生物素─链霉亲和素系统在检测巨细胞病毒抗体中的应用

本文用自制ＣＭＶ抗原和生物素－链霉亲和素系统试剂，通过最适条件的对比，建立了检测人血清中抗巨细胞病毒ＩｇＭ、ＩｇＡ类抗体的ＢＳＡ－ＥＬＩＳＡ，并与普通ＥＬＩＳＡ作比较，结果表明，ＢＳＡ－ＥＬＩＳＡ的非特异性吸附明显低于普通ＥＬＩＳＡ，与普通ＥＬＩＳＡ相比，抗体效价分别提高５和７倍，阳性率分别提高６８％和１５３．５％。在实际工作中曾用ＢＳＡ－ＥＬＩＳＡ检测临床样品１６５份，就所得结果进行分析。表明ＢＳＡ－ＥＬＩＳＡ检测ＣＭＶＩｇＭ和ＩｇＡ可提高特异性和灵敏度，值得推广应用。     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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Comparison of assays to detect cytomegalovirus shedding in the semen of HIV-infected men

We sought to determine the optimal assays for cytomegalovirus (CMV) shedding in semen. Over a 2-month period, 149 HIV-1-infected men who have sex with men each provided up to three semen specimens. Specimens were tested for CMV by culture, rapid assay (shell vial) and polymerase chain reaction (PCR). By culture, 30% of seminal plasma and 28% of seminal cell specimens grew CMV. By rapid assay, results were 38 and 33%, respectively. By PCR, 56% of seminal cell specimens demonstrated CMV: 20% in a single semen specimen; 33% in two specimens; and 34% in all three specimens. Overall, 69% of men had CMV detected by PCR in at least one seminal cell specimen. By quantitative PCR, 14% had ten, 14% had 100, 16% had 1000, and 12% had 10 000 copies in 6.25 μl of semen analyzed. Adjusting for initial CD4+ cell count, men with CMV shedding demonstrated by PCR at the first visit were approximately four times as likely to shed CMV at a subsequent visit (RR 4.28, CI: 2.30–7.95). CMV shedding was associated with decreased CD4+ cell counts in peripheral blood (P=0.05). It is concluded that the PCR assay provided the greatest sensitivity among the three detection methods.     

Area under the viraemia curve versus absolute viral load: utility for predicting symptomatic cytomegalovirus infections in kidney transplant patients

A novel approach to predicting symptomatic cytomegalovirus (CMV) infections combines the level and the duration of viraemia in a single parameter. Sixty-four kidney transplant recipients were monitored by quantitative shell vial culture, pp65 antigenaemia, and polymerase chain reaction (PCR) of leucocytes. The area under the curve (AUC) of each parameter was determined from the onset of viraemia to the beginning of antiviral treatment. The AUC values were significantly higher in symptomatic than in asymptomatic patients. For antigenaemia and PCR, optimal AUC thresholds for predicting symptomatic CMV infections were determined. They were superior to standard cutoff levels of absolute viral load in sensitivity, specificity, and positive and negative predictive value. In 8 of the 23 patients who became symptomatic, impending clinical features were indicated earlier by the AUC thresholds than by standard viral load. In conclusion, the concept of the AUC should facilitate identification of patients at risk of symptomatic CMV infection.     

In vitro biosynthesis of plasma proteins under ischemic conditions of closed-circuit perfusion of healthy and intoxicated rabbit liver

