66 resultados para Antibodies, Monoclonal, Humanized


Relevância:

20.00% 20.00%

Publicador:

Resumo:

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Purpose: UC is a disease of the entire urothelium, characterized by multiplicity and multifocality. The clonal relationship among multiple UCs has implications regarding adjuvant chemotherapy. It has been investigated in studies of chromosomal alteration and single gene mutation. However, these genetic changes can occur in unrelated tumors under similar carcinogenic selection pressures. Tumors with high MSI have numerous DNA mutations, of which many provide no selection benefit. While these tumors represent an ideal model for studying UC clonality, their low frequency has prevented their previous investigation. Materials and Methods: We investigated 32 upper and lower urinary tract UCs with high MSI and 4 nonUC primary cancers in 9 patients. We used the high frequency and specificity of individual DNA mutations in these tumors (MSI at 17 loci) and the early timing of epigenetic events (methylation of 7 gene promoters) to investigate tumor clonality. Results: Molecular alterations varied among tumors from different primary organs but they appeared related in the UCs of all 9 patients. While 7 patients had a high degree of concordance among UCs, in 2 the UCs shared only a few similar alterations. Genetic and epigenetic abnormalities were frequently found in normal urothelial samples. Conclusions: Multiple UCs in each patient appeared to arise from a single clone. The molecular order of tumor development varied from the timing of clinical presentation and suggested that residual malignant cells persist in the urinary tract despite apparent curative surgery. These cells lead to subsequent tumor relapse and new methods are required to detect and eradicate them.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

OBJECTIVES To determine whether the seroprevalence of antibodies to varicella zoster virus (VZV) in adults is similar to that reported in tropical populations elsewhere. METHODS We measured the seroprevalence of VZV IgG antibodies, using an enzyme immunoassay (EIA) in women attending an antenatal clinic in an urban centre in tropical Australia. RESULTS The overall seroprevalence of VZV antibodies in 298 women was 92% (95% CI 88-95), with no difference between women who spent their childhoods in the tropics and colleagues. None of the overseas-born women was seronegative. CONCLUSION The seroprevalence of VZV antibodies in this tropical population in Australia is as high as that reported from temperate regions, suggesting that social and cultural factors and population mobility are more important determinants of age distribution of VZV immunity than tropical climate.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Background Matrix metalloproteinases (MMPs) are a family of endopeptidases that digest the extracellular matrix (ECM). Overexpression of different MMPs has been shown to promote tumour cell invasion in vitro. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs that also possess growth-promoting properties. Aims To analyse the expression profile of MMP-2, MMP-9 and TIMP-2 in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Patient follow up was a minimum of 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-2, MMP-9 and TIMP-2. Results The results of the immunohistochemistry are set out below. marker tumour expression log-rank survival stromal expression log-rank survival MMP-2 9/72 (13%) p=0.10 34/72 (47%) p=0.34 MMP-9 79/152 (52%) p=0.04* 69/152 (45%) p=0.84 TIMP-2 28/90 (31%) p=0.04* 66/90 (73%) p=0.90 Two or more 16/59 (27%) p=0.007* There were no associations between expression and clinicopathological findings for any tumour marker. There was co-expression of MMP-2 and MMP-9 in tumour cells (p=0.01). Conclusions MMP-2, MMP-9 and TIMP-2 are expressed in NSCLC. MMP-9 and TIMP-2 tumour expression correlate with a poor outcome (both p=0.04) and are potential prognostic markers for NSCLC. Cumulative expression of two or more MMPs/TIMPs may also have increased prognostic significance. Proteases and their inhibitors are novel targets for therapeutic intervention and should be evaluated in NSCLC.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

This is the first report of an antibody-fusion protein expressed in transgenic plants for direct use in a medical diagnostic assay. By the use of gene constructs with appropriate promoters, high level expression of an anti-glycophorin single-chain antibody fused to an epitope of the HIV virus was obtained in the leaves and stems of tobacco, tubers of potato and seed of barley. This fusion protein replaces the SimpliRED™ diagnostic reagent, used for detecting the presence of HIV-1 antibodies in human blood. The reagent is expensive and laborious to produce by conventional means since chemical modifications to a monoclonal antibody are required. The plant-produced fusion protein was fully functional (by ELISA) in crude extracts and, for tobacco at least, could be used without further purification in the HIV agglutination assay. All three crop species produced sufficient reagent levels to be superior bioreactors to bacteria or mice, however barley grain was the most attractive bioreactor as it expressed the highest level (150 μg of reagent g-1), is inexpensive to produce and harvest, poses a minuscule gene flow problem in the field, and the activity of the reagent is largely undiminished in stored grain. This work suggests that barley seed will be an ideal factory for the production of antibodies, diagnostic immunoreagents, vaccines and other pharmaceutical proteins.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

