A 3-D interactive microbiology laboratory via virtual reality for enhancing practical skills.

Virtual Reality (VR) laboratories are a new pedagogical approach to support psychomotor skills development in undergraduate programmes to achieve practical competency. VR laboratories are successfully used to carry out virtual experiments in science courses and for clinical skills training in professional courses. This paper describes the development and evaluation of a VR-based microbiology laboratory on Head-Mounted Display (HMD) for undergraduate students. Student and faculty perceptions and expectations were collected to incorporate into the laboratory design. An interactive 3-dimensional VR laboratory with a 360° view was developed simulating our physical laboratory setup. The laboratory environment was created using Unity with the (created) necessary assets and 3D models. The virtual laboratory was designed to replicate the physical laboratory environment as suggested by the students and faculty. In this VR laboratory, six microbiology experiments on Gram staining, bacterial streaking, bacterial motility, catalase test, oxidase test and biochemical tests were placed on the virtual platform. First-year biomedical science students were recruited to evaluate the VR laboratory. Students' perception of the virtual laboratory was positive and encouraging. About 70% of the students expressed they felt safe using the VR laboratory and that it was engaging. They felt that the VR laboratory provided an immersive learning experience. They appreciated that they could repeat each experiment multiple times without worrying about mistakes or mishaps. They could personalise their learning by concentrating on the specific experiments. Our in-house VR-based microbiology laboratory was later extended to other health professions programmes teaching microbiology.

Protective effects of madecassoside, a triterpenoid from Centella asiatica, against oxidative stress in INS-1E cells.

Progressive decline in ß cell function and reduction in the ß cell mass is important in type 2 diabetes. Here, we tested the hypothesis that madecassoside's previously demonstrated in vivo protective effects on the ß cell in experimental diabetes were exerted directly. We investigated the effects of madecassoside in protecting a ß cell line (INS-1E) against a variety of agents. INS-1E cells were treated with madecassoside in the presence of high glucose (HG), a cytokine mixture, hydrogen peroxide (H2O2), or streptozotocin (STZ). HG, the cytokine mixture, H2O2 and STZ each produced a significant decrease in cell viability; this was significantly reversed by madecassoside. Pre-treatment with madecassoside reduced the number of apoptotic cells induced by HG, the cytokine mixture, H2O2, and STZ, and concentration-dependently reduced ROS production. Madecassoside also significantly enhanced glucose-induced insulin secretion. The results suggest that madecassoside's in vivo effects are exerted directly on the ß cell.

Effect of madecassoside in reducing oxidative stress and blood glucose in streptozotocin-nicotinamide-induced diabetes in rats.

OBJECTIVES: Madecassoside (MAD) is a triterpenoid constituent of Centella asiatica (L.) Urb., an ethnomedical tropical plant, extracts of which were shown to reduce blood glucose in experimental diabetes. This study examines MAD for its anti-hyperglycaemic effects and tests the hypothesis that it reduces the blood glucose in experimentally induced diabetic rats by protecting the ß-cells. METHODS: Diabetes was induced using streptozotocin (60 mg/kg, i.v.) followed by nicotinamide (210 mg/kg, intraperitoneal (i.p.)). MAD (50 mg/kg) was administered orally for 4 weeks, commencing 15 days after induction of diabetes; resveratrol (10 mg/kg) was used as a positive control. Fasting blood glucose, plasma insulin, HbA1c, liver and lipid parameters were measured, along with antioxidant enzymes and malondialdehyde as an index of lipid peroxidation; histological and immunohistochemical studies were also undertaken. KEY FINDINGS: MAD normalized the elevated fasting blood glucose levels. This was associated with increased plasma insulin concentrations. MAD alleviated oxidative stress by improving enzymatic antioxidants and reducing lipid peroxidation. Histopathological examination showed significant recovery of islet structural degeneration and an increased area of islets. Immunohistochemical staining showed increased insulin content in islets of MAD-treated rats. CONCLUSIONS: The results demonstrate an antidiabetic effect of MAD associated with preservation of ß-cell structure and function.

Self-Regulated Learning Practice of Undergraduate Students in Health Professions Programs.

