Subfractionated conversions of high density lipoproteins in vitro in sera with various lipid spectra

Using gradient gel electrophoresis, the interconversions of high density lipoproteins (HDL) upon heating for 24 hours at 37 degrees C of blood sera obtained from dyslipidemic patients, were studied. It was shown that during incubation of normolipidemic, hypercholesterolemic and hyperalphacholesterolemic sera, the portion of large particles of HDL2 subclass (HDL2b) is increased with a simultaneous decrease in the portion of the smallest particles of HDL3 subclass (HDL3b and HDL3c). Contrariwise, in hypertriglyceridemic and hypoalphacholesterolemic sera the portion of the largest HDL2b particles is decreased, while that of middle-sized HDL3a is increased. Hence, hypertriglyceridemia and hypoalphacholesterolemia are not associated with an intensive conversion of small HDL3 particles into large HDL2 ones, which may be the reason for the decreased cholesterol transport function of HDL.     

Detection of a nucleolar 7-2 ribonucleoprotein and a cytoplasmic 8-2 ribonucleoprotein with autoantibodies from patients with scleroderma

In studies on antinucleolar antibodies in sera from 24 patients with scleroderma, an autoimmune disease, one serum, designated "anti-To", contained antibodies against a nucleolar 7-2 ribonucleoprotein and a novel cytoplasmic 8-2 ribonucleoprotein. The 7-2 and 8-2 RNAs are distinct RNAs with a pppG terminus. They are partially conserved between rat and human species and are present in distinct ribonucleoprotein particles. Eight sera contained antibodies that precipitated particles containing nucleolar U3 RNA; these antibodies appear to be directed against preribosomal particles containing U3 ribonucleoprotein, rather than the U3 ribonucleoprotein particles alone. All these ribonucleoproteins required proteins for antigenicity. These antibodies will be of use in studies on the structure and function of these novel small ribonucleoproteins.     

Viral hepatitis with clinical and epidemiological manifestations similar to hepatitis A but of a different etiology

A case of viral hepatitis in man, appearing as the result of infection caused by pooled concentrated suspensions of fecal samples collected from patients having had repeated hepatitis infection during the period of 1-2 years, is described. Though this infection was similar to hepatitis A in many clinical and epidemiological signs, the possibility of its etiologic relationship with hepatitis virus A was positively excluded; there was also no evidence of the participation of hepatitis virus B in the process. Immunoelectron microscopy of excretions collected at the acute stage of the disease revealed the presence of spherical viral particles 27-30 nm in diameter. Antibodies capable of reacting with these particles were detected in the sera of patients having had 2 kinds of hepatitis and in the sera of patients having the 1 kind of hepatitis in the focus of infection where repeated cases of hepatitis had been observed. No such antibodies were found in the sera of patients with hepatitis A alone and in the set of standard sera specific to viruses causing hepatitis A and hepatitis non A, non B. The authors believe that 2 kinds of hepatitis with the fecal-oral mechanism of transmission exist and propose to name their causative agents hepatitis viruses A, type 1 and type 2.     

从实验感染动物粪便中分离出两株疑似戊型肝炎病毒

用Hep-G2细胞从肠道传播的非甲非乙型肝炎（ET－NANBH）病人粪便悬液感染恒河猴潜伏期粪便中分离出两株致细胞病变（CPE）的病毒（R、9,R25）。用免疫电镜（IEM）观察,这两种病毒均为球形,有实心和空心两种颗粒,直径约27－34nm,能被新疆和苏联ET－NANBH急性期血清特异性凝集,病毒颗粒间抗体桥明显,ET－NANBH急发型期血清对这两株病毒亦有一定中和作用,内地甲肝（HA）病人和正常人血清既不能凝集,也不能中和,是否为ET－NANBH病原体尚待进一步鉴定。     

A Study of Tuberculosis Antibodies by Bentonite Flocculation

Bentonite particles sensitized with concentrated human Old Tuberculin were used for detection of circulating antibodies to M. tuberculosis in human sera.There was no sharp distinction between the titres of patients with active and inactive tuberculosis, but progressively higher antibody titres accompanied increased severity of infection. The sera of 95.3% of the patients studied had a titre of 1:8 or higher. This level was considered to be the minimal significant titre indicative of the presence of metabolically active bacilli in old and new lesions. Tuberculin-negative controls and syphilitic sera were negative. A common zone of incidence in the 1:8-1:32 range was found where differentiation between tuberculin-positive healthy people and patients with tuberculosis was not possible; however, titres of 1:64 or more were found only in tuberculous patients. The antibody under study is heat-labile.     

