Magnetic assembly of 3D cell clusters: visualizing the formation of an engineered tissue.

OBJECTIVES: Contactless magnetic assembly of cells into 3D clusters has been proposed as a novel means for 3D tissue culture that eliminates the need for artificial scaffolds. However, thus far its efficacy has only been studied by comparing expression levels of generic proteins. Here, it has been evaluated by visualizing the evolution of cell clusters assembled by magnetic forces, to examine their resemblance to in vivo tissues. MATERIALS AND METHODS: Cells were labeled with magnetic nanoparticles, then assembled into 3D clusters using magnetic force. Scanning electron microscopy was used to image intercellular interactions and morphological features of the clusters. RESULTS: When cells were held together by magnetic forces for a single day, they formed intercellular contacts through extracellular fibers. These kept the clusters intact once the magnetic forces were removed, thus serving the primary function of scaffolds. The cells self-organized into constructs consistent with the corresponding tissues in vivo. Epithelial cells formed sheets while fibroblasts formed spheroids and exhibited position-dependent morphological heterogeneity. Cells on the periphery of a cluster were flattened while those within were spheroidal, a well-known characteristic of connective tissues in vivo. CONCLUSIONS: Cells assembled by magnetic forces presented visual features representative of their in vivo states but largely absent in monolayers. This established the efficacy of contactless assembly as a means to fabricate in vitro tissue models.

Agglutination of chicken lymphocytes by infectious bursal disease virus.

Four serotype 1 field strains of infectious bursal disease virus (IBDV) and one reference standard serotype 1 strain were tested for their ability to agglutinate peripheral blood lymphocytes of chickens. All the five strains agglutinated chicken lymphocytes. The agglutination was not inhibited by serotype 1 reference anti-IBDV serum. These results suggest the need for detailed analysis of IBDV ligand(s) and cell surface receptor(s) relationships.

DNA vaccination in the avian.

Inoculation of naked DNA represents a novel approach to vaccine and immune therapeutic development. DNA vaccines or genetic immunization offers several advantages over the conventional vaccines for specific immune activation. Although a large number of vaccines have been made and are being used in the poultry industry, there have been no major advances in vaccine technology for this animal industry sector for decades. The potential advantages of DNA vaccines, such as over coming maternal immunity, in ovo delivery and absence of requirement for a cold-chain, combined with immunological efficacy make this new vaccine technology very attractive for the poultry industry. This review lists all of the published reports of experimental DNA vaccines developed for use in poultry and focuses on the trends, potentials and remaining barriers in the development of this new revolution in poultry vaccinology.

A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization.

In this report, we show that dimethylsulfoxide (DMSO) enhances liposome-mediated transfection of nucleic acid in chicken macrophage cells and that this could be exploited for the transcutaneous delivery of naked DNA through the intact skin of chickens. We found that DMSO enhanced transfection efficiencies of lipofectamine and polyethyleneimine in HD-11 chicken macrophage cells. Based on this principle, we showed that transcutaneous delivery of a DNA plasmid-dimethylsulfoxide mixture (1:1) to untreated skin of chickens results in a wide distribution of the plasmid in the body. Distribution studies were done using plasmids encoding enhanced green fluorescent protein (EGFP) reporter gene and a bivalent DNA vaccine coding for infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) immunogenic protein genes. This bivalent vaccine induced mucosal and systemic immune responses, as evidenced by IgA and IgM production in the tears and serum of vaccinated chickens. Mucosal immune responses in the tears after topical vaccination were significantly higher (P < 0.05) than after i.m. delivery of the same DNA vaccine and were characterized by the absence of an IgG response. The biodistribution of plasmid indicated that topical delivery with DMSO resulted in a wide distribution and persistence of the plasmid until 15 weeks post-primary vaccination. Both delivery methods resulted in insert-specific message being made in several body tissues, but after topical delivery the virus-specific mRNA could be detected in the bone marrow of one out of three chickens until 15 weeks post-primary vaccination. Furthermore, transcutaneous delivery of this DNA vaccine using DMSO conferred protection from challenge with virulent IBDV (86% survival) and NDV (86% survival). This novel transcutaneous method of delivery of a DNA vaccine shows promise as being an easy and effective way to deliver nucleic acids through intact skin for vaccination or therapeutic purposes.