BACKGROUND: University students are expected to take charge of their learning without being dependent on teachers. Self-regulated learning (SRL) is the process by which students direct their learning to achieve their set targets and goals in a timely and controlled manner. This study was undertaken to explore the practice of SRL by undergraduate students from different programs in a health science focused university during COVID-19 pandemic. METHOD: Thirty-three undergraduate students of five health professions education programs were recruited to take part in focus group discussions to explore their SRL practice with online learning. Their responses were subjected to thematic analysis. RESULT: Our students appeared to practice SRL, going through the phases of forethought and goal setting, performance and self-reflection. They set goals for academic as well as personal development in the university. Academic goals like achieving target GPA or marks were achieved by following different study techniques, personal management including time management, and by creating a conducive learning environment. Personal development such as interpersonal skills, social networking was achieved through socializing and participating in extracurricular activities. The students also engaged in self-reflection and analysis of their own performance followed by designing strategies to manage the challenges they faced. CONCLUSION: Undergraduates of health professions programs appear to show evidence of practicing SRL. Although impacted by COVID-19 induced lockdown and online learning, they seem to have strategized and achieved their goals through individualized SRL processes. Promoting and fostering an atmosphere of SRL in universities to cater to the needs of the students would help them be more successful in their careers.

Role of microRNAs as biomarkers of cervical carcinogenesis: a systematic review.

We performed a systematic review to identify the role of microRNAs (miRNAs) as biomarkers in the progression of cervical precancerous lesions. A comprehensive search of the Cochrane Controlled Register of Trials, PubMed, ScienceDirect, and Embase databases was performed for articles published between January 2010 and June 2020. The following Medical Subject Headings (MeSH) terms were searched: "microRNA" and "cervical" and "lesion." All study designs that aimed to evaluate the correlation of miRNA expression with different precancerous cervical staging and/or cervical cancer were included, except for case reports and case series. Approximately 82 individual miRNAs were found to be significant in differentiating the stages of cervical carcinogenesis. Among the miRNAs, miR-21 is the most prevalent, and it is consistently upregulated progressively from normal cervical to worsening cervical lesion stages in both cell and serum samples. miR-205 has been shown to have a higher specificity than human papilloma virus testing in predicting the absence of high-grade squamous intraepithelial lesions (HSILs) in exfoliated cell samples. The tumor suppressor miRNAs miR-34, let-7, miR-203 miR-29, and miR-375 were significantly downregulated in low-grade squamous intraepithelial lesions, HSILs, and cervical cancer. We found significant dysregulated miRNAs in cervical carcinogenesis with their dynamic expression changes and ability to detect viral persistency, risk prediction of low-grade lesions (cervical intraepithelial neoplasia [CIN] 2) to high-grade lesions (CIN 3), and progression of CIN 3 to cancer. Their ability to discriminate HSILs from non-dysplastic lesions has potential implications in early diagnosis and reducing overtreatment of otherwise regressive early preinvasive lesions.

Inhibition of breast cancer xenografts in a mouse model and the induction of apoptosis in multiple breast cancer cell lines by lactoferricin B peptide.

Breast cancer has a diverse aetiology characterized by the heterogeneous expression of hormone receptors and signalling molecules, resulting in varied sensitivity to chemotherapy. The adverse side effects of chemotherapy coupled with the development of drug resistance have prompted the exploration of natural products to combat cancer. Lactoferricin B (LfcinB) is a natural peptide derived from bovine lactoferrin that exhibits anticancer properties. LfcinB was evaluated in vitro for its inhibitory effects on cell lines representing different categories of breast cancer and in vivo for its suppressive effects on tumour xenografts in NOD-SCID mice. The different breast cancer cell lines exhibited varied levels of sensitivity to apoptosis induced by LfcinB in the order of SKBR3>MDA-MB-231>MDA-MB-468>MCF7, while the normal breast epithelial cells MCF-10A were not sensitive to LfcinB. The peptide also inhibited the invasion of the MDA-MB-231 and MDA-MB-468 cell lines. In the mouse xenograft model, intratumoural injections of LfcinB significantly reduced tumour growth rate and tumour size, as depicted by live imaging of the mice using in vivo imaging systems (IVIS). Harvested tumour volume and weight were significantly reduced by LfcinB treatment. LfcinB, therefore, is a promising and safe candidate that can be considered for the treatment of breast cancer.

Enantiomeric pairs of ternary copper(ii) complexes and their aldol-type condensation products: synthesis, characterization, and anticancer and epigenetic properties.

