Novel human astroviruses: Novel human diseases?

Astroviruses are small, non-enveloped, single-stranded positive RNA viruses that belong to the Astroviridae family. While classical human astroviruses (HAstV) are a well-recognized cause of acute non-bacterial diarrhea among young children worldwide, novel astroviruses, named HAstV-MLB and HAstV-VA/HMO, have been identified recently in humans by molecular assays. They are phylogenetically more related to animal astroviruses than to classical human astroviruses, thus suggesting cross-species transmission. Serological studies demonstrated a surprisingly high seroprevalence in certain populations and highlighted a high infection rate in the early years of life. Although their pathogenic role has not yet been clearly determined, novel astrovirus RNA sequences have been identified in different biological specimens of symptomatic patients, including the feces, plasma, cerebrospinal fluid, and brain biopsies. Thus, there is evidence that they could contribute not only to digestive tract infection, but also to unexpected clinical syndromes, notably encephalitis and meningitis. Severe infections affect mainly immunocompromised patients. These findings indicate that novel astroviruses should be considered in the differential diagnosis of immunocompromised patients with meningitis or encephalitis of unknown origin.     

Cleavage of human IgM with human lysosomal elastase

Human lysosomal elastase, a serine proteinase stored in the azurophil granules of polymorphonuclear leucocytes, cleaves human monoclonal IgM producing two fragments and dialyzable peptides. An F(ab)₂μ-like fragment, called IgMe in this report, retains some reactivity with an anti-Fcμ-antiserum and is antigenically deficient with respect to both the subunit (IgMs) produced by reduction and alkylation of IgM and the similar fragment (IgMp) produced by papain digestion. The other fragment is very similar to Fabμ generated by papain digestion, as indicated by immunochemical identity and a similar molecular weight.     

Uptake of human eosinophil peroxidase by human neutrophils.

A cytochemical analysis was carried out for study of the interaction between human eosinophil peroxidase (EPO) and human neutrophils. To this end, neutrophils with a genetic deficiency of myeloperoxidase (MPO) were used to avoid the otherwise inevitable interference of the high endogenous MPO activity of normal neutrophils. The data show that human neutrophils incubated with EPO (1 GU/ml) rapidly bind the enzyme all over the cell surface and internalize it in small vesicles. Part of bound EPO concentrates in a limited area on the cell surface and is then internalized by means of coarse tubular channels. Fusion of the small vesicles to each other or possibly with the tubular channels gives rise ultimately to EPO-containing multivesicular bodies, which, after 30 minutes of incubation, are the only peroxidase-positive structures in the cytoplasm. Under identical experimental conditions, no binding of human MPO to the neutrophils was detected. At concentrations 10 times as high as those used for EPO, a minority of neutrophils bound MPO, but the binding pattern remained diffuse on the plasma membrane and the internalization was negligible. It seems, therefore, that the EPO trapping system of human neutrophils exhibits specificity at least among leukocyte peroxidases. Furthermore, it operates at much lower concentrations of EPO than those reported for EPO uptake by mast cells and basophils. The uptake of EPO by neutrophils may serve to sequester a potentially toxic agent, thus limiting damage to the tissue in eosinophil-rich inflammatory lesions.     

Blastogenic response of human lymphocytes to human cytomegalovirus.

A method was developed for measuring the blastogenic response of human lymphocytes to human cytomegalovirus (CMV). Viral and control antigens were prepared by extracting disrupted infected and uninfected cell cultures with an alkaline buffer. Lymphocytes from ten donors with complement-fixing (CF) antibody exhibited a blastogenic response, whereas cells from ten seronegative donors did not. A relationship between the stimulation index (SI) and the results of neutralization (NT), indirect haemagglutination (IHA) or CF tests was not observed. The maximum blastogenic response occurred after 5 to 7 days of incubation and was usually greater when the cultures were supplemented with homologous plasma instead of sera. The presence of CMV antibody in the supplementary sera did not appear to affect the reactivity of the lymphocytes.     

Effect of recombinant human interferon γ against human cytomegalovirus

Summary Escherichia coli-derived human interferon- (rIFN-) inhibited the replication of human cytomegalovirus (HCMV) synergistically when combined with IFN-. The induction of HCMV DNA polymerase was inhibited in rIFN--treated cells. It is suggested that the induction of 2–5 A synthetase does not play an important role in the anti-HCMV actions of IFNs.With 2 Figures     

The binding of human IgG subclasses to human monocytes

The direct binding of human IgG subclasses to human monocytes has been measured by autoradiography using radiolabeled myeloma proteins. Only IgGl and IgG3 were found to bind strongly to the monocyte surface. This binding could be inhibited both by fresh human serum and by soluble immune complexes.     

Persistence of human parvovirus B19 in human tissues

Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation.     

Development of human monoclonal antibodies against human cytomegalovirus.

Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.     

Detection of human cytomegalovirus DNA in human tonsillar lymphocytes

The human cytomegalovirus (HCMV) was first isolated in cell cultures from the oropharynx, which is thought to be a site of primary infection. Although HCMV can be recovered from the oropharynx during reactivation phases, its exact site of latency is not known. In the present study we demonstrated evidence suggesting the presence of latent HCMV in this anatomic region--in the palatine tonsils. Samples from 30 tonsils obtained by tonsillectomy were screened for the presence of HCMV. Out of the 30 tonsil donors, 23 were seropositive for HCMV. Three methods were used in attempts to demonstrate HCMV's presence in the tonsils: (1) viral isolation attempts on various cell cultures, (2) immunohistochemical staining--immunoperoxidase method--designed to detect viral antigens, and (3) DNA dot hybridization with a HCMV-DNA probe designed to detect viral DNA. Neither infectious HCMV nor other viruses were isolated in cell cultures. No viral antigens were detected by immunoperoxidase staining in the tonsillar tissue. Four out of the 30 tonsils studied were found to contain viral DNA. In one case in which the tonsillar mononuclear (MN) fraction was separated from the polymorphonuclear (PMN) fraction, only the first fraction contained the viral DNA.     