We are elaborating on the kinetics and mechanisms of septic rabbit liver to de novo biosynthesize acute-phase response (APR) proteins under in vitro conditions of deepening ischemia in reference to their in vivo prevalence in serum and cerebrospinal fluids (CSF) collected at predetermined times. The significance of the data is interpreted as relevant to grafting cadaveric liver into end-stage liver diseased patients and APR-induced ischemic heart diseases (IHD). Hepatic APR was induced by CCl(4)-intubation, and the administration of cholera toxin (CT) or scorpion venom (SV), or both, to rabbits. Hepatic functional efficiency, in terms of biosynthesis of APR proteins in closed circuit perfusion of the isolated intoxicated liver with oxygenated saline or L-15 media paralleled the two-dimensional immunoelectrophoresis (2D-IEP) spectrum of APR serum proteins at time of liver isolation. We are suggesting: (a) in vitro biosynthesis of plasma proteins by isolated perfused liver is the result of in vivo decoded and retained APR inflammatory signals; and (b) decoded inflammatory signals are expressed not withstanding the perfusate's organic composition. Furthermore, 90 min of ischemic perfusion in saline or L-15 medium precipitated mitochondrial aberrations which resulted in further deterioration of de novo biosynthesis of APR plasma proteins. Regardless of the nature of the inflammatory stimuli, mitochondrial aberrations rendered the perfused organ a biologically inert tissue mass that was incapable of resuming biological function upon perfusion with oxygenated L-15 medium. This is most likely due to ischemia-induced irreversible hepatic necrosis. Thus, in vitro aberrations of mitochondrial function(s) critically limit the capability of the isolated liver to resume its organic function to sustain biosynthesis of de novo plasma proteins. Extrapolation of these results to the surgical management of end-stage liver diseases points to the importance of the status and the handling protocol(s) of the cadaver donor liver prior to successful grafting. We conclude that although histology of a cadaver liver may reveal well-preserved hepatic cellular organelles with at least minimal intra- and intercellular communication required for viable hepatic function, we deem it essential to further define acceptable minimal capabilities to de novo biosynthesize plasma proteins by a cadaver liver as a measure of its functional viability and suitability for transplantation. Ultimately, this measure may improve the success of liver transplants with minimal surgical and drug interventions.     

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.     

Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment

The availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8+ T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8+ T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferon-gamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8+ T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0.005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0.005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.     

Development of allergy in children. I. Association with virus infections.

Children born into allergic families, with two allergic parents, are at high risk of developing allergy within the first 5 years of life. In order to observe possible external factors in the sensitization process, a prospective study of 13 such children was done, in which serial clinical and immunologic observations were made at 3- to 6-month intervals over a period of 1 to 4 yr. Eleven of these children are now clinically allergic; 5 have asthma. Immunologic evidence for allergic sensitization was observed in these 11 children by RAST, antigen-induced leukocyte histamine release, lymphoblastogenesis, and rise in serum IgE. Upper respiratory infections (URI) occurred in these 11 allergic children 1 to 2 months prior to the onset of allergic sensitization. In 10 of these 11 URI children, complement-fixing antibodies to viruses (parainfluenza, RSV, CMV) increased in the same blood samples in which immunologic allergic sensitization was first evidenced. This coincidence suggests that certain viruses may contribute to the allergic sensitization process.     

Diagnosis of cytomegalovirus pneumonitis on bronchoalveolar lavage fluid: comparison of cytology, immunofluorescence, and in situ hybridization with viral isolation.

Forty-three bronchoalveolar lavage (BAL) specimens from 40 immunocompromised patients were studied for the presence of cytomegalovirus (CMV) by rapid diagnostic methods. DNA in situ hybridization, cytology, and immunofluorescence were compared to conventional cell culture. Eleven (25%) of the 43 BAL samples grew CMV in culture. In situ hybridization detected 6 of these 11 for sensitivity, specificity, and predictive values of positive and negative of 55%, 94%, 75%, and 86%, respectively. Cytology had a sensitivity of 73% and specificity of 100%. Six Papanicolaou-stained cytospins were screened cytologically versus one hybridization cytospin, and the higher sensitivity of cytology may reflect this extensive sampling. The immunofluorescent method had a sensitivity equal to that of cytology (73%): however, the specificity (72%) was significantly less than that of either the probe or cytology. These data suggest that although in situ hybridization can be a rapid, useful method for detecting CMV in BAL specimens, cytology appears to be a more sensitive method.     

Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.