Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

A novel algorithm for mask detection and recognizing actions of human.

Face recognition has become a significant challenge today since an increasing number of individuals wear masks to avoid infection with the novel coronavirus or Covid-19. Due to its rapid proliferation, it has garnered growing attention. The technique proposed in this chapter seeks to produce unconstrained generic actions in the video. Conventional anomaly detection is difficult because computationally expensive characteristics cannot be employed directly, owing to the necessity for real-time processing. Even before activities are completely seen, they must be located and classified. This paper proposes an expanded Mask R-CNN (Ex-Mask R-CNN) architecture that overcomes these issues. High accuracy is achieved by using robust convolutional neural network (CNN)-based features. The technique consists of two steps. First, a video surveillance algorithm is employed to determine whether or not a human is wearing a mask. Second, Multi-CNN forecasts the frame's suspicious conventional abnormality of people. Experiments on tough datasets indicate that our approach outperforms state-of-the-art online traditional detection of anomaly systems while maintaining the real-time efficiency of existing classifiers.

Genomic analysis of a rare recurrent Listeria monocytogenes prosthetic joint infection indicates a protected niche within biofilm on prosthetic materials.

Listeria monocytogenes is a rare cause of prosthetic joint infections (PJI). In this study, we describe a case of recurrent L. monocytogenes infections, 39 months apart, following debridement and retention of a prosthetic hip. Despite numerous studies reporting persistent L. monocytogenes in human infections, the genomic and phenotypic changes that clinically relevant strains undergo in the host are poorly understood. Improved knowledge of how PJI occurs is needed to improve the management of prosthetic infections. We used a combination of long- and short-read sequencing to identify any potential genomic differences between two L. monocytogenes isolates that occurred over 39-month incubation in the host. The isolates, QI0054 and QI0055, showed three single nucleotide polymorphisms and three insertions or deletions, suggesting that the recurrent infection was caused by the same strain. To identify potential differences in the capacity for persistence of these isolates, their biofilm-forming ability and potential to colonize prosthesis-relevant materials was investigated both in microtitre plates and on prosthetic material titanium, stainless steel 316 and ultra-high molecular weight polyethylene. Whilst the L. monocytogenes isolate from the most recent infection (QI0055) was able to form higher biofilm in microtitre plates, this did not lead to an increase in biomass on prosthetic joint materials compared to the initial isolate (QI0054). Both clinical isolates were able to form significantly more biofilm on the two metal prosthetic materials than on the ultra-high molecular weight polyethylene, in contrast to reference strain Scott A. Transcriptomics revealed 41 genes overexpressed in biofilm state and 643 in planktonic state. Moreover, genes with mutations were actively expressed in both isolates. We conclude the isolates are derived from the same strain and hypothesize that L. monocytogenes formed biofilm on the prosthetic joint materials, with minimal exposure to stresses, which permitted their survival and growth.

Enhancing proteasomal processing improves survival for a peptide vaccine used to treat glioblastoma.

Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in several trials for glioblastoma. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model in mice. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti-PD-1 therapy cured 45% of the Y6-pepVIII-vaccinated mice but was ineffective for pepVIII-treated mice. Liquid chromatography-tandem mass spectrometry analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant in Y6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments may be a component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. We hypothesize that the immune response to a vaccine represents the collective contribution from multiple PCPS and linear products. Our work suggests a strategy to increase proteasomal processing of a vaccine that results in an augmented immune response and enhanced survival in mice.

NLOphoric imidazole-fused fluorescent anthraquinone dyes.

The Z-scan and DFT techniques were explored to investigate the non-linear optical properties of anthraquinone fused imidazole-based D-π-A dyes. With the help of UV-visible spectral analysis, pH study, CV analysis, HOMO-LUMO interaction, MEP plots, and quinoidal character the influence of intramolecular charge transfer characteristics and H-bonding process on electronic and photophysical studies of anthraquinone derivatives were understood. The dyes 2-(4-(diethylamino)-2-hydroxyphenyl)-3H-anthra[1,2-d]imidazole-6,11-dione (AQ1) and 2-(2-hydroxynaphthalen-1-yl)-1H-anthra[1,2-d]imidazole-6,11-dione (AQ2) displayed a single emission with pronounced Stokes shift and thermal stability (upto 290 °C). The dye AQ1 exhibited strong charge transfer character which is explained by the ICT process leading to high nonlinear susceptibility χ(3) in AQ1 3.43 × 10-13 e.s.u relative to the dye AQ2 which has only ESIPT core. But, the dye AQ2 6.27 J cm-2 showed better optical limiting value. The NLO properties of AQ1 and AQ2 were computed by DFT functionals based on Hartree Fork (HF) percentage exchange. The dye AQ1 exhibits noticeable NLO properties. The global hybrid functionals with HF composition beyond 50% (BHHLYP, M06-HF) and the long rang corrected functionals (CAM-B3LYP, ωB97, and ωB97X) demonstrated comparable NLO properties relative to B3LYP and PBE0. It was observed that the combined effect of ICT and the H-bonding cores enhance the NLO properties. The experimental findings (Z-scan) were successfully correlated with theoretical (DFT) results.

