Clinical performance of a polymerase chain reaction testing algorithm for diagnosis of HIV-1 infection in peripheral blood mononuclear cells

The clinical performance of a modified polymerase chain reaction (PCR) testing algorithm was evaluated for confirming the presence of HIV-1 proviral DNA in peripheral blood mononuclear cells. A whole cell lysate, rather than phenol-purified DNA, was used for PCR amplification, under systematically optimized conditions designed and verified within each PCR run to detect as few as 10 copies of proviral DNA. A sequential testing algorithm was designed requiring reactivity in duplicate (with corresponding non-reactivity in negative controls) with at least two sets of primers, before reporting a specimen as HIV-1-positive. In 196 specimens from patients staged according to the Walter Reed staging system, the PCR test sensitivity and the coculture isolation rate (in parentheses) were found to be: 97% (71%), 100% (85%), and 100% (76%) in stage 1, stage 2, and stage 3 specimens, respectively; and 100% (100%) in stage 4, 5, and 6 specimens. Results were uniformly negative for PCR and coculture isolation from 21 blind negative specimens and 105 (negative) donor leukopacks. These data indicate that this PCR testing algorithm is more accurate than tissue culture isolation methods, especially with early stage patients, and results in detection of HIV-1 in virtually 100% of seropositive individuals, with no false positives.     

Persistence of HIV-1 silent infection in seronegative subjects at high risk

Twenty regular sexual partners of HIV-1 infected subjects, without detectable human immunodeficiency virus (HIV-1) antibody and positive for HIV-1 genome by in situ hybridization (ISH), were selected and studied longitudinally for 6-36 months to estimate the duration of silent infection. During the follow-up period, 10 showed atypical Western Blot (WB) patterns. Two seronegative partners seroconverted. Rapid progress to AIDS was observed in 7 seropositive subjects.     

Non-radioactive PCR method for quantification of HIV-infected peripheral blood mononuclear cells in HIV-positive subjects

A nested PCR method for quantification of HIV-infected peripheral blood mononuclear cells (PBMC) which does not use radioactivity nor plasmids is described. Quantification was achieved by means of limiting dilutions and an ACH2 cell standard. The method was used to study 23 HIV-infected patients. A significant correlation was seen between the stage of HIV disease, as classified by CDC criteria, and the number of infected PBMC. Although the CD4+ cell count also correlated well with the stage of HIV disease, there was only weak correlation between the result obtained by the PCR method and the CD4+ cell count.     

器官移植术后外周血巨细胞病毒及其抗原和DNA的检测

目的　探讨及时诊断器官移植受者术后活动性巨细胞病毒（ 简称ＣＭＶ） 感染。方法　采集３２ 例器官移植受者术后８９ 份血标本,同时用酶免疫组织化学作抗原血症和病毒分离培养作病毒血症的检测。再以原位杂交（ＩＳＨ） 和聚合酶链反应（ＰＣＲ） 作ＤＮＡ 血症的检测。结果　８９ 份血标本中,抗原血症阳性３５ 份（３９－３％ ）,病毒血症阳性２５ 份（２８－１ ％） ,ＤＮＡ 血症的位杂交检测阳性３７ 份（４１－６ ％） 及ＤＮＡ血症ＰＣＲ 检测阳性５１ 份（５７－３％ ）。结论　ＤＮＡ血症原位杂交和ＰＣＲ 技术及抗原血症的检测方法,具有检测率高且时间早的优点,能作为临床快速检测ＣＭＶ的方法。本结果与移植受者的临床症状相关,可作为监视器官移植受者术后ＣＭＶ感染和指导抗病毒治疗的依据     

Detection by in situ hybridization of HIV and c-myc RNA in tumour cells of AIDS-related B-cell lymphomas

The incidence of acquired immune deficiency syndrome (AIDS)-related malignant lymphoma has increased since the disease was first described, but its pathogenesis is still not understood. There have been numerous molecular studies addressing the clonality of these proliferations, the presence of Epstein-Barr virus genome in the tumour cells, and rearrangements of the c-myc oncogene. However, very few in situ hybridization studies have been carried out. We analysed 24 cases of high-grade B-cell malignant lymphomas and two cases of polymorphic B-cell proliferation associated with human immunodeficiency virus (HIV) infection. Human immunodeficiency virus ribonucleic acids were detected in some of the tumour cells in 19 of the 24 cases of malignant lymphomas and in both cases of polymorphic B-cell proliferation, with the in situ hybridization technique and using a specific tritiated copy deoxyribonucleic acid probe. With the same technique, c-myc ribonucleic acid was detected in most of the tumour cells from all the 21 cases of malignant lymphomas tested but not in the polymorphic B-cell proliferation.     

Chemokine production by peripheral blood mononuclear cells in elderly subjects

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Iα, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Iα and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lα. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in ‘specific’ immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.     