Chiral enantiomers [Cu(phen)(l-ser)(H2O)]NO31 and [Cu(phen)(d-ser)(H2O)]NO32 (ser = serinato) underwent aldol-type condensation with formaldehyde, with retention of chirality, to yield their respective enantiomeric ternary copper(ii) complexes, viz. l- and d-[Cu(phen)(OCA)(H2O)]NO3·xH2O (3 and 4; phen = 1,10-phenanthroline; OCA = oxazolidine-4-carboxylate; x = 1/2, 0-2) respectively. These chiral complexes were characterized by FTIR, elemental analysis, circular dichroism, UV-visible spectroscopy, fluorescence spectroscopy (FL), molar conductivity measurement, ESI-MS and X-ray crystallography. The crystal structures of 1 and 3 showed both the cationic complexes to have a square pyramidal geometry. These complexes were about nine fold more potent than cisplatin against metastatic MDA-MB-231 breast cancer cells, inducing apoptotic cell death via ROS generation and a massive drop in mitochondrial membrane potential. The results of monitoring EZH1, EZH2 and H3K27me3 revealed that the mode of action of 1-4 also involved the downregulation of EZH2 and it seemed to be independent of the H3K27me3 status.

Cationic chitosan-propolis nanoparticles alter the zeta potential of S. epidermidis, inhibit biofilm formation by modulating gene expression and exhibit synergism with antibiotics.

Staphylococcus epidermidis, is a common microflora of human body that can cause opportunistic infections associated with indwelling devices. It is resistant to multiple antibiotics necessitating the need for naturally occurring antibacterial agents. Malaysian propolis, a natural product obtained from beehives exhibits antimicrobial and antibiofilm properties. Chitosan-propolis nanoparticles (CPNP) were prepared using Malaysian propolis and tested for their effect against S. epidermidis. The cationic nanoparticles depicted a zeta potential of +40 and increased the net electric charge (zeta potential) of S. epidermidis from -17 to -11 mV in a concentration-dependent manner whereas, ethanol (Eth) and ethyl acetate (EA) extracts of propolis further decreased the zeta potential from -17 to -20 mV. Confocal laser scanning microscopy (CLSM) depicted that CPNP effectively disrupted biofilm formation by S. epidermidis and decreased viability to ~25% compared to Eth and EA with viability of ~60-70%. CPNP was more effective in reducing the viability of both planktonic as well as biofilm bacteria compared to Eth and EA. At 100 µg/mL concentration, CPNP decreased the survival of biofilm bacteria by ~70% compared to Eth or EA extracts which decreased viability by only 40%-50%. The morphology of bacterial biofilm examined by scanning electron microscopy depicted partial disruption of biofilm by Eth and EA extracts and significant disruption by CPNP reducing bacterial number in the biofilm by ~90%. Real time quantitative PCR analysis of gene expression in treated bacteria showed that genes involved in intercellular adhesion such as IcaABCD, embp and other related genes were significantly downregulated by CPNP. In addition to having a direct inhibitory effect on the survival of S. epidermidis, CPNP showed synergism with the antibiotics rifampicin, ciprofloxacin, vancomycin and doxycycline suggestive of effective treatment regimens. This would help decrease antibiotic treatment dose by at least 4-fold in combination therapies thereby opening up ways of tackling antibiotic resistance in bacteria.

Root canal irrigants influence the hydrophobicity and adherence of Staphylococcus epidermidis to root canal dentin: an in vitro study.

OBJECTIVES: To determine the effect of root canal irrigants on the hydrophobicity and adherence of Staphylococcus epidermidis (S. epidermidis) to root canal dentin in vitro. MATERIALS AND METHODS: Root dentin blocks (n = 60) were randomly divided into 4 groups based on the irrigation regimen: group 1, saline; group 2, 5.25% sodium hypochlorite (NaOCl); group 3, 5.25% NaOCl followed by 17% ethylenediaminetetraacetic acid (EDTA); group 4, same as group 3 followed by 2% chlorhexidine (CHX). The hydrophobicity of S. epidermidis to root dentin was calculated by cell surface hydrophobicity while the adherence was observed by fluorescence microscopy, and bacteria were quantified using ImageJ software (National Institutes of Health). Statistical analysis of the data was done using Kruskal-Wallis test and Mann-Whitney U test (p = 0.05). RESULTS: The hydrophobicity and adherence of S. epidermidis to dentin were significantly increased after irrigating with group 3 (NaOCl-EDTA) (p < 0.05), whereas in group 4 (NaOCl-EDTA-CHX) both hydrophobicity and adherence were significantly reduced (p < 0.05). CONCLUSIONS: The adherence of S. epidermidis to dentin was influenced differently by root canal irrigants. Final irrigation with CHX reduces the bacterial adherence and may impact biofilm formation.