Metagenomic insights into the fungal assemblages of the northwest Himalayan cold desert.

Psychrophilic fungi are a critical biotic component in cold deserts that serves a central role in nutrient recycling and biogeochemical cycles. Despite their ecological significance, culture-independent studies on psychrophilic mycobiome are limited. In the present study, the fungal diversity patterns across the Drass, an Indian cold desert in the Himalaya, were indexed by targeted amplicon pyrosequencing (ITS). In the Drass dataset, Ascomycota was represented by 92 genera, while 22 genera represented Basidiomycota. The most abundant genus was Conocybe (20.46%). Most of the identified genera were reported in the literature to be prolific extracellular hydrolytic enzyme producers. To identify whether the Drass fungal assemblages share similarities to other cold deserts, these were further compared to Antarctic and Arctic cold deserts. Comparative analysis across the three cold deserts indicated the dominance of Dikarya (Ascomycota and Basidiomycota). The observed alpha diversity, Shannon index as well as Pielou's evenness was highest in the Antarctic followed by Drass and Arctic datasets. The genera Malassezia, Preussia, Pseudogymnoascus, Cadophora, Geopora, Monodictys, Tetracladium, Titaea, Mortierella, and Cladosporium were common to all the cold deserts. Furthermore, Conocybe was represented predominantly in Drass. Interestingly, the genus Conocybe has not been previously reported from any other studies on Antarctic or Arctic biomes. To the best of our knowledge, this is the first fungal metagenome study in Drass soil. Our analysis shows that despite the similarities of low temperature among the cold deserts, a significant differential abundance of fungal communities prevails in the global cold deserts.

Analysis of the Whole CDR3 T Cell Receptor Repertoire after Hematopoietic Stem Cell Transplantation in 2 Clinical Cohorts.

A major cause of morbidity and mortality for patients who undergo hematologic stem cell transplantation (HSCT) is acute graft-versus-host disease (aGVHD), a mostly T cell-mediated disease. Examination of the T cell receptor (TCR) repertoire of HSCT recipients and the use of next-generation nucleotide sequencing have raised the question of whether features of TCR repertoire reconstitution might reproducibly associate with aGVHD. We hypothesized that the peripheral blood TCR repertoire of patients with steroid-nonresponsive aGVHD would be less diverse. We also hypothesized that patients with GVHD who shared HLA might also share common clones at the time of GVHD diagnosis, thereby potentially providing potential clinical indicators for treatment stratification. We further hypothesized that HSCT recipients with the same HLA mismatch might share a more similar TCR repertoire based on a potentially shared focus of alloreactive responses. We studied 2 separate patient cohorts and 2 separate platforms for measuring TCR repertoire. The first cohort of patients was from a multicenter Phase III randomized double-blinded clinical trial of patients who developed aGVHD (NCT01002742). The second cohort comprised samples from biobanks from 2 transplantation centers and the Center for International Blood and Marrow Transplant Research of patients who underwent mismatched HSCT. There were no statistically significant differences in the TCR diversity of steroid responders and nonresponders among patients with aGVHD on the day of diagnosis. Most clones in the repertoire were unique to each patient, but a small number of clones were found to be both exclusive to and shared among aGVHD nonresponders. We were also able to show a strong correlation between the presence of Vß20 and Vß29 and steroid responsiveness. Using the Bhattacharya coefficient, those patients who shared the same HLA mismatch were shown to be no more similar to one another than to those who had a completely different mismatch. Using 2 separate clinical cohorts and 2 separate platforms for analyzing the TCR repertoire, we have shown that the sampled human TCR repertoire is largely unique to each patient but contains glimmers of common clones of subsets of clones based on responsiveness to steroids in aGVHD on the day of diagnosis. These studies are informative for future strategies to assess for reproducible TCR responses in human alloreactivity and possible markers of GVHD responsiveness to therapy.

Generation of regulable EGFRvIII targeted chimeric antigen receptor T cells for adoptive cell therapy of glioblastoma.

Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Epidermal growth factor receptor variant III (EGFRvIII) was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation, production of cytokines and extent of cell lysis. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use.

Next-generation sequencing (NGS) of immune receptors has become a standard tool to assess minimal residual disease (MRD) in patients treated for lymphoid malignancy, and it is being used to study the T-cell repertoire in many clinical settings. To better understanding the potential clinical utility and limitations of this application outside of MRD, we developed a BIOMED-2 primer-based NGS method and characterized its performance in controls and patients with graft-versus-host disease (GVHD) after allogeneic hematopoietic transplant. For controls and patients with GVHD, replicate sequencing of the same T-cell receptor ß (TRB) libraries was highly reproducible. Higher variability was observed in sequencing of different TRB libraries made from the same DNA stock. Variability was increased in patients with GVHD compared with controls; patients with GVHD also had lower diversity than controls. In the T-cell repertoire of a healthy person, approximately 99.6% of the CDR3 clones were in low abundance, with frequency <10-3. A single library could identify >93% of the clones with frequency ≥10-3 in the repertoire. Sequencing in duplicate increased the average detection rate to >97%. This work demonstrates that NGS reliably and robustly characterizes TRB populations in healthy individuals and patients with GVHD with frequency ≥10-3 and provides a methodologic framework for applying NGS immune repertoire methods to clinical testing applications beyond MRD.

Bacterial diversity of Drass, cold desert in Western Himalaya, and its comparison with Antarctic and Arctic.

Drass is the coldest inhabited place in India and the second coldest, inhabited place in the world, after Siberia. Using the 16SrDNA amplicon pyrosequencing, bacterial diversity patterns were cataloged across the Drass cold desert. In order to identify the ecotype abundance across cold desert environment, bacterial diversity patterns of Drass were further compared with the bacterial diversity of two other cold deserts, i.e., Antarctic and Arctic. Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Gemmatimonadetes were among the highly abundant taxonomic groups present across all the three cold deserts and were designated as the core phyla. However, Firmicutes, Nitrospirae, Armatimonadetes (former candidate division OP10), Planctomycetes, TM7, Chloroflexi, Deinococcus-Thermus, Tenericutes and candidate phyla WS3 were identified as rare phyla in Drass, Antarctic and Arctic samples. Differential abundance patterns were also computed across all the three samples, i.e., Acidobacteria (32.1 %) were dominant in Drass and Firmicutes (52.9 ± 17.6 %) and Proteobacteria (42 ± 1.3 %) were dominant in Antarctic and Arctic reference samples, respectively. Alpha diversity values Shannon's (H) and Simpson's (1-D) diversity indices were highest for Antarctic samples, whereas richness estimators (ACE and Chao1) were maximum for Drass soil suggesting greater species richness in bacterial communities in Drass than the Antarctic and Arctic samples.

Use of a stop-flow programmable shunt valve to maximize CNS chemotherapy delivery in a pediatric patient with acute lymphoblastic leukemia.

BACKGROUND: The requirement for frequent intraventricular drug delivery in the setting of shunt dependence is particularly challenging in the treatment of central nervous system infection, neoplastic disease, and hemorrhage. This is especially relevant in the pediatric population where both hematogenous malignancy requiring intrathecal drug delivery and shunt-dependent hydrocephalus are more prevalent. Intrathecal and intraventricular chemotherapy agents can be prematurely diverted in these shunt-dependent patients. CASE DESCRIPTION: We report the use of a stop-flow programmable shunt valve to maximize delivery of intraventricular chemotherapy in a child with acute lymphoblastic leukemia and disseminated intravascular coagulation who presented with spontaneous intracerebral and intraventricular hemorrhages. The patient then developed posthemorrhagic hydrocephalus and eventually progressed to shunt dependence but still required frequent intraventricular chemotherapy administration. A ventriculoperitoneal shunt, equipped with a valve that allows for near cessation of cerebrospinal fluid flow (Certas(®), Codman, Raynham, MA), and a contralateral Ommaya reservoir were inserted to maximize intraventricular dissemination of chemotherapy. CONCLUSIONS: To the best of our knowledge, this is the first reported case of the use of a high-resistance programmable valve being used to virtually cease cerebrospinal fluid flow through the distal catheter temporarily in order to maximize intraventricular drug dissemination in a pediatric patient with acute lymphoblastic leukemia.

Targeting a glioblastoma cancer stem-cell population defined by EGF receptor variant III.

