Serodiagnosis of acute hepatitis A

Hepatitis A antibody (anti-HAV) was detected by specific radioimmunoassay (RIA) method in sera from 10 patients with acute icteric hepatitis. Anti-HAV was detectable in many subjects very early before the onset of jaundice, but the diagnosis of type A hepatitis in all patients was confirmed by the demonstration of seroconversion during convalescence. Since the initial antibody detected by RIA is predominantly IgM, while IgG specific anti-HAV appears later reaching peak levels within 1 to 2 months, we treated serum specimens of these patients with 2-mercaptoethanol (2ME) in order to differentiate acute-from convalescent-phase hepatitis A sera. Inactivation of IgM fraction with 2ME produced a significant reduction of anti-HAV titer only in acute-phase sera, so that this procedure may be used for early diagnosis of acute type A hepatitis.     

Studies on transmission of hepatitis A virus to squirrel monkeys

Non-human primates have been playing an essential role in the study of hepatitis A virus (HAV) biology, pathogenesis and for testing candidate HAV vaccines. This study was to determine the suitability of squirrel monkeys (Saimiri sciureus) as animal model for HAV infection. Animals were inoculated, either intragastrically or intravenously, with a Brazilian HAV isolate (HAF-203). Alanine aminotransferase (ALT) and anti-HAV antibodies (IgM and total) were monitored. Feces were daily collected for HAV antigen and HAV RNA detection. Samples of liver tissue were obtained by biopsy before inoculation at peak ALT levels and/or when anti-HAV antibodies developed, and at necropsy for morphological examination. Monkeys inoculated by the intravenous route rapidly developed significant elevations of serum ALT, anti-HAV antibodies, and liver histologic changes, while the only evidence of HAV infection in intragastrically inoculated animals was the seroconversion. Moreover, squirrel monkeys excreted very low levels of HAV detectable in only few fecal samples after amplification by RT-PCR, different from humans and other non-human primate species that eliminate large quantities of virus during the late incubation period. The unusual onset of hepatitis A in experimentally infected squirrel monkeys represent an important obstacle for its use as animal model for the study of this viral infection. However, they can represent a valuable tool for the obtention of hyperimmune sera for HAV, in the view of the very high titer of anti-HAV developed (10⁵) 24 days after a single intravenous inoculation.     

Antibodies to meningococcal antibodies in the sera of patients and donors

The comparative study of sera taken from healthy persons (pooled sera of 100 donors, 6 individual serum specimens) and sera taken from patients with meningococcal meningitis (pooled sera of 10 patients with meningococcal infection, group A, and 6 individual serum specimens from patients with meningococcal infection, groups A, B, C) was carried out by the method of immunoblotting. All proteins from healthy donors were found to contain antibodies to meningococcal iron-regulated protein (IRP) of 85 kD, designated as TbpB. In 30% of donor sera the presence of antibodies to meningococcal IRP of 34 kD (FbpA) was registered. Moreover, donor sera were found to contain antibodies to meningococcal IRP of 45 kD. The sera taken from convalescents were found to have the increased content of antibodies to IRP of 70 and 85 kD and somewhat lesser content of antibodies to proteins of 98, 44 and 34 kD. As regards other (non iron-regulated) proteins, in the process of convalescence the most intensive antibody production was observed with respect to minor protein with a molecular weight of 50 kD, as well as proteins of class 5, characterized by molecular weights of 30 kD and less.     

Immunoenzyme determination of IgA specific anti-HAV in the serological diagnosis of hepatitis A

A capture enzyme immuno assay for the detection in the serum of specific IgA antibodies to HAV is described. A total of 203 sera from patients and controls were tested. IgA anti-HAV were present only in sera from patients with recent hepatitis A. 23 patients were followed prospectively and IgA anti-HAV were at detectable levels for at least six months after the onset. The detection of IgA anti-HAV is proposed as an important test to differentiate hepatitis A with persistent hypertransaminasemia from non-A, non-B patients.     

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.     

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.     

Serum antibodies to the hepatitis A virus in persons following a history of the infection

Enzyme immunoassay was used for titration of serum antibodies in subjects with a history of clinically pronounced or asymptomatic hepatitis A infection. Titers of hepatitis A virus antibodies (anti-HAV) essentially decreased during 3-4 years after the disease, then the rate of this decrease slowed down and antibody titers stabilized at low levels. After clinically pronounced hepatitis A anti-HAV levels were considerably higher than after the asymptomatic form of this infection.     

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65- kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.     

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.     