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.     

Immortalization of human keratinocytes with human papilloma virus DNA

Human keratinocytes, derived from the cervix or foreskin, can be immortalized with the HPV-16 or HPV-18 E6 and E7 genes. Two methods of introducing the viral oncogenes into keratinocytes i.e. calcium phosphate transfection and retroviral transduction, are described below, both of which have been optimized for human keratinocytes. While the calcium phosphate transfection method can be used in a normal tissue culture facility, transduction with a retroviral vector containing oncogenes, requires a containment facility and appropriate laboratory practice.     

Immunocytochemical localization of human choriogonadotropin in human malignant trophoblast. Model for human choriogonadotropin secretion.

Human choriogonadotropin (HCG) and its subunits have been studied immunocytochemically using a recently developed unlabeled antibody method. Factors influencing the antigenicity of HCG and its subunits were explored, and optimum conditions were determined. HCG has been successfully localized at the cellular level using paraffin-embedded tissues. Several antibodies to HCG or its subunits were evaluated for specificity using rigorous immunologic controls. This methodology was applied to placental tissue, in vitro choriocarcinoma, and in vitro breast carcinoma. HCGbeta and HCGalpha were localized in the syncytial trophoblast of first trimester placenta with cytotrophoblast cell islands and Langhans' cells being negative. Conditions of stimulated and unstimulated HCG production have been explored in the BeWo line of malignant trophoblast. Stimulation was accomplished by treatment with dibutyryl cyclic AMP and theophylline for varying periods of time. Only 6 per cent of the cells could be shown to be producing HCGbeta and HCGalpha in the unstimulated condition. Stimulation increased this percentage to 70 and 18 per cent, respectively, after 24 hours of treatment. Ultrastructural localization has shown that the mechanism of synthesis and secretion of HCG follows a pathway that is not as extensively developed as that of the pituitary hormones.     

Latent infection of human ovarian teratocarcinoma cells with human cytomegalovirus

Summary Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA₁) cells, and maintained for more than 200 days. Infected cultures maintained at 34°C (PA₁CMV₃₄) and 37°C (PA₁CMV₃₇) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32°C (PA₁CMV₃₂) and 39°C (PA₁CMV₃₉) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p. i.) respectively. PA₁CMV₃₂ cultures shifted to 37°C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA₁ cells at 32°C. In contrast, PA₁CMV₃₉ cells did not produce infectious virus even when cultured at 37°C for more than 100 days after the temperature shift.With 1 Figure     

Reaction of human smooth muscle antibody with human platelets.

Platelets, prepared from fresh human platelet-rich plasma smeared on slides and stained with human serum containing smooth muscle antibodies (SMA) in indirect IFL, showed a bright cytoplasmic fluorescence with numerous projections extending from the surface. A prerequisite for obtaining a positive reaction with SMA-positive serum was that a chelating agent was present in the suspending medium when preparing the smears. The projections could be demonstrated also by anti-HeLa cell (anti-species) serum. This indicates that the projections had a membraneous cover. Staining of live platelets was always negative. Platelets treated with cytochalasin B for 1 hr were smooth and spherical and did not show any surface projections.     

人胸腺组织原代培养细胞与人类免疫缺陷病毒间的相互作用

背景：人类免疫缺陷病毒能够结合人胸腺的细胞CD4受体,从而导致CD4细胞活性降低。 目的：观察人胚胎胸腺组织原代培养与人类免疫缺陷病毒相互作用关系。 方法：取人胎胸腺组织进行原代细胞培养,取胸腺的细胞与人类免疫缺陷病毒1 ⅢB混合培养设为实验组,设置不加人类免疫缺陷病毒培养的胸腺的细胞为对照组。 结果与结论：透射电镜观察显示,培养40 h,人类免疫缺陷病毒诱导的胸腺的细胞内即可发现大量艾滋病病毒颗粒。MTT法和免疫组织化学染色检测结果显示,培养40 h~22 d,HIV诱导胸腺的细胞吸光度值均显著低于对照组(r=0.9733,P < 0.05),人类免疫缺陷病毒诱导胸腺的细胞膜上和细胞浆Fas-L阳性表达率均升高(P < 0.05)。说明人类免疫缺陷病毒可致原代培养的人胚胎胸腺的细胞活性降低。     

结直肠癌组织中人细小病毒B19的检测

目的探讨人细小病毒B19感染与结直肠癌发生的关系。方法运用原位杂交对50例石蜡包埋结直肠癌患者的肿瘤组织,癌周结直肠组织以及10例正常成人结肠组织中B19病毒进行检测。激光捕获显微切割肿瘤细胞及癌周正常肠上皮细胞,巢式PCR扩增B19DNA。结果50例结直肠癌标本中,原位杂交示B19阳性信号在肿瘤组织为78％（39／50）,癌周结直肠组织为40％（20／50）,正常结肠组织中5例（n=10）,经统计分析肿瘤与癌周组织B19感染差异有显著性（P〈0．01）,正常结肠组织与癌周组织间未见统计学差异（P=1．000）。显微切割进一步证实B19病毒DNA存在于肿瘤细胞内。结论结直肠组织中人细小病毒B19感染较常见,主要存在于结直肠癌上皮细胞内,该病毒可能在结直肠癌的发生过程中起一定作用。     

Antibody production by human X human hybridomas in serum-free medium

Four human X human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human X human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies.