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.

Fasciola hepatica:development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.

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.

Immunohistochemistry in the era of personalised medicine

Immunohistochemistry (IHC) plays a central role in the histopathological classification of diseases, including cancer. More recently, the importance of immunohistochemical staining is increasing. IHC usage in diagnostics is invaluable; however, the genetic and therapeutic significance of biomarker immunostaining has become equally relevant.

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

A cell surface antigen associated with meiosis in male Staurdoderus scalaris (Orthopteran)

Immunofluorescence has identified seven monoclonal antibodies reactive with the surface of meiotic cells and absent in premeiotic cells. Analysis by immunogold electron microscopy indicated that these antigens were present on the external surface of the cells and were coincident with the presence of synaptonemal complexes in the nucleus. On immunoblots a common glycosylated protein of 205 kDa was recognized, in addition to smaller subunits, suggesting the presence of a protein complex comprised of smaller peptides.

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.

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.

Localisation of actin in the liver fluke, Fasciola hepatica

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

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).

The addition of gemtuzumab ozogamicin to low-dose Ara-C improves remission rate but does not significantly prolong survival in older patients with acute myeloid leukaemia:results from the LRF AML14 and NCRI AML16 pick-a-winner comparison

The treatment of older patients with acute myeloid leukaemia, who are not considered suitable for conventional intensive therapy, is unsatisfactory. Low-dose Ara-C(LDAC) has been established as superior to best supportive care, but only benefits the few patients who enter complete remission. Alternative or additional treatments are required to improve the situation. This randomised trial compared the addition of the immunoconjugate, gemtuzumab ozogamicin (GO), at a dose of 5 mg on day 1 of each course of LDAC, with the intention of improving the remission rate and consequently survival. Between June 2004 and June 2010, 495 patients entered the randomisation. The addition of GO significantly improved the remission rate (30% vs 17%; odds ratio(OR) 0.48 (0.32-0.73); P=0.006), but not the 12 month overall survival (25% vs 27%). The reason for the induction benefit failing to improve OS was two-fold: survival of patients in the LDAC arm who did not enter remission and survival after relapse were both superior in the LDAC arm. Although the addition of GO to LDAC doubled the remission rate it did not improve overall survival. Maintaining remission in older patients remains elusive.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment

Background We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor alpha (TNF-alpha) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD).

Design Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab.

Results Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-gamma-secreting Th1 cells and IFN-alpha-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab.

Conclusions New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL23 antibody therapy is a highly effective therapy for these lesions.