A study of the relationship of virus-like particles and Australia antigen in liver

The ultrastructural and antigenic characteristics of the HB Ag-associated liver particles and the relationship of the liver particles to serum HB Ag were studied by three correlative and comparative observations. (1) Ultrastructural tissue study in 29 patients defined the morphologic characteristics of the unique noncoated virus-like particles that were found in liver cell nuclei and sometimes in the cytoplasm. Their presence in liver tissue may be of diagnostic significance for active disease. The coated particles were shown to be morphologically identical to the Dane particles in serum, and were found in the cisternae of proliferative endoplasmic reticulum. In addition, the cisternae of the endoplasmic reticulum contained peculiar filaments and their probable cross sections with an owl eye appearance. The outer covering of the coated particles was shown to be continuous with the filaments, suggesting that the latter were probably the crystalloid coat material. (2) Phosphotungstic acid negative stain of liver tissue homogenates and ultrastructural sections of the pellets from the homogenates provided a convenient linkage for comparative morphologic interpretation of the tissue and smear findings. Evidence indicated the hepatic origin of the HB Ag particles. (3) Immunoagglutination electron microscopy indicated that the HB Ag particles in tissue homogenates and sera were antigenically identical, whereas the noncoated particles, although morphologically similar to the core component of the Dane particles, were most likely antigenically different from the coat material of HB Ag. The observations support the concept that the Dane particles have dual antigenic specificities. Possible explanations are given for the absence of pure core particles in the serum.     

Electron Microscopy and Immunoelectronmicroscopy of Cytoplasmic Hepatitis B Antigen in Hepatocytes

The localization of hepatitis B antigen (HB Ag) and the nature of the virus-like particles in hepatocytes of patients with HB antigenemia are controversial. In many reports, numerous virus-like particles have been demonstrated in hepatocytic nuclei; the few reported in the cytoplasm are insufficient in number to explain the intense cytoplasmic fluorescence after staining with fluoresceinated antibody to HB Ag (HB Ab). We found numerous tubular and circular structures, measuring 20 to 30 nm in diameter, in the cisternae of the excess smooth endoplasmic reticulum (ER) of varying numbers of hepatocytes in 13 of 16 HB Ag carriers and in 4 of 9 patients with HB Ag-positive chronic hepatitis corresponding to cytoplasmic HB Ag-specific fluorescence. Direct immunoelectronmicroscopy using peroxidase-labeled HB Ab revealed that the intracisternal bodies and the surrounding membranes contain HB antigenic determinants. These bodies are an ultrastructural correlate of cytoplasmic HB Ag. It is suggested that they are virally coded coat material rather than the mature hepatitis B virus or its core.     

The detection of hepatitis B antigen in hepatic parenchyma by the fluorescent antibody technic.

Tissue sections from 42 specimens of liver were examined by indirect immunofluorescence microscopy for the presence of hepatitis B antigen (HB Ag). In all cases the serologic status of HB Ag was known. Fourteen of the specimens were also examined by electron microscopy. In four biopsies from three patients positive cytoplasmic fluorescence was detected using antisera prepared in animals and 20-nm. nuclear particles were found by electron microscopy. These patients were all seropositive for HB Ag, all had chronic aggressive hepatitis or active cirrhosis, and all were receiving immunosuppressive therapy at the time of examination. Nuclear fluorescent staining was demonstrated when one of these biopsies was re-examined using a human antiserum.     

Physicochemical Fractionation of Extracellular Cornea-Damaging Proteases of Pseudomonas aeruginosa

Fractionation of the culture supernatant fluids of a cornea-virulent strain of Pseudomonas aeruginosa by ammonium sulfate precipitation, diafiltration, isoelectric focusing, ion-exchange chromatography, gel filtration, and sucrose density gradient centrifugation failed to separate the rabbit cornea-damaging activity and the in vitro protease activity of the preparations. Three proteases having similar molecular weights (approximately 20,000) and isoelectric points of approximately 4.6, 5.8, and 8.8 were obtained free of detectable amounts of other known extracellular pseudomonal enzymes. Heating a mixture of the three proteases for 15 min at 80 C resulted in complete loss of protease and cornea-damaging activities. The sterile culture filtrate of a nonproteolytic but lethal toxin-producing strain of P. aeruginosa did not contain cornea-damaging activity. Cultivation of the proteolytic strain in broth containing 4.7% ammonium sulfate yielded a culture supernatant fluid free of protease and cornea-damaging activities. The results obtained support the conclusion that a cornea-virulent strain of P. aeruginosa can produce, in vitro, at least three different extracellular proteases capable of eliciting rapid and extensive damage to rabbit corneas.     