Correction: Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms.

[This corrects the article DOI: 10.1371/journal.pone.0174888.].

Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms.

Propolis obtained from bee hives is a natural substance with antimicrobial properties. It is limited by its insolubility in aqueous solutions; hence ethanol and ethyl acetate extracts of Malaysian propolis were prepared. Both the extracts displayed antimicrobial and anti-biofilm properties against Enterococcus faecalis, a common bacterium associated with hospital-acquired infections. High performance liquid chromatography (HPLC) analysis of propolis revealed the presence of flavonoids like kaempferol and pinocembrin. This study investigated the role of propolis developed into nanoparticles with chitosan for its antimicrobial and anti-biofilm properties against E. faecalis. Bacteria that grow in a slimy layer of biofilm are resistant to penetration by antibacterial agents. The use of nanoparticles in medicine has received attention recently due to better bioavailability, enhanced penetrative capacity and improved efficacy. A chitosan-propolis nanoformulation was chosen based on ideal physicochemical properties such as particle size, zeta potential, polydispersity index, encapsulation efficiency and the rate of release of the active ingredients. This formulation inhibited E. faecalis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 µg/ml concentration. When tested on pre-formed biofilms, the formulation reduced bacterial number in the biofilm by ~40% and ~75% at 200 and 300 µg/ml, respectively. The formulation not only reduced bacterial numbers, but also physically disrupted the biofilm structure as observed by scanning electron microscopy. Treatment of biofilms with chitosan-propolis nanoparticles altered the expression of biofilm-associated genes in E. faecalis. The results of this study revealed that chitosan-propolis nanoformulation can be deemed as a potential anti-biofilm agent in resisting infections involving biofilm formation like chronic wounds and surgical site infections.

Depletion of regulatory T-cells leads to moderate B-cell antigenicity in respiratory syncytial virus infection.

OBJECTIVES: The regulation of the immunopathology of respiratory syncytial virus (RSV) by regulatory T-cells (CD4(+)CD25(+)Foxp3(+); Tregs) is not understood. METHODS: To deduce the same, Tregs were depleted in BALB/c mice by injecting anti-CD25 antibody followed by RSV infection (anti-CD25-RSV mice). RESULTS: In this model, a decrease in anti-fusion (F) antibody and neutralizing activity, and an increase in anti-nucleocapsid (N) antibody in serum, were seen. Decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity, increased IgG2a, and an influx of activated CD8(+) T-cells into the lungs were also observed. Co-culture of splenic CD45RA(+) B-cells from RSV-infected normal mice with CD4(+) cells isolated from anti-CD25-RSV mice (B/CD4) increased anti-F antibody secretion. The inclusion of CD25(+) Tregs isolated from isotype Ig-RSV mice into the B/CD4 co-culture substantially enhanced the frequency of anti-F antibody production. However, the same effect was not seen in the co-culture of CD45RA(+) B-cells with dendritic cells (DCs) (B/DCs) or CD8(+) cells (B/CD8) that were obtained from anti-CD25-RSV mice. The transfer of enriched B-cells from anti-CD25-RSV mice into RSV-infected SCID mice increased severe lung inflammation associated with the increased viral load and eosinophil number. CONCLUSIONS: These results indicate that Tregs modulate B-cell activity, particularly in producing F-specific neutralizing antibodies, to regulate RSV-mediated exacerbated diseases.

Heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy.

Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90ß siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90ß resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90ß and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.

Human SCARB2 transgenic mice as an infectious animal model for enterovirus 71.

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.

Human SCARB2-mediated entry and endocytosis of EV71.

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.

Immunoprotectivity of HLA-A2 CTL peptides derived from respiratory syncytial virus fusion protein in HLA-A2 transgenic mouse.

Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33-41), 13 (F214-222), 14 (F273-281), and 23 (F559-567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.

Functional interaction between Env oncogene from Jaagsiekte sheep retrovirus and tumor suppressor Sprouty2.