Evaluation of an immunochromatography strip assay for the detection of Salmonella sp. from poultry.

The present study was conducted to evaluate the sensitivity and specificity of an immunochromatography-based diagnostic kit for Salmonella. The analytical sensitivity of the test when using pure colonies of different Salmonella species was in the range of 1 X 10(4) to 1 x 10(5) colony-forming units per milliliter. The strip detected 19 of 22 strains of Salmonella spp. but failed to detect S. worthington, S. choleraesuis var. kunzendorf and S. johannesburg. The strip did not detect 27 different enteric bacteria, including Escherichia coli O157:H7, Campylobacter jejuni, Shigella sonnei, and Vibrio parahaemolyticus. In direct testing of feces (n = 66) from chickens infected with Salmonella typhimurium, the strip had a sensitivity of 12.3% and a specificity of 100%. Evaluation of the strip assay (n = 510) after sample pre-enrichment in 2% buffered peptone water (BPW) yielded a sensitivity of 93.8% and specificity of 89% when compared to isolation and identification with xylose-lysine-tergitol 4 (XLT4) selective plating media. Subsequent enrichment in Hajna tetrathionate (TT) broth yielded a higher sensitivity (94.7%) and specificity (96.8%). The agreement (kappa) between the strip test and isolation was 0.004 in direct fecal testing, 0.82 in BPW, and 0.89 in TT broth. The assay could detect Salmonella sp. as early as 18-48 hours during pre-enrichment and enrichment compared to isolation on XLT4, which required an overnight incubation step for the presumptive isolation and identification of Salmonella.

Pathogenesis and tissue distribution of a variant strain of infectious bursal disease virus in commercial broiler chickens.

The detection of either infectious virus, viral antigen, and/or viral RNA in different tissues of commercial broilers inoculated at 1 day of age with E/Del variant strain of infectious bursal disease virus (IBDV) was investigated at 2, 4, and 6 wk postinoculation (PI). Virus was readily isolated from homogenates of bursa, cecal tonsils, and bone marrow at 2 and 4 wk PI. Virus isolation coupled with immunoperoxidase assay or reverse transcription-polymerase chain reaction for IBDV-specific RNA extended the window of IBDV detection in the bursa of Fabricius to 6 wk PI. Serology indicated an active early virus infection; however, viral pathology was observed later and beginning at 4 wk PI. This study indicates that variant strains of IBDV may be present in commercial broilers longer than previously thought, and cecal tonsils and bone marrow may serve as nonbursal lymphoid tissues supporting virus replication at later time points PI.

Effect of a variant infectious bursal disease virus (E/Del) on Salmonella typhimurium infection in commercial broiler chickens.

The effect of infectious bursal disease virus (IBDV) on Salmonella typhimurium (ST) infections in broilers was investigated in terms of Salmonella shedding and persistence, pathogenicity, and isotype specific humoral immune responses. Thirty-six, 1-day-old, straight-run commercial broiler chickens that were Salmonella negative by polymerase chain reaction (PCR) and culture were divided into two groups of 18 chicks each (ST and ST-IBDV). One group (ST-IBDV) of chicks received the E/Del strain of IBDV (10(5.0) median tissue culture infective dose [TCID50]/ml) through the ocular and cloacal routes divided into doses of 50 microl each at 2 days of age. Both groups were then inoculated with 10(8) colony-forming units (CFU)/ml nalidixic acid-resistant ST in the drinking water at 3 days of age. Environmental Salmonella counts were higher in the ST-IBDV group at 2 and 3 wk postinfection (PI) compared to the ST group. ST carriage in the cecal contents between the ST and ST-IBDV groups was not statistically different. The ST-IBDV group had a single mortality at 10 days postinfection compared to none in the ST group. The ST-IBDV group had significantly lower bursa to body weight ratios at 4 and 6 wk, as well as higher bursal lesion scores than the ST group at 2, 4, and 6 wk PI. The ST group had significant increase in serum IgG from 2 to 6 wk PI in comparison to the ST-IBDV group, which had no significant changes over time. Both IgA and IgM were significantly increased at 4 and 6 wk relative to 2-wk levels. There was an IBDV-induced failure of anti-Salmonella IgG seroconversion over time in ST-IBDV. Both groups continued to shed high levels of Salmonella up to the end of the study despite high antibody levels in the ST group and an unimpaired IgM and IgA production in the ST-IBDV group, indicating a limited influence of humoral immunity on Salmonella clearance.

