Sequential respiratory syncytial virus cytomegalovirus pneumonia following bone marrow transplantation

A 6-month-old child with familial hemophagocytic lymphohistiocytosis (FHL) experienced early sequential pneumonia due to respiratory syncytial virus (RSV) and cytomegalovirus (CMV) following bone marrow transplantation (BMT). The patient was deficient in natural killer (NK) cell activity (as found frequently in patients with FHL), and this risk factor may have played a major role in the concomitant infection by the two viral pathogens. Rapid diagnostic methods for both viruses are essential and early specific treatment may serve to ameliorate RSV- and CMV-induced lung injury in these life-threatening infections. © 1995 Wiley-Liss, Inc.     

巢式PCR检测龈下菌斑中HCMV、EBV、HSV-1与慢性牙周炎活动性的关系

目的　建立临床检测龈下菌斑标本中人巨细胞病毒 (HCMV)、Epstein Barr病毒 (EBV)、单纯疱疹病毒 1型 (HSV 1 )巢式PCR方法 ,研究这 3种病毒与慢性牙周炎活动性的关系。方法　收集6 2例慢性牙周炎患者 (男性 2 7例、女性 35例 ,平均年龄 5 3岁 )的牙周炎活动部位、牙周炎静止部位的龈下菌斑 ,提取DNA后使用巢式PCR检测HCMV、EBV、HSV 1 ,比较分析其在同一病人不同部位的检出率。结果　牙周炎活动部位的HCMV检出率为 38.7%,EBV的检出率为 5 8%,HSV 1的检出率为30 .6 %,两种以上病毒合并感染的检出率为 4 0 .3%;牙周炎静止部位的HCMV检出率为 1 4 .5 %,EBV为 2 2 .6 %,HSV 1为 1 1 .3%,两种以上病毒合并感染的检出率为 1 1 .3%。这 3种病毒及其合并感染在牙周炎活动部位的检出率均高于牙周炎静止部位 ,差异有统计学意义 (P <0 .0 5 )。结论　提示HC MV、EBV、HSV 1与慢性牙周炎的活动性相关。     

Effect of double-stranded RNA and type I interferons on cytomegalovirus reproduction in human fibroblasts

D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR D. K. L'vov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 7, pp. 80–83, July, 1991.     

用聚合酶链反应和放射免疫法检测肾移植术后人巨细胞病毒感染

将５０例肾移植术后发热病人的尿标本接种细胞，用聚合酶链反应（ＰＣＲ）技术检测第１代细胞培养上清中的人巨细胞病毒（ＨＣＭＶ）ＤＮＡ。结果１９例为ＨＣＭＶＤＮＡ阳性，阳性率为３８．００％，用固相放射免疫法（ＲＩＡ）检测ＨＣＭＶ抗原，阳性的有１５例，阳性率为２７．７７％。将５４倒尿标本接种人胎肺二倍体单层细胞，分离到６株ＨＣＭＶ。用ＥＬＩＳＡ检测６５例病人单份血清的ＨＣＭＶ－ＩｇＭ抗体，阳性的有１７例，检测４５例病人双份血清ＩｇＧ抗体，１１例的第２份血清的ＨＣＭＶ－ＩｇＧ抗体滴度比第１份有４倍以上升高。上述结果表明，ＰＣＲ检测结果与ＲＩＡ的检测结果基本符合；病人血清抗体的阳性率与ＰＣＲ检测的阳性率基本符合。     

人巨细胞病毒UL136基因在临床低传代分离株中多态性分析

目的 研究人巨细胞病毒(human cytomegalovirus，HCMV)UL136基因在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系。方法 对48株经荧光定量PCR方法检测HCMV DNA为阳性的临床低传代分离株进行HCMV ULl36全序列PCR扩增，对于扩增阳性的12株PCR产物进行ULl36基因全序列测定及结果分析。结果 48株临床低传代分离株ULl36 PCR扩增，12株阳性，阳性率25％，以HCMV Toledo株作为参考株，进行序列比较分析表明，12株临床分离株ULl36开放阅读框架(open reading frame，ORF)长度均与Toledo株相同，为723bp，编码241个氨基酸的蛋白。DNA序列变异均为碱基替换，不同临床分离株ULl36基因与Toledo株进行同源性比较，结果在核苷酸水平为97．7％～99．3％，氨基酸水平为96．6％～99．1％。ULl36编码蛋白的氨基酸变异率为0．83％～3．3％。二级结构预测分为两种构象。大多数HCMV ULl36蛋白翻译后修饰位点在所有分离株中均高度保守，仅几个位点在一些分离株中存在缺失或新增。Toledo株及12株临床分离株核苷酸及氨基酸序列系统进化树分析表明：45J最接近Toledo株。结论 12株临床低传代分离株HCMV ULl36基因DNA及其编码产物的氨基酸序列比较保守，但仍存在一定多态性。未发现不同临床分离株ULl36基因多态性与HCMV先天性感染的表现关系。     

原位PCR技术检测石蜡包埋脑组织中人巨细胞病毒DNA

应用原位聚合酶链反应（ＩＳＰＣＲ）技术检测了２５例尸检畸形胎儿石蜡包埋脑组织中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ，并与普通ＰＣＲ及原位杂交（ＩＳＨ）进行了比较。ＩＳＰＣＲ、ＰＣＲ及ＩＳＨ检测阳性率分别为４４％，３６％及２０％。与ＩＳＨ相比较，ＩＳＰＣＲ不仅检出阳性率高，而且信号强度增强。研究结果提示，ＩＳ－ＰＣＲ是诊断ＨＣＭＶ感染的快速、敏感、特异的实用方法。     

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.     

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.     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.