Viruslike particles containing knob-associated histidine-rich protein are secreted into the culture medium of Plasmodium falciparum in vitro cultures

This report describes the isolation of a viruslike particle from in vitro cultures of the human malaria parasite P. falciparum. Electronmicroscopic observations suggest that the particles are liberated into the culture medium by budding from the erythrocyte membrane. The density of the free particles is 1.16, they contain nucleic acid and two distinct molecular species of the knob-associated Histidine-rich protein. Proteins of the particles are recognized by sera from malaria patients. The previously described knobs may correspond to viral coats inserted in the membrane.     

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.     

An Antigen in Human Breast Cancer Sera related to the Murine Mammary Tumour Virus

THE possibility of an oncogenic virus in human breast cancer has been increased by recent findings. Virus-like particles resembling the mammary tumour virus (MTV) were found in electron micrographs of human breast cancer tissue¹ and particles physically and morphologically similar to MTV particles have been seen in human milk². These particles were found more frequently in the milk of American women with a history of breast cancer in their immediate families and in the milk of Parsi women in Bombay than in the milk of nonselected American women³. Parsi women are three times more likely to have breast cancer than other women in Bombay³. The detection of RNA-dependent DNA-polymer-ase activity in such particles isolated from human milk emphasized the possibility that these particles represent an oncogenic RNA virus⁴. In addition to the physical and morphological resemblance of human milk particles and MTV an immunological relationship between these two kinds of particles seems probable; sera from breast cancer patients neutralize the biological activity of MTV whereas normal human sera did not do so⁵. We report data supporting the hypothesis of an immunological cross relationship between MTV and human breast cancer.     

Exposure to Mimivirus Collagen Promotes Arthritis

Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.     

The demonstration of subtype (D or Y)-specific determinants on the surface of the presumed hepatitis B virus.

Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.     

Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes

Anti-La sera from patients with autoimmune disorders precipitate a set of nuclear and cytoplasmic small RNA-protein complexes. Up to now, it has been thought that the La antigen is associated only with RNAs transcribed by RNA polymerase III, including precursors of tRNA and 5 S ribosomal RNA. Here we report that anti-La sera also react with ribonucleoprotein particles containing small nuclear RNA U1, which is transcribed by RNA polymerase II. Anti-La sera from 12 out of 12 patients tested were found to precipitate U1 RNA-protein complexes from HeLa cell nuclear extracts, under conditions where nonimmune sera do not. Ribonucleoprotein particles containing a second small nuclear RNA, U2, do not react appreciably with anti-La sera although they are present in HeLa cell nuclei at the same concentration as U1 RNA. Anti-La sera also react with U1 RNA-protein complexes in mouse and frog cells, but not in Drosophila or Chironomus, two organisms which lack the La antigen. Hybridization of cloned U1 DNA with anti-La-reactive RNA from HeLa cell nuclear extracts reveals mature U1 RNA, whereas anti-La-reactive cytoplasmic RNA contains a series of hybridizing bands that represent molecules 1-7 nucleotides longer than U1 and which may include precursors of nuclear U1 RNA (Madore, S. J., Wieben, E. D., and Pederson, T. (1984) J. Cell Biol., 188-192). Pulse-chase experiments suggest that the association of La antigenicity with these cytoplasmic U1 RNA molecules is transient. These results are discussed in relation to the presence of uridylate-rich sequences in the 3' termini of U1 RNA precursors and mature U1 RNA, which are similar to La antigen binding sites in several RNAs transcribed by RNA polymerase III.     

Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. They consist of uridylate-rich small nuclear RNA complexed with several proteins. snRNP particles U1, U2, U4/U6, and U5 all contain a common protein core consisting of proteins B'/B, D, D', E, F, and G. In addition to this core, U1 snRNP particles contain proteins 70K, A, and C, whereas U2 snRNP particles contain proteins A' and B". Almost any of the small nuclear RNA-associated polypeptides is targeted by autoantibodies in the sera from patients with SLE or related connective tissue diseases. We immunized a genetically non-autoimmune mouse with recombinant human B" protein and obtained three mAb reactive with native U2 snRNP particles. Two of these mAb particles cross-reacted with U1 snRNP, 9A9 and 11A1, via epitopes present on the U2 snRNP B" protein as well as on the U1 snRNP-specific A protein. A third mAb 4g3, reacted exclusively with U2 snRNP via a unique epitope on protein B". Two epitopes mapped at the carboxy-terminal region of the B" protein, whereas binding of the third mAb involved both amino- and carboxy-terminal amino acids of the B" protein. Epitope mapping, employing a DNAse I fragment library of the B" cDNA, revealed that the three mAb-reactive sites were discontinuous. Autoantibodies in sera from patients with SLE and other connective tissue diseases competed for binding with the mAb, implying that the mAb define a major autoantibody-reactive region on protein B".     

Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body

A panel of sera from 892 autoimmune patients was screened by indirect immunofluorescence on mammalian cells. Seventy-three sera were identified that recognize the nucleolus. Three of these sera appear to stain the nucleolus in yeast, suggesting that they recognize highly conserved antigens. These three sera also immunoprecipitate mammalian U3 snRNA-containing particles, which reside in the nucleolus and have been implicated in rRNA processing. Double immunofluorescence experiments with anti-nucleolus and anti-tubulin antibodies revealed a novel form of non-random nuclear organization in yeast. The spindle pole body and the nucleolus — both of which are associated with the nuclear envelope — preferentially localize at opposite ends of the nucleus. Organization of these and other components into specific regions of the nucleus may be important for optimizing their proper function.     

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

Microprecipitation test for the detection of adenovirus antibody

A microprecipitation test (MPT) for the detection of adenovirus antibody has been developed. The new procedure combines precipitation of virus particles with specific antibody, separation of unreacted components from the resulting electroneutral virus-antibody complexes by electrophoresis, and detection of these complexes with a protein stain. Type-specific antibody was detected in rabbit antisera but, under similar conditions, antibody in convalescent human sera reacted with adnovirus antigens of types 4 and 7. Paired sera from 57 patients with suspected adenovirus infection were examined for significant rises in antibody activity by the microprecipitation test and by complement fixation.     

肠道传播非甲非乙肝炎在恒河猴中的传代研究

用ET-NANBH感染的两只猴(R5和R6)含病毒颗粒的粪便悬液和肝组织悬液分别接种7只和4只恒河猴,分别有5只猴和4只猴在攻毒后20—49天内ALT开始升高,持续时间为7—10天,肝组织学的特征性改变为肝细胞的嗜酸性变和嗜酸小体的形成。在猴ALT升高前2—3天和升高后一周内均检查到大量的27—34nm的病毒样颗粒,这些颗粒只与ET-NANBH病人血清、黑猩猩和猴感染后急性期和恢复期血清发生特异性聚集。在猴感染后血清中未检出抗-HAV、抗-HAV-IgM、HBsAg和抗-HBe-IgM。结果提示:恒河猴是研究ET-NANBH较适宜的动物模型;病人和猴粪便中的27—34nm的病毒样颗粒是ET-NANBH的病原因子。     

Small round structured virus associated with an outbreak of acute gastroenteritis in Chiba, Japan

In an outbreak of acute gastroenteritis which originated in a restaurant in Chiba, Japan, in December, 1987, small round structured virus (SRSV) particles were observed by electron microscopy in 14 of 16 stool specimens from patients. The particles were 30 to 35 nm in diameter, possessed amorphous surface structure surrounded by fine projections and had a buoyant density of 1.36 to 1.37 g/ml in cesium chloride. Serological responses to the SRSV were found by immune electron microscopy and Western blot (WB) assay in paired sera of 12 of 19 patients. Furthermore, WB analysis revealed that the antibody against SRSV was cross-reactive to other SRSV, Tokyo 86/510.     

Interconversions of HDL subfractions in normolipidemic and hypoalphacholesterolemic sera incubated with fibroblasts and hepatoma cell line Hep-G2

Gradient gel electrophoresis was used to study the changes in the HDL subfractional spectrum according to the particle size during their interaction with hepatoma Hep-G2 cells and human skin fibroblasts. Incubation of normolipidemic sera with Ch-loaded fibroblasts resulted in a drop in small particle (HDL3a' HDL3b, HDL3c) and, a rise in large particle (HDL2b, HDL2a) proportions. In contrast, incubation of the same sera with hepatoma cells caused a decrease in HDL2b proportion and a HDL3a release. Incubation of hypoalphacholesterolemic sera with fibroblasts showed a decrease in HDL3b, HDL3c particles and an increase only in HDL2a. The proportion of HDL2b rose considerably when the sera were incubated with hepatoma cells. It is concluded that not only low HDL sera levels, but also the formation of defective HDL2b particles which interact less effectively with liver cells, are characteristic of hypoalphacholesterolemia.     

Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K.

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.     

Discrimination between M2 and M4 antimitochondrial antibodies in primary biliary cirrhosis

10 sera were studied from patients with primary biliary cirrhosis (PBC), that were anomalous in their reactivity against mitochondrial antigens as detected by Western blotting. They had low reactivity against the major, M2 reactive antigen (Mr for beef heart mitochondria, 74 Kd) but reacted against an antigen of Mr 52 Kd (species independent) which was apparently inaccessible in submitochondrial particles (SMP) on ELISA and which was not present in chloroform-released ATPase preparations. In all respects this differed from the characteristics of the M2 antigens and it is concluded that these sera are detecting predominantly the M4-reactive antigen.To whom correspondence should be addressed.