Non-A,non-b hepatitis virus: identification of a core antigen-antibody system that cross reacts with hepatitis b core antigen and antibody

Using immunodiffusion, a major cross-reactivity had been previously demonstrated between hepatitis B(HBe/3 Ag) and the antigen reported in the serum of non-A, non-B hepatitis patients, therefore redesignated NANBe Ag. By direct immunofluorescence a new antigen associated with, but distinct from, NANBe Ag has now been identified in the liver of 14/26 patients with NANB chronic active hepatitis. The homologous antibody was detected in the serum of these 14 patients. Behaving like HBc Ag and cross reacting with it by immunofluorescence, the new antigen was termed NANBc Ag. Anti NANBc also became detectable in serial acute phase and convalescence sera from 5/5 NANB Ag-positive posttransfusion hepatitis cases. Further characterization of NANBe and NANBc antigens achieved by fractionation of a NANB virus-infected liver showed NANBc Ag to be expressed on 22–25 nm HB core-like particles containing DNA polymerase activity. Cross-reactivity between NANBc and HBc antigens was confirmed by immunodiffusion. Liver-derived NANBe Ag identical to serum NANBe Ag exhibited the same physical properties as HBe/3 Ag and could be similarly released by disruption of the non-A, non-B virus cores. These results indicate that hepatitis B and NANB virions not only share the same structure and DNA polymerase activity but are also antigenically related and belong to the same new class of DNA viruses.     

Response in guinea-pigs to inoculation with mixtures of hepatitis B antigen at variable ratios

Equivalence proportions of hepatitis B (HB) antigen (Ag) and antibody (Ab) positive plasmas were determined by liquid-phase radioimmunoassay (RIA). Variable proportions of HB Ab preincubated with HB Ag resulted in HB Ag:Ab ratios ranging from greater than to less than unity. Pairs of guinea-pigs were immunized intramuscularly with these mixtures and bled at approximately 3-week intervals. Sera were tested by RIA for presence of HB Ag and Ab.

Animals injected with a four-fold equivalence excess of HB Ab did not respond with Ab to HB Ag. Only at an equivalence ratio (1:1) for HB:Ag did one of two animals demonstrate a stable and consistent immune response. This began at the 11th week after inoculation. The earliest immune response detected in both guinea-pigs of a pair was when HB Ag was given at a four-fold equivalence excess over the Ab. This response was noted as early as 5 weeks. Quantitatively, the immune responses correlated very well with the HB Ag dose, i.e. higher Ag:Ab ratios resulted in greater anti-HB responses.

Very low levels of antigenaemia were evidenced by test specimens inhibiting the precipitation of ¹²⁵I-labelled HB in RIA and these levels persisted for 4½ months. Antigenaemia was noted only for those animals which received mixtures of lower ratios of Ag:Ab as inoculum. Excess Ab caused immunological suppression rather than the enhancement that might be expected to occur with Ag:Ab complexes alone.

     

Serial propagation and purification of chicken Anaemia agent in mdcc-MSB1 cell line

The TK-5803 strain of an agent which induces chicken anaemia was propagated and titrated in cultures of the MDCC-MSB1 cell line. When the isolates (tentatively designated MSBI-TK-5803 strain) from the original material were inoculated into 1-day-old susceptible chicks, they showed a severe pathogenicity, inducing anaemia with aplasia of the bone marrow and atrophy of the lymphoid organs. On density gradient separation, the peak of their infectivity titre appeared at a density of 1.35 to 1.36 g/cm(3) and numerous virus particles were demonstrated in the same fraction. The agent was resistant to heating for 30 min at 60 degrees C and 50% chloroform for 15 min at 4 degrees C, and passed through a 25 nm membrane filter, but it was completely inactivated after heating for 30 min at 70 degrees C or more. The purified virus particles were spherical or hexagonal in shape and 19.1 +/- 0.2 nm in diameter.     