Recombinant Newcastle disease virus as a vaccine vector.

Veterinary vaccines remained conventional for more than fifty years. Recent advances in the recombinant genetic engineering techniques brought forward a leap in designing vaccines for veterinary use. A novel approach of delivering protective immunogens of many different pathogens in a single virus vector was made possible with the introduction of a "reverse genetics" system for nonsegmented negative-sense RNA viruses. Newcastle disease virus (NDV), a nonsegmented negative-sense virus, is one of the major viruses of economic importance in the poultry industry throughout the world. Despite the availability of live virus vaccines of good potency, the intrinsic ability of attenuated strains to revert in virulence makes control of this disease by vaccination difficult. Armed with the knowledge of virulence factors of this virus, it is now possible to produce genetically stable vaccines and to engineer mutations that enhance immunogenicity. The modular nature of the genome of this virus facilitates engineering additional genes from several different pathogens or tumor-specific antigens to design contemporary vaccines for animals and humans. This review will summarize the developments in using NDV as a vaccine vector and the potential of this approach in designing next generation vaccines for veterinary use.

Cell mediated immunity to Plasmodium vivax infection: in vitro inhibition of parasite growth by monocyte derived macrophages.

OBJECTIVE: To study the ability of soluble blood stage or cell associated antigens of Plasmodium vivax to stimulate human peripheral blood mononuclear cells (PBMC) and produce factors capable of causing inhibition of parasite growth in vitro was the objective of this investigation. METHOD: A local isolate of P vivax was either synchronized by triple sorbitol lysis for antigen preparation or used as unsynchronized culture for parasite inhibition, employing a macrophage inhibition assay. The soluble or cell associated antigens of P vivax were added to human monocyte derived macrophages with P vivax parasitized red blood cells. The percent inhibition of parasite growth was examined after 72 hrs by microscopy of Giemsa stained smears of red blood cells from the experimental and control groups. RESULTS: The differences in parasite inhibition were compared using Wilcoxon rank sum test for paired differences. Unstimulated PBMC supernatants did not inhibit parasite growth. Significant inhibition of parasite growth (90%) was seen after incubating P vivax infected erythrocytes with PBMC supernatants resulting from stimulation with soluble antigens (T = 3; P < 0.05). However, the cell associated antigens of P vivax did not stimulate PBMC to activate macrophages for parasite killing in vitro (T = 14, P < 0.05). CONCLUSION: We conclude that the soluble blood stage antigens of P vivax can stimulate human PBMC to produce factors capable of activating macrophages to function as effector cells in P vivax malaria.

Quantitative counter-immunoelectrophoresis for estimation of antibodies to infectious bursal disease virus.

Quantitative counter-immunoelectrophoresis was standardized to detect antibodies to the avian infectious bursal disease virus. This technique correlated well with the conventional quantitative agar gel precipitation test in estimating antibodies to IBDV. The use of blood dried on filter paper as an alternative to serum is discussed. QCIE is simple, easy to perform and faster than QAGP.

In vitro and numerical support for combinatorial irreversible electroporation and electrochemotherapy glioma treatment.

