A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection.     

Further study of hepatitis C virus RNA detection in formalin-fixed, paraffin-embedded liver tissues by ligation-dependent polymerase chain reaction

AIM: Refractive cataract surgery using corneal incisions is aiming at neutralization of preoperative astigmatism. PATIENTS AND METHODS: 61 patients with preoperative astigmatism of 2.25 +/- 0.98 were included in the treatment. A self-sealing corneal tunnel incision measuring 4.0 to 4.1 mm in external diameter and 6.5 to 7.0 mm in internal diameter (stretch incision) was performed on the steeper axis. After capsulorhexis and phacoemulsification a 5 mm PMMA lens was implanted without suturing. Keratometry and corneal topography were performed preoperatively, 3 days and 1 year respectively following surgery. The statistical analysis was based on the Wilcoxon signed ranks test. RESULTS: Surgical induced astigmatism (IA) following superior incisions in cases of astigmatism with the rule (n = 29) amounted to 1.93 +/- 0.97, while lateral incisions in cases of astigmatism against the rule (n = 29) led to an IA of 1.35 +/- 0.73. Axial shifts by more than 30 degrees were 23% following superior incisions and 17%, after lateral incisions. We observed. astigmatic reduction of 1.3 D after superior incisions and 0.7 D following lateral incisions. CONCLUSION: By 4 mm corneal cataract incisions on the steeper axis a high preoperative astigmatism can be reduced significantly without additional keratotomies.     

Reactivity of anti-AraC-resistant cell monoclonal antibody, YU-311, in formalin-fixed paraffin-embedded specimens of various hematopoietic disorders

YU-311 is a monoclonal antibody reacting with cytosine arabinoside (AraC)-resistant human leukemic cell line and identifies a 92 kDa membrane protein. We have examined YU-311 reactivity with various hematopoietic disorders by an immunohistochemical method and evaluated a correlation between YU-311 expression and refractoriness to chemotherapy, retrospectively. YU-311 reacted with AraC-resistant human leukemia cell lines, in which a 92 kDa membrane protein was identified by Western blotting, whereas drug-resistant cell lines to other than AraC failed to express YU-311 antigen. The frequency of YU-311 positivity was significantly increased in relapsed cases. Only five cases were positive for YU-311 at diagnosis and 24 cases at relapse. Unexpectedly, only eight cases of relapsed leukemia/lymphoma expressed YU-311 and P-glycoprotein simultaneously. Most of the YU-311-positive relapsed cases showed clinical refractoriness for chemotherapy and then failed to induct complete remission or relapsed at short periods with short disease-free duration. These findings indicate that YU-311 expression is closely associated with some aspects of drug resistance, especially with AraC resistance.     

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues

Little is known about the molecular background to senescence in T-lymphocytes. In fibroblast systems replicative senescence has been shown to correlate with a number of changes in the expression of the proteins normally regulating progression through the G1 phase of the cell cycle, and recently the Ink4 inhibitor p16 was implicated as a central regulator of replicative senescence in human fibroblasts. It has, however, been claimed that p16 is not expressed in T-lymphocytes. In the present study we have analysed G1 regulating proteins in ageing human T-lymphocytes. We show that PHA and IL-2 stimulated T-lymphocytes cease to proliferate after around 20 population doublings, these cells can not thereafter be restimulated to growth, and were also found to exhibit markers for senescence. We found that T-lymphocytes accumulate p16 and p15 protein during successive population doublings and display high levels of these proteins as they enter into replicative senescence. There was also an increased binding of p16 to the Cdk6 kinase in senescent cells, and a decreased Cdk6 as well as Cdk2 kinase activity. The levels of other G1 regulating proteins were also altered in the senescent cells, such as slightly elevated levels of p21/WAF1, and downregulation of Cdk2 and cyclinD3. The levels of p27/ Kip1 is down regulated in proliferating cells but rise to approximately 15% of the levels in un-stimulated quiescent cells. As a high proportion of T-cell childhood acute lymphoblastic leukaemias have deletions of both p15 and p16, our data suggest that inactivation of these genes makes it possible for leukemic cells to avoid senescence.     

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.     

Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.     