Chemokine production by peripheral blood mononuclear cells in elderly subjects

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.     

套式PCR和原位杂交技术检测肝病患者单个核细胞内TTV DNA

目的　了解慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA存在情况。方法　采用套式PCR以及原位杂交技术检测外周血单个核细胞(PBMC)内TTVDNA。结果　套式PCR检测26例慢性乙型肝炎(中度)患者PBMC内TTVDNA阳性7例,阳性率269%,非常显著高于健康对照(χ2=143,P<0.001);21例原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA阳性4例,阳性率190%,显著高于健康对照(χ2=486,P<0.05)。7例PBMC内TTVDNA阳性的慢性乙型肝炎(中度)患者PBMC原位杂交,其中4例细胞内阳性。结论　慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC细胞浆内存在TTVDNA,且阳性率相当高。     

外周血单个核细胞在狼疮性肾炎病人体内高效表达IL-6的观察

为了确定狼疮性肾炎（ＬＮ）病人体内是否存在内源性ＩＬ－６表达增高及其来源，作者应用ＥＬＩＳＡ法检测５８例ＬＮ病人的血清及外周血单个核细胞（ＰＢＭＣ）内ＩＬ－６蛋白的含量，用原位杂交方法结合ＩＢＡＳ２．０图像分析系统，检测其中２０例ＬＮ病人ＰＢＭＣ在未受任何刺激时ＩＬ－６ｍＲＮＡ表达强度，并分析三者之间的关系。结果表明该三者水平在ＬＮ病人体内均异常增高，活动期尤其明显，三者之间互呈直线正相关。提示ＬＮ病人ＰＢＭＣ内源性过度表达和合成ＩＬ－６是其外周血ＩＬ－６水平增高的原因之一，异常增高的循环ＩＬ－６对狼疮性全身性病理损害，尤其对肾损害具有重要的作用。此外，血清ＩＬ－６过高还提示ＬＮ处于活动期，对临床指导治疗有一定意义。     

In vitro activation of peripheral mononuclear cells by zinc in HIV-infected patients and healthy controls.

Zinc is a mitogen for peripheral blood mononuclear cells (PBMC). The optimal mitogenic concentration was found to be 0.05 mmol/l (327 micrograms/dl), four times higher than physiological serum levels. Maximal proliferation was observed after 6 days. Limited dilution technique revealed a frequency of zinc reactive cells of 1:3467 (median; range 1:1628-1:6235). Cord blood mononuclear cells from four of six healthy children could be stimulated to proliferate by zinc. A normal zinc-induced proliferative response could be demonstrated in all six HIV-infected patients in the Walter-Reed-stage I, in nine of 11 patients in Walter-Reed II and in only two of five patients in Walter-Reed III. In Walter-Reed IV to VI all eight patients showed a weak response to zinc (less than 50% of the healthy day control). Decreased zinc serum levels were found in 10 of 28 patients and in one of 16 controls. There was a significant correlation of a diminished zinc-induced proliferation with lower serum levels of zinc and a reduced proportion of CD4 helper cells in HIV-1-infected men. Because of a suppression of mitogenesis by high dose of zinc an excessive intake of zinc as used by some HIV-1-infected patients can presently not be recommended. The value of zinc-induced proliferation for monitoring HIV-infected patients has still to be established.     

Detecting Epstein-Barr virus DNA from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in Taiwan

Epstein-Barr virus (EBV) has been found by many serology studies to be associated with systemic lupus erythematosus (SLE). However, the results of DNA studies have been conflicting. Therefore, instead of antibody to EBV, we studied the association between EBV DNA and SLE. In this case-control study in Taiwan, we enrolled 87 SLE patients and 174 age- and sex-matched controls. Peripheral blood mononuclear cells of SLE patients and matched controls were tested for EBV DNA by polymerase chain reaction (PCR) and Southern blot. Of the 87 SLE patients, 71 (81.6%) were found to be positive for EBV DNA, while 85 (48.9%) of the 174 controls (odds ratio 4.64, 95% confidence interval 2.50–8.62, P<0.0001) were positive. While the EBV DNA-positive rate did not decline with age in SLE patients (P>0.05), it did decline with age in controls (P<0.05). Furthermore, based on a real-time quantitative PCR study, we have found a significant difference between EBV viral load in SLE and controls (P=0.008). Therefore, in our molecular study of DNA level, we found evidence for the association of EBV infection and SLE, suggesting that EBV contributes, if not to the development of SLE, then to disease perpetuation.     

Presence of Epstein-Barr virus harbouring small and intermediate-sized cells in Hodgkin'S disease. Is there a relationship with Reed-Sternberg cells?