Irreversible electroporation (IRE) achieves targeted volume non-thermal focal ablation using a series of brief electric pulses to kill cells by disrupting membrane integrity. Electrochemotherapy (ECT) uses lower numbers of sub-lethal electric pulses to disrupt membranes for improved drug uptake. Malignant glioma (MG) brain tumors are difficult to treat due to diffuse peripheral margins into healthy neural tissue. Here, in vitro experimental data and numerical simulations investigate the feasibility for IRE-relevant pulse protocols with adjuvant ECT drugs to enhance MG treatment. Cytotoxicity curves were produced on two glioma cell lines in vitro at multiple pulse strengths and drug doses with Bleomycin or Carboplatin. Pulses alone increased cytotoxicity with higher pulse numbers and strengths, reaching >90% by 800 V/cm with 90 pulses. Chemotherapeutic addition increased cytotoxicity by >50% for 1 ng/mL concentrations of either drug relative to 80 pulses alone with J3T cells at electric fields ≥400 V/cm. In addition to necrosis, transmission electron microscopy visualizes apoptotic morphological changes and Hoescht 33342 staining shows apoptotic cell fractions varying with electric field and drug dose relative to controls. Numerically simulated treatment volumes in a canine brain show IRE combined with ECT expands therapeutic volume by 2.1-3.2 times compared to IRE alone.

Mathematical model of the role of intercellular signalling in intercellular cooperation during tumorigenesis.

OBJECTIVES: Intercellular cooperation has been hypothesized to enhance cell proliferation during cancer metastasis through autocrine signalling cascades and mathematical models can provide valuable insights into underlying mechanisms of metastatic tumorigenesis. Here, we present a model that incorporates signal-stimulated cell proliferation, and investigate influences of diffusion-driven heterogeneity in signal concentration on proliferation dynamics. MATERIALS AND METHODS: Our model incorporates signal production through both autocrine and paracrine pathways, and signal diffusion and loss for a metastasizing cell population at a host site. We use the signalling pathway of IL-6 for illustration where this signalling species forms an intermediate complex with its receptor IL-6R. This in turn forms a heterodimeric complex with transmembrane protein gp130, ultimately resulting in production of downstream signals. Cell population dynamics are taken to follow a modified logistic equation for which the rate term is dependent on local IL-6 concentration. RESULTS AND CONCLUSIONS: Our spatiotemporal model agrees closely with experimental results. The model is also able to predict two phenomena typical of metastatic tumorigenesis - host tissue preference and long periods of proliferation dormancy. It confirms that diffusivity of the signalling species in a host tissue plays a significant role during the process. Our results show that the proliferation-apoptosis balance is tipped in favour of the former for host sites that have relatively smaller signal diffusivities.

Changes in the Cytokine and Toll-Like Receptor Gene Expression Following Infection of Indigenous and Commercial Chickens With Infectious bursal disease virus.

A comparative study of cytokine and toll-like receptor (TLR) mRNA expression in 3 weeks old indigenous and commercial chickens infected with a very virulent strain of Infectious bursal disease virus (IBDV) was performed using a custom-made microarray chip. In uninfected indigenous chickens, the basal levels of interleukin (IL) 15 were lower and IL 16 was higher than their commercial counterparts. In the IBDV infected indigenous chickens, only IL16 gene expression was down regulated, while TLR3 expression was up regulated significantly. In the IBDV infected commercial chickens IL15, IL16 and TLR3 were down regulated. But, IL1-ß, IL2, IL8, IL12, IL17, interferon (IFN)-α and ß were significantly increased compared with the control. In IBDV infected indigenous chickens, IL15, IFN-γ, beta-defensin and TLR3 were up regulated compared to virus-infected commercial chickens. The results suggested that up regulation of TLR3, a ligand for double-stranded (ds) RNA probably could account for the possible clinical resistance in these birds. There was a 5.2 fold difference by quantitative real-time RT-PCR between indigenous and commercial chickens in TLR3 mRNA expression. Therefore, TLR3, a receptor for dsRNA could be a putative molecule that could play a role in differential innate and adaptive immune responses to IBDV in commercial and indigenous chickens.

Efficient fluorescence-based imaging methods for quantitating infectivity and oncolytic efficacy of Newcastle disease virus.

A high throughput quantitation protocol is desired to determine the replication of various recombinant oncolytic viruses in vitro. Plaque assay is the classic method for viral infectivity quantitation but is laborious and time consuming; moreover it does not report the oncolytic efficacy of a virus. In this paper, three new imaging methods for quantitating viral infectivity are derived and evaluated: fluorescence intensity, infection counts, and infection degree. Infection of oncolytic Newcastle disease virus in human tumor and normal cells was followed over a time course by plaque assay and the imaging methods. For the latter, brightfield and green channel images were acquired at various fixed locations in the cell culture, and later analyzed. One of the imaging methods was found to be highly correlated with viral titer; the other methods are complementary to plaque assay and provide additional information like oncolytic efficacy, syncytium formation etc. The new methods significantly reduce the time and material costs required by plaque assay, and provide an efficient system for quantitating and characterizing infectivity and efficacy of oncolytic viruses.

