies, Viral↗