BACKGROUND: Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus capable of transforming target cells in vitro and in vivo. The Envelope (Env) gene from JSRV and from related retroviruses can induce oncogenic transformation, although the detailed mechanism is yet to be clearly understood. Host cell factors are envisaged to play a critical determining role in the regulation of Env-mediated cell transformation. RESULTS: JSRV Env-mediated transformation of a lung adenocarcinoma cell line induced rapid proliferation, anchorage-independent growth and tumor formation, but completely abrogated the migration ability. An analysis of the signaling scenario in the transformed cells suggested the involvement of the ERK pathway regulated by Sprouty2 in cell migration, and the PI3K-Akt and STAT3 pathways in proliferation and anchorage-independence. On the other hand, in a normal lung epithelial cell line, Env-mediated transformation only decreased the migration potential while the other functions remained unaltered. We observed that Env induced the expression of a tumor suppressor, Sprouty2, suggesting a correlation between Env-effect and Sprouty2 expression. Overexpression of Sprouty2 per se not only decreased the migratory potential and tumor formation potential of the target cells but also made them resistant to subsequent Env-mediated transformation. On the other hand, over expression of the functional mutants of Sprouty2 had no inhibitory effect, confirming the role of Sprouty2 as a tumor suppressor. CONCLUSIONS: Our studies demonstrate that Env and Sprouty2 have a functional relationship, probably through shared signaling network. Sprouty2 functions as a tumor suppressor regulating oncogenic transformation of cells, and it therefore has the potential to be exploited as a therapeutic anti-cancer agent.

Generation and characterization of JSRV envelope transgenic mice in FVB background.

Jaagsiekte sheep retrovirus (JSRV) that causes contagious ovine pulmonary adenocarcinoma (OPA) in sheep carries an oncogenic Envelope gene (Env), which is capable of transforming target cells in vitro and in vivo. We cloned full-length JSRV Env cDNA into an expression vector, SPC/SV40, where the transgene was driven by lung-specific surfactant protein C (SPC) promoter, to obtain SPC-JSRV Env construct. SPC-JSRV Env was microinjected into immunocompetent FVB/N mice embryos to generate Env transgenic mice. We obtained two lines of transgenic mice, both of which were capable of developing spontaneous lung tumors from 1 month onwards and the tumor incidence rate was about 56% at the age of 7 months in Env Transgenic line 1 and about 71% at the age of 6 months in Env Transgenic line 2. We were able to correlate higher tumor incidence rate and tumorigenicity in Env Transgenic line 2 to higher level of expression of Env transgene compared to Env Transgenic line 1. Immunohistochemical analysis showed that the tumor was primarily composed of type II pneumocytes where SPC promoter is known to be active similar to natural infection of JSRV in sheep. Analysis of cellular mitogenic signal transduction pathways revealed significant induction of p44/42 ERK pathway in the transgenic mice lungs with tumors compared to the lungs from non-transgenic FVB/N mice. Tumors in our transgenic mice pose similarities to human lung adenocarcinoma and therefore our mice could serve as a model system for evaluating the mechanisms of lung tumorigenesis in vivo.

Immunogenic properties of RSV-B1 fusion (F) protein gene-encoding recombinant adenoviruses.

Two recombinant adenoviruses designated rAd-F0DeltaTM and rAd-F0 carrying the transmembrane truncated and full length of the F gene of the RSV-B1 strain, respectively, were engineered. Comparative immunogenicity studies in BALB/c mice showed that each vector was capable of inducing RSV-B1-specific antibodies that cross-reacted with the RSV-long and RSV-A2 viruses. The anti-RSV-B1 antibodies were neutralizing, and exhibited strong cross-neutralizing activity against the RSV-long and RSV-A2 isolates as well. Analysis of the cellular responses revealed that animals immunized with rAd-F0DeltaTM and rAd-F0 elicited CD4(+) T-cell responses of the Th1 and Th2 phenotypes, as well as F protein-specific CTLs. Production of Th2 cytokines (IL-4, IL-5 and IL-13) by splenocytes of the rAd-F0DeltaTM and rAd-F0 immunized mice was markedly lower than those released by animals administered with heat-inactivated RSV-B1 (HIRSV-B1). Comparison of the overall humoral and cellular responses suggests that rAd-F0DeltaTM is significantly more immunogenic than rAd-F0. The anti-viral immunity generated by both recombinant adenovirus vectors has conferred protection against live RSV-B1 challenge as judged by the lower viral load recovered in the lungs, a faster rate of recovery of body weight loss, and a lower count of eosinophils as compared to eosinophilia in mice immunized with HIRSV-B1. Results from these studies suggest that rAd-F0DeltaTM or rAd-F0 possess immunogenic properties that meet the requirements expected from potential RSV vaccine candidates.