Immunoenzyme analysis of antigen-specific determination of HBsAG-containing circulating immune complexes (CIC HBsAG/IgM and CIC HBsAG/IgG) in blood serum of patients with HBV-infection]

A complex enzyme immunoassay (ELISA) has been designed for antigen-specific determination of HBsAg-containing circulating immune complexes (CIC HBsAg/IgM and CIC HBsAg/IgG) in human blood sera in parallel with registration of free HBsAg and specific antibodies to viruses of hepatitis A, B and D. It is shown that effective formation of HBsAg-containing CIC serologically is registered predominantly as a mutually incompatible marker with detection of free HBsAg (in 70-85% of the cases). CIC HBsAg/IgM and CIC HBsAg/IgG may be registered both in parallel and as mutually exclusive markers. Effective formation of HBsAg-containing CIC in the presence of anti-HBsAg occurs in case of a mild course of viral hepatitis of epidemic and sporadic type, while in severe forms of VH-free HBsAg is predominantly detected thus pointing either to ineffective formation of HBsAg-containing CIC or to their continuous registration with demonstration of the effect of delay of witching of anti-HBsM over to anti-HBsG (or CIC HBsAg/IgM to CIC HBsAg/IgG). It was also found that in case of epidemic VH in Tajik SSR (1987) serologically marked as VH both A and B convalescent phase was characterized by parallel disappearance (or lowering of the titer levels) of HBsAg-containing CIC and class M antibodies to both hepatitis A (anti-HAV M) and B (anti-HBcM, anti-HBsM) along with the containing parallel registration of relevant G-antibodies (anti-HAV G/anti-HBcG). This observation requires further studies both in terms of close association of viruses of hepatitides A and B and with regards to possible antigenic mimicry.     

Synthesis of a Neu2en5Ac analog hapten and isolation of monoclonal antibody to Neu2en5Ac.

Neu2en5Ac is a minor component of body fluids and is abundant in sialuria, but no antibody to detect it has been reported. 5-Acetamido-2,6-anhydro-9-glutaramido-3,5,9-trideoxy-D-glycero-D- galacto-non-2-enonic acid has been synthesized and conjugated with keyhole limpet hemocyanin (KLH) for immunization. A hybridoma named SIC172 was obtained that produces a monoclonal antibody (MAb) to Neu2en5Ac. SIC172 MAb in culture supernatant bound strongly to the hapten conjugated to BSA in ELISA, but slightly to fetuin, a glycoprotein which is rich in Neu5Ac. SIC172 MAb (IgG3(kappa)), purified with a protein A/G affinity column, bound strongly to fetuin. Neu2en5Ac competed with the MAb in binding in amounts as low as 3 microM, while the competition of Neu5Ac appeared at amounts of more than 300 microM. SIC172 MAb is a unique MAb specific to Neu2en5Ac and might be useful for detecting Neu2en5Ac, which occurs naturally and in sialuria.     

Antibodies to the hepatitis A virus in the healthy population of Moscow

Serum specimens collected from 1002 persons in Moscow were tested for the presence of antibodies to hepatitis A virus (anti-HAV antibodies) by solid-phase enzyme immunoassay. The prevalence of these antibodies increased progressively with age from 10% in children aged 5-9 years to over 90% in the age groups of 40-49 years and over, the 50% immunity level being established at the age of 18 years. 79% of infants under 1 year were found to be immune, which was obviously due to the placental transfer of antibodies from mother to child. In a considerable part of seropositive persons over 30 years high or medium antibody titers were detected. These age groups showed a stable proportion of the low, medium and high level of anti-HAV antibodies. The prevalence of such antibodies was not related to sex. The presence of an ample amount of anti-HAV antibodies was determined in all of 18 tested lots of commercial serum immunoglobulin obtained from 3 different manufacturers.     

Infection of human cytomegalovirus in cultured human gingival tissue

Background

Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs.

Results

We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 μg/ml) even after two consecutive infections but increased to ~50 μg/ml after the third infection. Competition with free M2e peptide indicated that ~20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a ≥ 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 μg/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV.

Conclusion

The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.     

