Demonstration of antibodies in archival sera from Canadian seals reactive with a European isolate of phocine distemper virus

Sera from seals infected during the 1988 European epizootic of phocine distemper virus and sera from Canadian seals collected since 1972 have been tested for the presence of antibodies to morbillivirus. Approximately one third of the Canadian sera have been shown to contain anti-morbillivirus antibodies; the possibility that these populations of seals provided a source of infection for European seals is discussed.

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.

Fasciola hepatica:development of monoclonal antibodies and their use to characterize a glycocalyx antigen in migrating flukes

Using mice harbouring early Fasciola hepatica infections, six monoclonal antibodies were prepared against a tegumental antigen present in T1 granules and glycocalyx of flukes. Blocking tests indicated that all monoclonals bound the same T1 epitope (or epitopes in close proximity on the antigen molecule), but this was not the determinant recognized by sheep and cattle. Localization of antibody binding at light and electron microscope levels showed that T1-type antigen also occurred in metacercarial tegument and in glycocalyx of gut cells and excretory ducts in juvenile and adult flukes. This indicates that the natural host-antibody response to F. hepatica may be to one antigen early in the infection. Protein A-gold labelling of monoclonal treated fluke sections revealed that the epitope was probably a polypeptide, unmodified by glycosylation in Golgi bodies. When isolated by immunoadsorption and separated electrophoretically under reducing conditions T1-type antigen was found to consist of a polypeptide mol. wt. 50 000, possibly linked to smaller entities mol. wt. 25-40 000. Tissue-specific variations in the antigen molecule might be conferred by linkage of different polypeptides or carbohydrate side-chains to an antigenic core polypeptide. A component of T1-type antigen was found to have mol. wt. of 25 000, possibly resembling a polypeptide of mol. wt. 24 000 from Schistosoma mansoni tegument.

Purification of measles virus with preservation of infectivity and antigenicity

Measles virus Edmonston strain was purified by ultrafiltration followed by two successive sedimentations through sucrose. Purified virus retained infectivity and, when used as an immunogen, elicited high titred antibody to measles antigens by conventional serology. The measles preparations were examined by SDS-PAGE followed by staining. In addition, following PAGE, the purity of these preparations was assessed immunochemically using antisera directed to measles and host cell antigens. The results of these studies demonstrate the utility of the purification method for the preparation of milligram quantities of relatively pure measles virus.

Antibodies to the structural polypeptides of measles virus following acute infection and in SSPE

A solid-phase radioimmunoassay was used to determine the specificity of IgG antibodies from normal sera, sera and CSF from patients with SSPE for the structural polypeptides of measles virus. The polypeptide specificity of antibodies from these sources were qualitatively similar; these results indicate antigenic cross-reactivity between SSPE-derived (Mantooth) and non-SSPE-derived strains of measles virus and stimulation of antibody formation by comparable antigens.

Fasciola hepatica:studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.

Characteristics of a cetacean morbillivirus isolated from a porpoise (Phocoena phocoena)

A virus isolated from a porpoise during the 1988 seal epizootic was shown to be a morbillivirus. In order to determine the relationship of the virus to phocine distemper virus (PDV) a battery of monoclonal antibodies raised against canine distemper virus (CDV), PDV or the porpoise isolate were assessed for their ability to bind to CDV, PDV or porpoise virus epitopes in indirect immunofluorescence assays and ELISAs. The porpoise isolate contained several unique epitopes and several epitopes present on CDV and PDV were absent on the porpoise isolate. The data presented in this study indicate that the porpoise virus is an antigenically distinct morbillivirus and as such has been tentatively named as delphinoid distemper virus (DDV).

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

Common community-acquired infections and subsequent risk of multiple myeloma: a population-based study

The role of bacteria and viruses as aetiological agents in the pathogenesis of cancer has been well established for several sites, including a number of haematological malignancies. Less clear is the impact of such exposures on the subsequent development of multiple myeloma (MM). Using the population-based U.S. Surveillance Epidemiology and End Results-Medicare dataset, 15,318 elderly MM and 200,000 controls were identified to investigate the impact of 14 common community-acquired infections and risk of MM. Odds ratios (ORs) and associated 95% confidence intervals (CIs) were adjusted for sex, age and calendar year of selection. The 13-month period prior to diagnosis/selection was excluded. Risk of MM was increased by 5-39% following Medicare claims for eight of the investigated infections. Positive associations were observed for several infections including bronchitis (adjusted OR 1.14, 95% CI 1.09-1.18), sinusitis (OR 1.15, 95% CI 1.10-1.20) pneumonia (OR 1.27, 95% CI 1.21-1.33), herpes zoster (OR 1.39, 95% CI 1.29-1.49) and cystitis (OR 1.09, 95% CI 1.05-1.14). Each of these infections remained significantly elevated following the exclusion of more than 6 years of claims data. Exposure to infectious antigens may therefore play a role in the development of MM. Alternatively, the observed associations may be a manifestation of an underlying immune disturbance present several years prior to MM diagnosis and thereby part of the natural history of disease progression.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts

Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy.

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

Immunoselection of breast and ovarian cancer cells with trastuzumab and natural killer cells:selective escape of CD44high/CD24low/HER2low breast cancer stem cells

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44(high)CD24(low) "cancer stem cell-like" phenotype and expressed significantly less HER2 compared with non-stem cells. Likewise, the CD44(high)CD24(low) population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44(high)CD24(low) cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants.

CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication. (C) 2003 Elsevier B.V. All rights reserved.