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. in situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative. In EBV-PCR positive non-malignant lymph nodes, only a few EBER-1 and -2 positive cells could be observed. As in infectious mononucleosis, these cells frequently expressed the B-cell marker CD20. Although we cannot exclude the fact that the majority of the smaller EBV-positive cells in HD belong to reactive EBV-infected lymphocytes, our data favour the hypothesis that at least some of these smaller cells may belong to the reservoir of neoplastic cells in HD.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

Comparison of immunocytochemistry and in situ hybridization in the cytodiagnosis of genital herpetic infection.

Over a 62-month period, 53 patients were found to have cervicovaginal smears that contained cells consistent with, or equivocal cells for, a herpes simplex virus (HSV) infection. The Papanicolaou-destained smears from these cases were restrained in situ hybridization (ISH) with a biotinylated cloned DNA probe and immunocytochemistry (ICC) assay and were compared for the detection of HSV in cervicovaginal smears by two methods. Cytological findings classified the 53 slides into two groups, i.e., cytologically herpes positive (33 patients) and equivocal cases (20 patients). Each group was subdivided into two groups: group A was confirmed by ICC, and group B was confirmed by ISH technique. Of the 33 cellular samples containing cells considered to be consistent with a herpes infection, 15 (88%) of 17 were positive by means of ICC technique (group A), 6 (43%) of 14 were positive by ISH technique (group B). Of the 20 smears showing equivocal cell changes thought unlikely to be caused by an HSV infection, 6 (60%) of 10 were positive by ICC (group A), 2 (29%) of 7 were positive by ISH (group B). With the ISH technique, five smears showed dislodged cells from glass slides due to enzyme treatment and denaturation. The results revealed that the ICC technique is a rapid and reliable procedure and thus recommended for routine diagnosis of HSV infection. Moreover, ICC is easier to perform and interpret and is less expensive than ISH. Therefore, the ICC may be preferable to ISH for detecting HSV in routine Papanicolaou diagnostic work.     

Association of pKi-67 with satellite DNA of the human genome in early G1 cells

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.     

Characterization of human immunodeficiency virus (HIV1C) variants in peripheral blood mononuclear cells and spermatozoa

The presence of distinct viral variants in different cells and secretions of the same person influences the transmission of HIV as well as the response to the host defense and to therapy. Sperm-associated virus is also a risk factor for sexual transmission of HIV. Characterization of the C2-V3 region of HIV1C env gene by the Heteroduplex Mobility Assay (HMA) and sequencing demonstrated the presence of distinct variants in the peripheral blood mononuclear cells (PBMCs) and the sperm of the same individual (n = 6). The translated amino acid sequences of HIV variants in the PBMCs of all the study participants (n = 12) and spermatozoa of the six participants characterized showed the presence of distinct variants with different numbers of N-linked glycosylation (NLG) sites. Infectivity of PBMCs of these persons by co-culture with PBMCs from healthy individuals as detected by the p24 levels in the culture supernatant did not show a correlation with the blood plasma viral load. Interestingly, the infectivity of the sperm samples from four of the five individuals showed positive correlation with the viral load in seminal plasma. The study suggests the presence of distinct viral variants in the sperm and PBMCs of the same person with differential infectivity, and the NLG sites may be associated with the affinity of HIV to receptor/co-receptor usages as well as affinity toward neutralizing antibodies which may influence the risk of sperm associated virus in sexual transmission of HIV and transmit the virus further to distal cells.     

用PCR制备地高辛精标记的单链探针进行原位杂交

用聚合酶链反应（ＰＣＲ）方法制备地高精标记的单链探针，用以原位检测低丰度的β肾上腺素能受体ＭＲＮＡ在大鼠肺组织中扮布。结果表明本方法制备的探针的检测敏感性显著高于用随机引物法标记的双链探针。已克隆于载体的ＣＤＮＡ或ＲＴ－ＰＣＲ产物均可作为模板合成单链探针，江ＤＡＴＰ、ＤＣＴＰ、ＤＧＴＰ浓度各为２００μｍｏｌ／Ｌ，ｄＴＴＰ＋Ｄｉｇ－ｄＵＴＰ浓度降至５０－７５ μｍｏｌ／Ｌ，扩增产量无显著变化，ｄＴＴ     

JC virus genomes in progressive multifocal leukoencephalopathy: detection using a sensitive non-radioisotopic in situ hybridization method

JC virus genomes have been localized in formalin-fixed, paraffin-embedded brain tissues of two cases of known progressive multifocal leukoencephalopathy by in situ hybridization utilizing a biotinylated JC virus DNA probe. A three-stage immunoperoxidase system with gold-silver amplification of the diaminobenzidine substrate was used to visualize biotinylated nucleic acid hybrids. Dot-blot quantification of this visualization system indicates that subpicogramme amounts of biotinylated DNA can be detected. Optimal detection of the virus genomes in the brain tissues required a microwave irradiation step prior to hybridization. JC virus genomes were observed in the nuclei of enlarged oligodendrocytes and of some bizarre astrocytes. No other cell types were found to harbour the genomes.