Detection of a new Mycobacterium species in wild striped bass in the Chesapeake Bay.

Investigation into recent declines in striped bass health in the Chesapeake Bay in Maryland resulted in the isolation of a putative new species of Mycobacterium. This isolate was obtained from fish showing skin ulcers and internal granulomas in various organs. The isolate was slow growing at 28 degrees C; was nonchromogenic; showed no activities of nitrate reduction, catalase activity, Tween 80 hydrolysis, tellurite reduction, or arylsulfatase reduction; grew best at low salt concentrations; and was urease and pyrazinamidase positive. By PCR a unique insertional sequence was identified which matched nothing in any database. Analysis of the nearly complete 16S rRNA gene sequence also indicated a unique sequence which had 87.7% sequence homology to Mycobacterium ulcerans, 87.6% homology to Mycobacterium tuberculosis, and 85.9% homology to Mycobacterium marinum. Phylogenetic analysis placed the organism close to the tuberculosis complex. These data support the conclusion that the isolate probably represents a new mycobacterial species.

Rocket immunoelectrophoresis in the diagnosis of infectious bursal disease.

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.

In ovo delivery of DNA to the avian embryo.

We have developed a simple method of transfecting avian embryos in ovo with various plasmid vectors that results in protein expression in the embryo. Using the chloramphenicol acetyl transferease (CAT) reporter gene, we were able to show that transfecting avian embryos with a plasmid/neutral lipid/dimethylsulfoxide (DMSO) mixture delivered to the air cell, is better than transfecting naked DNA or cationic lipid encapsulated DNA, using DMSO (P < 0.05). This method resulted in CAT expression in several avian embryonic tissues of all the embryos inoculated. We found that both the cytomegalovirus (CMV) and chicken beta actin promoters worked significantly better (P < 0.05) than the Rous sarcoma virus promoter in vitro for reporter gene expression after cationic liposome-mediated transfection. However, after in ovo delivery with neutral lipid encapsulation and DMSO mediated delivery, no significant difference (P > 0.05) between the various promoters could be determined. We believe this neutral lipid encapsulation method may represent an important platform for delivery of DNA to the avian embryo.

Estimation of antibodies to Newcastle disease virus in tears.

Micro-haemagglutination inhibition tests (Micro-HI) were used to measure the level of maternal IgG in the tears of chicks and also to measure the levels of HI antibodies in the tears and serum after vaccination with "F" strain of Newcastle disease virus (NDV) and in the face of an outbreak of Newcastle disease. There was a 1.4 fold difference between the maternal IgG concentration in the serum and tears. The ratio of serum IgG to lachrymal IgG after maternal transfer was 4 to 5:1 on day 4 to 9 and decreased to 2.6:1 on day 12 post-hatch. The intra-ocular vaccination of chicks with "F" strain of NDV resulted in the highest titre of HI antibodies in the tears though there was no significant difference in the response of chicks vaccinated through intranasal, oral and intravenous routes. In the face of an ND outbreak, the level of HI antibodies in the tears during the acute phase was very high and persisted at the same level for 14 days.

Characterisation of Newcastle disease viruses isolated in India.

Eleven Newcastle disease virus (NDV) isolates obtained from outbreaks of disease in chickens (9) and Japanese quail (2) in Tamil Nadu, India were characterised in pathogenicity tests, antigenically, using mouse monoclonal antibodies (MAbs), and other established tests devised to distinguish between different strains. All 11 isolates were shown to be highly virulent for chickens. In indirect immunoperoxidase tests used to assess the ability of a panel of 28 MAbs to bind to infected cell cultures, 10 of the isolates showed an identical reaction pattern, the other isolate (No. 4) failed to react with one MAb which bound to cells infected with the other isolates. Isolates 9 was unstable at pH 3 while the other 10 were stable. All other properties were shared by the 11 isolates.