The majority of non-small cell lung cancer (NSCLC) patients present with advanced disease and with a 5 year survival rate of <15% for these patients, treatment outcomes are considered extremely disappointing. Standard chemotherapy regimens provide some improvement to ~40% of patients. However, intrinsic and acquired chemoresistance are a significant problem and hinder sustained long term benefits of such treatments. Advances in proteomic and genomic profiling have increased our understanding of the aberrant molecular mechanisms that are driving an individual's tumour. The increased sensitivity of these technologies has enabled molecular profiling at the stage of initial biopsy thus paving the way for a more personalised approach to the treatment of cancer patients. Improvements in diagnostics together with a wave of new targeted small molecule inhibitors and monoclonal antibodies have revolutionised the treatment of cancer. To date there are essentially three targeted agents approved for clinical use in NSCLC. The tyrosine kinase inhibitor (TKI) erlotinib, which targets the epidermal growth factor receptor (EGFR) TK domain, has proven to be an effective treatment strategy in patients who harbour activating mutations in the EGFR TK domain. Bevacizumab a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) can improve survival, response rates, and progression-free survival when used in combination with chemotherapy. Crizotinib, a small-molecule drug, inhibits the tyrosine kinase activity of the echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion protein, resulting in decreased tumour cell growth, migration, and invasiveness in patients with locally advanced or metastatic NSCLC. The clinical relevance of several other targeted agents are under investigation in distinct molecular subsets of patients with key "driver" mutations including: KRAS, HER2, BRAF, MET, PIK3CA, AKT1,MAP2K1, ROS1 and RET. Often several pathways are activated simultaneously and crosstalk between pathways allows tumour cells to escape the inhibition of a single targeted agent. This chapter will explore the clinical development of currently available targeted therapies for NSCLC as well as those in clinical trials and will examine the synergy between cytotoxic therapies.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Background The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Method An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Background Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. Methods The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. Results Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. Conclusions These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Background Infection with human herpesvirus 8 (HHV-8) has been consistently linked to Kaposi's sarcoma, but its mode of transmission, association with other cancers, and interaction with the human immunodeficiency virus type 1 (HIV-1) are largely unknown. Methods Between January 1992 and December 1997, we interviewed 3591 black patients with cancer in Johannesburg and Soweto, South Africa. Blood was tested for antibodies against HIV-1 and HHV-8 in 3344 of the patients. Antibodies against HHV-8 were detected with an indirect immunofluorescence assay. The intensity of the fluorescent signal correlated well with the titers of antibodies (P<0.001). The relations among the presence of anti–HHV-8 antibodies, sociodemographic and behavioral factors, type of cancer, and the presence or absence of coexistent HIV-1 infection were examined with the use of unconditional logistic-regression models. Results Among the 3293 subjects with cancers other than Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 32 percent, which did not differ significantly from the standardized seroprevalence among black blood donors. Among these 3293 patients, the prevalence of antibodies against HHV-8 increased with increasing age (P<0.001) and an increasing number of sexual partners (P=0.05) and decreased with increasing years of education (P=0.007); it was not strongly associated with HIV-1 infection. Anti–HHV-8 antibodies were more frequent among black than white blood donors (P<0.001). Among the 51 patients with Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 83 percent, significantly higher than the prevalence among those without Kaposi's sarcoma (P<0.001). For 16 other specific types of cancer, including multiple myeloma (108 cases) and prostate cancer (202 cases), the variation in the standardized seroprevalence of antibodies against HHV-8 was not remarkable. At a given intensity of fluorescence of anti–HHV-8 antibodies, Kaposi's sarcoma was more frequent among HIV-1–positive patients than among those who were HIV-1–negative (P<0.001). Conclusions Among black patients with cancer in South Africa, the seroprevalence of anti–HHV-8 antibodies is high and is specifically associated with Kaposi's sarcoma, particularly at high titers.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p\0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.