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII(+)/CD133(+) defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII(+) cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII(-) cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII(+)/CD133(+) coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII(+)/CD133(+) population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. METHODS: A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. RESULTS: Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. CONCLUSIONS: Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

Stromal platelet-derived growth factor receptor α (PDGFRα) provides a therapeutic target independent of tumor cell PDGFRα expression in lung cancer xenografts.

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression.

Late effects in adult survivors of pediatric cancer: a guide for the primary care physician.

Because of significant medical advances in the past 50 years, the number of adult survivors of childhood/adolescent cancer has increased dramatically. Unfortunately, more than 60% of these survivors will have at least 1 long-term side effect from treatment. This growing population requires dedicated care by their primary physicians because they have specific risk factors depending on their initial cancer diagnosis and the treatment modalities they received. Internists and family physicians play an integral role in providing appropriate screening, treatment, and counseling to prevent morbidity and mortality in these patients.

Zebrafish transgenic lines co-expressing a hybrid Gal4 activator and eGFP in tissue-restricted patterns.

We have used a Tol2-derived trapping vector, carrying a hybrid Gal4 gene and a UAS:eGFP reporter cassette, to identify 16 transgenic zebrafish lines expressing the fluorescent marker eGFP in tissue-restricted patterns during development. Most lines show co-expression of eGFP and a hybrid Gal4 transcription activator containing a truncated VP16 domain that facilitate induction of other UAS-transgenes (UAS:RFP). Notably, many of the transgenic lines are expressed in particular areas of the central nervous system, such as the retina. We mapped the genomic positions of most of the activated insertions, and for three retina-specific lines we also demonstrate that eGFP reports the expression of particular endogenous genes. One of the identified zebrafish genes shows expression in ventral retina, and encodes a protein containing a repulsive guidance molecule (RGM) domain, suggesting a role in axonal guidance during optic nerve formation. Among the lines labeling other tissues, three show early co-expression of eGFP and Gal4-VP16 in blood vessels, erythrocytes and other hematopoietic cells. Interestingly, the activated insertion in the erythrocyte line was mapped to a site near the globin cluster on chromosome 3. All the reported lines co-expressing eGFP and the hybrid Gal4 activator may have potential as genetic tools to study developmental processes.

Development of an EGFRvIII specific recombinant antibody.

BACKGROUND: EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. RESULTS: In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M⁻¹ as determined by enzyme-linked immunosorbent assay (ELISA). CONCLUSION: This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

Smac mimetic increases chemotherapy response and improves survival in mice with pancreatic cancer.

Failure of chemotherapy in the treatment of pancreatic cancer is often due to resistance to therapy-induced apoptosis. A major mechanism for such resistance is the expression and activity of inhibitors of apoptosis proteins (IAP). Smac (second mitochondria-derived activator of caspase) is a mitochondrial protein that inhibits IAPs. We show that JP1201, a Smac mimetic, is a potent enhancer of chemotherapy in robust mouse models of pancreatic cancer. Combination of JP1201 with gemcitabine reduced primary and metastatic tumor burden in orthotopic xenograft and syngenic tumor models, induced regression of established tumors, and prolonged survival in xenograft and transgenic models of pancreatic cancer. The effect of JP1201 was phenocopied by XIAP small interfering RNA in vitro and correlated with elevated levels of tumor necrosis factor alpha protein in vivo. The continued development of JP1201 and other strategies designed to enhance therapy-induced apoptosis in pancreatic cancer is warranted.

Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells.

Here we report that organic copper complexes can potently and selectively inhibit the chymotrypsin-like activity of the proteasome in vitro and in vivo. Several copper compounds, such as NCI-109268 and bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ)(2)], can inhibit the chymotrypsin-like activity of purified 20S proteasome. In human leukemia cells, proteasome inhibition occurs within 15min after treatment, followed by apoptosis. Neither proteasome inhibition nor apoptosis occurs in non-transformed, immortalized human natural killer cells under the same treatment. Furthermore, proteasome inhibition and apoptosis induction were detected in prostate cancer cells treated with the ligand 8-OHQ alone following pre-treatment with copper(II) chloride. None of these events occurred in cells treated with copper(II) chloride alone, 8-OHQ alone (without growth in copper-enriched media), or nickel(II) chloride pre-treatment followed by 8-OHQ. Furthermore, we found that copper-mediated inhibition of purified 20S proteasome cannot be blocked by a reducing agent and that organic copper compounds do not generate hydrogen peroxide in the cells, suggesting that proteasome inhibition and apoptosis induction are not due to copper-mediated oxidative damage of proteins. Our results suggest that certain types of organic ligands could bind to tumor cellular copper, forming potent proteasome inhibitors and apoptosis inducers at copper concentrations found in tumor tissues.