Insect immunity. III. Purification and partial characterization of immune protein P5 from hemolymph of Hyalophora cecropia pupae.

We previously showed that in pupae of Hyalophora cecropia, eight hemolymph proteins (P1 through P8) were selectively synthetized after immunization (Faye et al., Infect, Immun. 12:1426-1438, 1975). We also showed that a gross fractionation was obtained by a series of ammonium sulfate precipitations (designed A through D) and that protein P5 was enriched in fraction A. Starting from fraction A, we have now purified protein P5 by using dialysis, isoelectric focusing, and hydroxylapatite chromatography. The final product gave a single band in both gradient gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the latter method, proteins P5 and P8 were found to be enriched in fraction A, but they were absent in fraction Ac prepared from nonimmunized pupae. Protein P5 was found to have a pI of 6.6 and a molecular weight of 24,000 in sodium dodecyl sulfate and 96,000 in tris (hydroxymethyl) aminomethane-borate, pH 9.0. These data suggest a structure for P5 composed of four subunits of equal size. Protein P5 stimulated the killing of Escherichia coli by hemolymph fractions B and D, but it had neither killing nor phenol oxidase activity of its own.     

Incidence and nature of cytoplasmic hepatitis B antigen in hepatocytes.

Hepatitis B antigen (HB Ag) in the hepatocytic cytoplasm is detected by immunofluorescence after reaction with fluoresceinated antiserum to HB Ag or by electron microscopy as numerous 20- to 30-nm. tubular and circular structures in dilated cisternae of excess endoplasmic reticulum. On light microscopy, these hepatocytes can be recognized because their cytoplasm has a ground-glass appearance and stains with Gomori's aldehyde fuchsin. Aldehyde fuchsin-positive ground-glass hepatocytes were detected in all 14 asymptomatic carriers of HB Ag and in 16 of 60 HB Ag-seropositive patients with chronic hepatitis, but not in HB Ag-seropositive acute viral hepatitis or in various other HB Ag-seronegative liver diseases. These cells are helpful in identifying on light microscopy HB Ag carriers and a portion of patients with HB Ag-positive chronic hepatitis. Nuclear HB Ag did not stain with aldehyde fuchsin. Nucleic acids were not detected in the ground-glass cytoplasm by special stains at the light or electron microscopic level. We suggest that the tubular and circular structures in the hepatocytic cytoplasm are coat material of the hepatitis B virus or virally coded host cell reaction product rather than the complete hepatitis B virus.     

Cloning and expression of the Pseudomonas aeruginosa exoenzyme S toxin gene

The gene for exoenzyme S, an ADP-ribosyl transferase, was cloned from Pseudomonas aeruginosa strain DG1 using an oligonucleotide probe based on the partial N-terminal amino acid sequence to screen a library of DG1 SstI fragments inserted into pKT230 in Escherichia coli DH1. A positive clone, designated pPD3, hybridized with the oligonucleotide probe and contained a 15 kb SstI insert. In E. coli minicells pPD3 expressed a single protein of Mr 68,000. This protein was localized primarily in the periplasm in E. coli. A 3.6 kb HindIII-BamHI fragment was subcloned into the vector pT7-4 which contains the promoter from bacteriophage T7 to construct pT7-4HB. In E. coli strains expressing the T7 RNA polymerase on a second plasmid, the Mr 68,000 protein was expressed and shown to react with antibodies to exoenzyme S. No enzymatic activity was detected in cell sonicates or culture supernatants of E. coli (pPD3). Cell sonicates of E. coli (pT7-4HB) however were cytotoxic to HeLa cells and this cytotoxicity was neutralizable with anti-exoenzyme S antiserm. Thus, exoenzyme S expressed in E. coli is toxic but not enzymatically active. When plasmids carrying the exoenzyme S gene were introduced into P. aeruginosa, there was a significant increase in ADP-ribosyl transferase activity, indicating that the plasmid encoded protein is enzymatically active in P. aeruginosa.     

Lack of immune tolerance to hepatitis B antigen in offsprings of guinea pigs injected with HB ag during pregnancy.

Immune tolerance to hepatitis B antigen has been examined in the guinea pig. The offsprings of guinea pigs injected with purified HB Ag during pregnancy were found capable of producing HB antibodies. Purified HB Ag is suitable for producing immune serum for the systemic screening of blood donors for HB Ag.     

