John S. Lumsden

Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class.

Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fcγ portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

BackgroundEpithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.MethodsWe recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.ResultsING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.ConclusionA moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Antimicrobial Resistance in Generic Escherichia coli Isolates from Wild Small Mammals Living in Swine Farm, Residential, Landfill, and Natural Environments in Southern Ontario, Canada

ABSTRACT To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.

MASS MORTALITY ASSOCIATED WITH KOI HERPESVIRUS IN WILD COMMON CARP IN CANADA

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×101 to 2.4×106 copies μg−1 host DNA. This is the first report of KHV in Canada.

Viral haemorrhagic septicaemia virus IVb experimental infection of rainbow trout, Oncorhynchus mykiss (Walbaum), and fathead minnow, Pimphales promelas (Rafinesque)

Viral haemorrhagic septicaemia virus (VHSV) in the Great Lakes has had a dramatic impact on fish husbandry because of the implications of the presence of a reportable disease. Experimental infections with VHSV IVb were conducted in rainbow trout, Oncorhynchus mykiss (Walbaum), and fathead minnows, Pimphales promelas (Rafinesque), to examine their susceptibility and the clinical impact of infection. Triplicate groups of rainbow trout (n = 40) were injected intraperitoneally (i.p.) with 100 microL 10(6.5)50% tissue culture infective doses (TCID(50)) or waterborne exposed to graded doses (10(4.5), 10(6.5), and 10(8.5) TCID(50) mL(-1)) of VHSV IVb. Duplicate groups of fathead minnows (n = 15) were i.p. injected with (10(6.5) TCID(50) 100 microL) or waterborne exposed (10(6.5) TCID(50) mL(-1)). All experiments were performed with single-pass well water maintained at 12 degrees C. Following either i.p. or waterborne exposure, VHSV RNA was detectable in both rainbow trout and fathead minnows by nested reverse transcription polymerase chain reaction (nRT-PCR) as early as 4-7 days post-infection (p.i.). Infected fathead minnow and rainbow trout exhibited lesions characteristic of VHS at 9 and 15 days p.i., respectively. Route of exposure had little effect on the onset of clinical signs. Cumulative mean mortality in rainbow trout was 4.4%, 2.6%, 2.6% and less than 1% in the i.p., high, medium and low dose waterborne exposures, respectively. Cumulative average mortality of 50% and 13% occurred in i.p. and waterborne-exposed fathead minnows, respectively. VHSV was detected from pooled rainbow trout tissue by RT-PCR and virus isolation at 38 days p.i., but not at 74 days p.i., regardless of the exposure route. Immunohistochemistry (IHC) with a rabbit antibody to VHSV IVb revealed the viral tissue tropisms following infection, with the identification of viral antigen in myocardium and necrotic branchial epithelium of both species and in gonadal tissue of fathead minnows. Rainbow trout, but not fathead minnows, are relatively refractory to experimental infection with VHSV IVb.

Characteristics of Flavobacterium branchiophilum, the Cause of Salmonid Bacterial Gill Disease in Ontario

Abstract We characterized six filamentous, gram-negative bacterial isolates, each recovered from a naturally occurring salmonid bacterial gill disease (BGD) outbreak in Ontario. The morphological, physiological, biochemical, and antigenic traits of the Ontario isolates were similar to those of Flavobacterium branchiophilum (ATCC 35035, ATCC 35036) recovered from salmonid BGD in Japan and Oregon. We conclude that the Ontario BGD isolates are strains of F. branchiophilum

Use of Hydrogen Peroxide to Treat Experimentally Induced Bacterial Gill Disease in Rainbow Trout

Abstract Bacterial gill disease, experimentally produced in rainbow trout Oncorhynchus mykiss exposed to Flavobacterium branchiophilum, was effectively treated by using 1-h static baths containing hydrogen peroxide as an alternative to treatment with chloramine-T. The optimal concentration of hydrogen peroxide depended on the number of treatments, the time intervals between them, and the stage of the disease. When administered as two treatments at 48-h intervals, 250 mg hydrogen peroxide/L was more effective than 10 mg chloramine-T/L. The effectiveness of 100 mg hydrogen peroxide/L was more variable, but treatment consistently and significantly reduced percent cumulative mortality (PCM) compared with untreated control groups; treatment with 25 mg/L was less effective. Clearance of gill-associated F. branchiophilum antigen was greatest with chloramine-T or 250 mg hydrogen peroxide/L; 100 mg hydrogen peroxide/L was again more variable. Three 1-h static baths at 24-h intervals reduced the concentration of hy...

Antimicrobial Susceptibility of Flavobacterium psychrophilum Isolates from Ontario

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an important disease in the Ontario fish farming industry and in finfish aquaculture in temperate waters worldwide. The development of antimicrobial resistance by F. psychrophilum is a concern because management of outbreaks of BCWD often requires the use of antibiotics. Seventy-two isolates of F. psychrophilum collected over a 16-year period from farmed salmonids with clinical signs of BCWD were tested for susceptibility to 10 antimicrobial agents using cation-adjusted Mueller-Hinton broth in custom Trek Sensititre susceptibility plates for aquaculture. The minimum inhibitory concentrations (MICs) for the isolates were determined by means of a broth microdilution antimicrobial susceptibility testing method established by the Clinical and Laboratory Standards Institute. Most of the F. psychrophilum isolates had decreased susceptibility to two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim-sulfadimethoxine [> or =0.5/9.5 .tg/mL for 93% of isolates] and trimethoprim-sulfamethoxazole [> or = 0.25/4.8 microg/mL for 89% of isolates]). High MIC values (> or =2 microg/mL) were obtained for florfenicol and oxytetracycline in 53% and 61% of the isolates, respectively, and 83% of the isolates were relatively susceptible (< or =16 microg/mL) to erythromycin. The MIC values were also high for ampicillin, oxolinic acid, and gentamicin.

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin.

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.

Identification of cold temperature regulated genes in Flavobacterium psychrophilum

ABSTRACT Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.