Pathogenicity studies of an Arkansas variant infectious bursal disease virus

A variant infectious bursal disease virus (IBDV), IBDV-s977, was blind passaged in cell culture, plaque purified, and attenuated by serial passage at a high multiplicity of infection (MOI) in chick embryo fibroblasts (CEF). Cell culture passages of virus caused less bursal atrophy and splenomegaly than did the original isolate and retained immunogenicity; however, virus tended to persist for a longer time in the bursa and spleen of birds infected with the highest CEF passages. Antibody to both low MOI and high MOI passages of IBDV-s977 poorly neutralized virus that was isolated from bursal tissue 28 days postinfection (PI). The spleens of chickens infected with the eighteenth CEF passage were negative for virus at 3 and 7 days PI but had high titers of virus at 14 and 28 days PI. There was also more virus in the bursa of birds infected with the fifteenth and eighteenth CEF passages at 28 days PI than at 7 or 14 days PI. Defective interference (DI) was demonstrated when cell cultures were coinfected with a constant amount of low MOI virus and serial dilutions of high MOI virus. There was an increase in interference score with increased passage number in CEF, and there was more interference in virus passaged at a high MOI. There was an inverse relationship between interference score and bursal lesion score and splenomegaly at 7 days PI, indicating that DI particles may be involved in virus attenuation. There was a positive relationship between interference and viral persistence in the bursa and spleen at 28 days PI. Antiserum to s977 was shown to enhance the nonlytic replication of s977 in CEF, presumably within macrophages, providing a possible mechanism for the pathotypic variation seen in emerging strains of IBDV.     

Seroprevalence of infectious bursal disease virus in free-living wild birds in Japan

Serum samples collected from 739 free-living wild birds of 44 species from Gifu, Mie and Hyogo Prefectures in Japan during the period 1989 to 1997 were tested for antibodies to infectious bursal disease virus (IBDV) serotypes 1 and 2 by a virus neutralization test. Serological evidence of infection with serotypes 1 and 2 was found in 15 (2%) of the sera of 6 species and 36 (4.9%) of the sera of 11 species, respectively. Antibodies to IBDV were detected from both sedentary and migratory species. These findings suggest that free-living wild birds have an important role in the natural history of IBDV. These findings raise the possibility that the IBDV prevalent in the breeding grounds of these birds in other countries could be imported by the migratory species. This is the first report of an extensive serological survey of IBDV in wild birds.     

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease

The right hand end Nde I fragment 3 (90.8-100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.     

Immunoperoxidase staining of hepatitis C virus in formalin-fixed, paraffin-embedded needle liver biopsies

The localization of hepatitis C virus (HCV) in the liver has not been well clarified. We report successful indirect immunoperoxidase staining of the HCV core antigen using polyclonal antibodies raised in rabbits and conventional formalin-fixed, paraffin-embedded needle biopsy sections of liver. The core antigen was distributed in a fine granular pattern diffusely, perisinusoidally, or focally within the hepatocellular cytoplasm of livers from patients with HCV infection. The staining tended to show a more heterogeneous pattern in terms of intensity and distribution in cases of more advanced disease. Hepatocellular carcinoma cells were also frequently stained. HCV immunostaining will provide important information on the pathogenesis and treatment of HCV-related liver diseases.     

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

The nucleotide sequences of the genome segments A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3039 nucleotides (1012 deduced amino acids) and 2640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

Some characteristics of a cellular receptor for virulent infectious bursal disease virus by using flow cytometry

A flow cytometric virus binding assay that directly visualizes the binding of infectious bursal disease virus (IBDV) to its target cells was established. The chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for IBDV infection, bound high levels of the virus. Another B lymphoblastoid cell line, LSCC-1104-B1, bound low levels of the virus, although it was nonpermissive. No virus binding was detected in nonpermissive T lymphoblastoid cell lines. In the binding assay to heterogeneous cell populations of chicken lymphocytes, IBDV (a highly virulent OKYM strain) bound to 94% cells in the lymphocytes prepared from the bursa of Fabricius, 37% cells in those prepared from the spleen, 3% cells in those prepared from the thymus, and 21% cells in those prepared from the blood. Most of the cells, which bound the virus, were surface immunoglobulin M (SIgM)-positive, but a small number of them were SIgM-negative. Additionally, the binding of IBDV to the LSCC-BK3 cells was affected by treatment of the cells with proteases and N-glycosylation inhibitors. These findings may indicate that the IBDV host range is mainly controlled by the presence of a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by SIgM-bearing cells.     