An automated passive haemagglutination technique suitable for the detection of hepatitis B virus antigen and antibody in blood donors

A method is described for preparing purified hepatitis B antigen (HB Ag) viral antigen without density gradient centrifugation. A method for sensitizing human group O red cells with this preparation is given, together with the technical details of an automated passive haemagglutination technique suitable for the mass screening of blood donors for HB virus and anti-HB antibody.     

Non-a, non-b hepatitis: identification of hepatitis-B-like virus particles in serum and liver

Hepatitis B virus-like particles including: small spheres and filaments 15--25 nm in diameter together with a 35--40 nm Dane particle-like virion have been identified in sera of patients with non-A, non-B hepatitis. In a coded serological study, such particles were detected transiently in 3/4 acute, and persistently in 7/8 chronic cases of non-A, non-B hepatitis with non-A, non-B antigenemia. Only 2/12 similar cases without non-A, non-B antigens (Ag) in serum had detectable particles but neither patients with drugs, or type A hepatitis, nor cases of obstructive jaundice. The particles did not express hepatitis B surface (HBs) or non-A, non-B Ag at their surface but were associated, in three patients, with significant endogenous DNA polymerase activity. Furthermore, particles similar to hepatitis B cores (BHc) and also associated with DNA polymerase activity were demonstrated by sucrose gradient ultracentrifugation of a liver homogenate obtained from a patient who had died of non-A, non-B hepatitis. The non-A, non-B hepatitis virion described here appears, therefore, as a hepatitis B-like virus. The exact kinship between these two agents is currently being investigated.     

An evaluation of low voltage counterimmuno-electrophoresis for the detection of hepatitis-B antigen (HB Ag)

A newly available low voltage counterimmunoelectrophoresis (CIEP) system for the detection of HB Ag (Hapindex, Ortho Diagnostics) was compared with a conventional CIEP method used at this laboratory. A total of 1216 sera were tested. The Hapindex system was found to be at least as sensitive as the conventional CIEP. No false positives were found in this series. The elimination of any preparative work makes the Hapindex system particularly suitable for laboratories not testing large numbers of sera for HB Ag. It also eliminates many of the contamination hazards inherent in the conventional method.     

Hepatitis b surface antigen (hbsag) in peritoneal fluid of hbsag carriers undergoing peritoneal dialysis

Samples of ascitic fluid and outflow dialysate were collected from HB_sAg carriers undergoing peritoneal dialysis and tested for HB_sAg by solid-phase radioimmunoassy. The surface antigen was detected in every sample from HB_sAg carriers. This finding was not dependent upon the presence of occult blood in the sample. Surface antigen particles and possibly Dane particles were also observed in HB_sAg-positive samples by immunoelectron microscopy. These results identify the outflow dialysate of HB_sAg carriers undergoing peritoneal dialysis as a potential source of hepatitis B virus transmission.     

Characterization of genomic alterations in hepatoblastomas. A role for gains on chromosomes 8q and 20 as predictors of poor outcome

As data on the genomic alterations in hepatoblastoma (HB) are limited, 34 HB tumors and three HB cell lines were screened for DNA copy number changes by comparative genomic hybridization. The average number of chromosomal imbalances per tumor was 2.3 +/- 0.5 (mean +/- SEM) with gains sevenfold more frequent than losses. The most frequent gains of chromosomal material in HB tumors were on 2q (44%), 1q (41%), 2p (29%), 20 (24%), 22q (18%), 8q (15%), 8p and 12q (9% each), as well as 7q, 12p, and 17 (6% each) and the only recurrent loss was on 4q in 12% of cases. Highly amplified sequences were identified in four tumors and mapped to 2q24 in two cases, to 8q in two cases (once to 8q11.2-q13 and once to 8q11.2-q21.3) as well as to 10q24-q26 in one case. In one cell line, highly amplified DNA sequences were mapped to 7p and 8q. Comparison to previously published data on this series of HB revealed that the number of chromosomal imbalances was significantly higher in HB tumors with loss of heterozygosity on 11p (P = 0.03), whereas in five of 10 HB biopsies without chromosomal imbalances, beta-catenin gene mutations were found. HB patients were divided into a good (no evidence of disease) and a poor (died of disease) outcome group according to their clinical course after standard therapy. Two alterations were found to be significantly associated with poor outcome: gain on 8q (P = 0.007) and gain on 20 (P = 0.009). In summary, our analysis allowed the identification of gains on chromosomes 1q and 2 as hallmark DNA copy number changes in HB with 2q24 as a critical chromosomal band. Furthermore, this study provided evidence that gains on 8q and 20 play a role as markers of prognostic significance in HB.     

Ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa.

In mice, active protection against Pseudomonas aeruginosa could be induced with two fractions derived from a crude preparation of ribosomes from P. aeruginosa. The two fractions were obtained by gel filtration chromatography of the crude ribosomal preparation on Sepharose CL-2B. In fraction I, less than 1% of the ribonucleic acid (RNA) applied to the column was recovered. Fraction II contained RNA and protein in a ratio of 1.94. The presence of ribosomes in this fraction was confirmed by analysis on a sucrose density gradient. The protection by fraction I was not affected by treatment with ribonuclease; in contrast, incubation of fraction II with ribonuclease completely abolished active protection. Fraction I contained lipopolysaccharide (LPS) as was indicated by the presence of 2-keto-3-deoxyoctonic acid. No LPS was found in fraction II. The adjuvant dimethyl dioctadecyl ammonium bromide enhanced the protection by fraction II; however, immunity by a low dose of fraction I was abolished by dimethyl dioctadecyl ammonium bromide. Protection by fractions I and II appeared to be restricted to the homologous serotype of P. aeruginosa. These results indicate that RNA is required for protection by fraction II. Active protection by fraction I is likely due to LPS.     

Identification of a Streptococcus salivarius Cell Wall Component Mediating Coaggregation with Veillonella alcalescens VI

Cell walls of Streptococcus salivarius HB aggregated Veillonella alcalescens V1, but cell walls of the mutant S. salivarius HB-V5 did not. We found no correlation between the presence of fimbriae on streptococcal walls and the ability to aggregate Veillonella strains. Treatment of the walls with lysozyme solubilized a fraction which possessed Veillonella-aggregating activity. Solubilized cell wall preparations of strain HB contained three major (glyco)proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at least four antigens as determined by immunoelectrophoresis with antiserum prepared against strain HB walls. A specific antiserum, which was obtained by adsorption of anti-HB serum on strain HB-V5 cells, contained monospecific antibody that reacted with the solubilized strain HB wall preparation. Similar fractions prepared from strain HB-V5 cell walls did not possess aggregating activity and lacked one protein band (protein I) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and one antigen (antigen b) after immunoelectrophoresis. The same antigen was absent when lysozyme-solubilized wall preparations of strain HB were reacted with anti-HB-V5 serum. Crossed-immunoisoelectric focusing indicated that this specific (glyco)protein and this antigen were identical and had an isoelectric point of 4.60. Protein I and antigen b were specifically adsorbed when solubilized strain HB cell walls were incubated with V. alcalescens V1 but were not adsorbed by nonaggregating Veillonella parvula ATCC 10790 cells. Culture supernatants of strain HB contained V. alcalescens V1-aggregating activity. Antigen b was present in the culture supernatant, but was not found in cultures of strain HB-V5. A total of 18 S. salivarius isolates possessing the streptococcal group K antigen released aggregating activity and antigen b into the culture medium, but 11 strains which lacked the K-antigen did not.     

Alginate production by clinical nonmucoid Pseudomonas aeruginosa strains.

The slime material from a revertant nonmucoid variant, derived by serial passage of a heavily mucoid Pseudomonas aeruginosa strain isolated from a patient with bacteremia, was found to contain 16% uronic acids, 48.5% carbohydrates, 11% protein, and 2% lipids. Chromatographic analysis by ion exchange chromatography revealed that this extracellular material consisted of three fractions, one uronic acid fraction with properties similar to those of the alginate fraction of the parental strain and two other fractions consisting of neutral sugars and proteins in approximately a 5:1 ratio. In addition, the slime material from six other clinical macroscopic nonmucoid P. aeruginosa strains was found to contain alginate. These results demonstrate that alginate production in various amounts is a property shared by all P. aeruginosa strains.