ELISA for detection of IgM and IgG antibodies to sandfly fever sicilian virus

• &#x02022;An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific human immunoglobulin G and M antibodies to sandfly fever Sicilian (SFS) virus. Acute and early convalescent serum pairs with ⩾ 7 days between the 2 specimens were available from 20 patients and all showed significant optical density (OD) increase and significant titre rise (⩾ 4-fold) by IgG ELISA. However, negative or borderline-positive sera were found as late as 11 days after onset of symptoms when tested by IgG ELISA.
• &#x02022;Specific IgM antibodies were detected during the first week of symptoms, and maximum OD values were obtained during the first 4 weeks after onset of disease. The IgM OD values declined over the following 3–9 months. All sera collected later than 14 months post-onset were negative by IgM ELISA.
• &#x02022;The combination of early antibody response and the need to test only one serum specimen gives IgM ELISA an advantage over IgG ELISA in patient diagnosis.
• &#x02022;The IgG ELISA was also evaluated as a seroepidemiological tool and compared to a plaque reduction neutralization test (PRNT) using sera from a normal Cypriot population. Of 183 sera tested, 34 (19%) were positive in plaque reduction neutralization tests (PRNT) and 113 (62%) by IgG ELISA. A number of PRNT-negative sera were strongly positive by IgG ELISA and also by indirect immunofluorescence test, which may suggest the presence of a virus related to SFS in Cyprus which has not yet been isolated.

     

Downregulation of protein phosphatase 2A activity in HeLa cells at the G2-mitosis transition and unscheduled reactivation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA)

In the cell cycle the transition from G2 phase to cell division (M) is strictly controlled by protein phosphorylation-dephosphorylation reactions effected by several protein kinases and phosphatases. Although much indirect and direct evidence point to a key role of protein phosphatase 2A (PP2A) at the G2/M transition, the control of the enzyme activity prior to and after the transition are not fully clarified. Using synchronized HeLa cells we determined the PP2A activity (i.e. the increment sensitive to inhibition by 2nM okadaic acid) in immunoprecipitates obtained with antibodies raised against a conserved peptide sequence (residues 169-182, Ab(169/182)) of the PP2A catalytic subunit (PP2A C). Two different substrates were offered: the phospho-peptide KR(p)TIRR and histone H1 phosphorylated by means of the cyclin-dependent protein kinase p34(cdc2). The results indicate that in HeLa cells the specific activity of PP2A towards both substrates goes through a minimum in late G2 phase and stays low until metaphase. Treatment of G2 cells with TPA (10(-7) M) caused a reactivation of the downregulated PP2A activity within 20 min, i.e. the same time frame within which TPA was shown earlier to block HeLa cells at the transition from G2 to mitosis [Kinzel et al., 1988. Cancer Res. 48, 1759-1762]. Activation of PP2A was also induced by TPA in mitotic cells. The low activity of PP2A in mitotic cells was accompanied by a strong reaction of mitotic PP2A C with anti-P-Tyr antibodies in Western blots, which was reversed by treatment of mitotic cells with TPA. The results suggest that the activity of cellular PP2A requires downregulation for the transition from G2 phase to mitosis. Unscheduled reactivation of PP2A induced by TPA in late G2 phase appears to inhibit the progress into mitosis.     

An immunological approach to enrich a mitotic stimulator and to reveal G2-phase-specific proteins in Physarum polycephalum

Purified antibodies from an antiserum against S-phase proteins of the myxomycete Physarum polycephalum were attached to protein-A-Sepharose CL-4B. A late G2-phase extract that contained a mitosis-stimulating protein was applied to this immunoadsorbent, and the mitosis-stimulating protein was enriched by a factor of ten. This protein, which is present in the cell in low amounts, is synthesized in late G2 phase and obviously degraded in a later stage of the cycle. Immunoadsorption of a G2-phase extract with anti-S-antibodies decreased the 700 main proteins to 20 as demonstrated by two-dimensional gel electrophoresis. No difference in protein pattern could be observed on two-dimensional gels between S-phase and G2-phase extracts before and after immunoadsorption with anti-S-antibodies. This indicates that there are no G2-phase-specific proteins among the 700 most abundant proteins of Physarum polycephalum.     

Antibodies to meningococcal iron regulated protein FbpA and minor proteins in the sera of patients with meningococcal meningitis

Altogether 7 blood serum specimens taken from 3 patients with meningococcal meningitis were studied by the method of immunoblotting. The study revealed that on day 7 and especially on day 10 from the onset of the disease antibodies to periplasmatic iron-regulated protein FbpA with a molecular weight of 34 kD appeared in the serum of one patient. In the sera of two other patients the appearance of antibodies to minor iron-regulated proteins with molecular weights of 43 kD and 46 kD, absent at the acute stage of the disease, were detected on day 7. As the process of convalescence was progressing, in all patients under observation an increase in the specific immune response to proteins of class 5 with molecular weights of 20-14 kD was observed.