Immunohistochemical diagnosis of typhus rickettsioses using an anti-lipopolysaccharide monoclonal antibody

A monoclonal antibody directed against an epitope on the lipopolysaccharide of typhus-group rickettsiae was developed for the purpose of detecting this heat-stable, proteinase-resistant antigen in formalin-fixed, paraffin-embedded tissues. Rickettsia prowazekii organisms were identified in endothelium and macrophages in sections of the brains of three Egyptian men who died of epidemic louse-borne typhus in Cairo during World War II and in the brain from a recent case of typhus fever acquired in Burundi. R. typhi organisms were identified in endothelial cells from a fatal case of murine typhus and in experimentally infected mice. This approach is applicable not only to the study of archival tissues and experimental animal models but also could be used to establish a timely diagnosis of typhus-group rickettsiosis by immunohistochemical examination of cutaneous biopsies of rash lesions during the acute stage of illness.     

Comparison of neutralizing epitopes among infectious bursal disease viruses using radioimmunoprecipitation

Monoclonal antibodies (MAbs) were used to analyze the relatedness of neutralizing epitopes of infectious bursal disease virus (IBDV) strains. The MAb B69 neutralized the homologous D78 virus but not the MD and Del-A variant strains of IBDV. MAbs 33E8 and R63 neutralized D78 and variant strains MD and Del-A. A cDNA clone consisting of a 1000-base-pair fragment of the VP2 gene from the Del-A IBDV strain was translated using an in vitro system. Six peptides were observed following translation, which represented a full-length product (35.5 kilodaltons) and five truncated products. The translated peptides were radioimmunoprecipitated using MAbs B69, 33E8, and R63. Only MAb R63 immunoprecipitated the in vitro translation products from Del-A. MAbs B69 and 33E8 did not immunoprecipitate the in vitro-translated VP2 peptides. The D78 and Del-A viruses were radiolabeled in vivo using 35S-methionine. Proteins from these viruses were examined by radioimmuno-precipitation with MAbs B69, 33E8, and R63. Although the background made interpretation of the results difficult for MAb B69, this MAb clearly immunoprecipitated VP2 of D78. MAb 33E8 immunoprecipitated the D78 VP3 protein, and R63 immunoprecipitated the VP2 protein. MAb R63 also immunoprecipitated the 35S-methionine-labeled VP2 protein from Del-A. It was concluded that the neutralizing epitope represented by 33E8 was located on VP3 and that the epitope represented by R63 is located on both classic and variant viruses in the region of VP2 that is generally thought to contain sequence variability among IBDV strains.     

Improved detection of CD5 epitope in formalin-fixed paraffin-embedded sections of benign and neoplastic lymphoid tissues by using biotinylated tyramine enhancement after antigen retrieval

To evaluate the effectiveness of the immunohistochemical staining of B- and T-cell lymphomas with Leu-1 (clone L17F12 CD5 antibody, Becton Dickinson, San Jose, Calif) in formalin-fixed paraffin-embedded sections, we stained 12 specimens reflecting cases of chronic lymphocytic leukemia/small lymphocytic lymphoma, 7 of mantle cell lymphoma, 13 of T-cell lymphomas, and 9 of various B-cell neoplasms that do not ordinarily express CD5, using a streptavidin-horseradish peroxidase method with biotinylated tyramine enhancement after antigen retrieval. We were able to detect CD5 reactivity of neoplastic cells in 9 (75%) of 12 cases of chronic lymphocytic leukemia, 6 (86%) of 7 cases of mantle cell lymphoma, and 13 (100%) of 13 of the T-cell lymphomas. B-cell neoplasms (9/9) not typically associated with CD5 expression showed no reactivity of tumor cells. We conclude that the Leu-1 (CD5) antibody, routinely used for cryopreserved tissues, is also effective in formalin-fixed paraffin-embedded sections using an antigen retrieval and streptavidin-horseradish peroxidase method with biotinylated tyramine.     

Infectious bursal disease virus changes the potassium current properties of chicken embryo fibroblasts

Homophobia is a socially accepted, culturally based belief, which is heavily influenced by an individual's or a community's inherent attitudes, beliefs and values. This conceptual analysis of homophobia has endeavoured to review existing literature on homophobia and subsequently identify and examine the phobic constituents of the concept. References to homophobia are mostly from the 1970-1980 period and there is much unacknowledged conceptual baggage that accompanies the term, which results in restrictive and inappropriate ideas about this concept. This is mainly the consequence of comparisons of homophobia to other phobias, which directly infers fear of homosexuals, while in reality homophobia is more of a biased disgust at homosexuals' lifestyles. This paper attempts to re-conceptualize homophobia so that empirical research can begin to test the critical attributes of the concept. This forms the basis for the development of a comprehensive social psychological theory of attitudes towards homosexuals. Such a theory would transcend the unilateral and unidimensional concept of homophobia as a fear and help the understanding of attitudes and feelings towards homosexuals.     

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.     

Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.