Putative Antigen Research Articles

Dalfampridine: Review of its Efficacy in Improving Gait in Patients with Multiple Sclerosis

Multiple sclerosis (MS) is a progressive immune-mediated neurodegenerative disease of human central nervous system (CNS), which causes irreversible disability in young adults. The cause and cure for MS remain unknown. Pathophysiology of MS includes two arms: inflammatory demyelination and neurodegeneration. The inflammatory demyelination of MS which is mainly promoted by a massive activation of the immune system against putative CNS antigen(s) leads to loss of oligodendrocyte/myelin complex which slows down or halts impulse conduction in denuded axons. Practically, loss of myelin significantly reduces signal conduction along the demyelinated axons through alterations in the distribution of axonal ion channels. Dalfampridine (4-aminopyridine or 4-AP) is an oral potassium channel blocker, which was recently approved by FDA for symptomatic treatment of MS. Dalfampridine, which acts at the central and peripheral nervous systems, enhances conduction in demyelinated axons and improves walking ability of MS patients. A number of clinical trials have evaluated the safety and efficacy of fampridine in MS patients with the degree of gait improvement as the main outcome. The objective of this manuscript is to provide an overview of the pharmacology, pharmacokinetics, clinical trials, side effects and interactions of dalfampridine used in treatment of MS patients.

A CD8+ T-Cell Clone Directed Against AML Blasts that Recognizes a Tumor Specific Antigen Expressed Also by Solid Tumors

AbstractAbstract 4289The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells.To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive.We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines.In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures:No relevant conflicts of interest to declare.

Genetic diversity and the ensuing strain specific immunity to asexual erythrocytic stage vaccine candidate antigens, MSP-1, AMA-1 and DBP, of Plasmodium vivax in Sri Lanka

Characterization of genetic diversity among locally prevalent parasite strains and the ensuing strain-specific immunity will be particularly important in a specific geographical setting for development of vaccine constructs and for planning future vaccination strategies. The present study characterized the prevailing genetic diversity of three major asexual stage putative vaccine antigens of Plasmodium vivax, 42 kDa fragment of the Merozoite Surface Protein -1 (MSP-142), Apical Membrane Antigen-1 domain II (AMA-1 DII) and the Duffy Binding Protein region II (PvDBPII), in patients infected with P. vivax malaria from two endemic areas, Anuradhapura and Kataragama, in Sri Lanka, where unstable malaria prevails with low transmission, and from a malaria non-endemic area, Colombo, where the patients contracted the infections while visiting areas endemic to vivax malaria. Based on nucleotide sequences, extensive allelic polymorphism was evident in the N-terminal region of MSP-142(Pvmsp-133), Pvama- 1dII and PvdbpII, due to recombination, mutations, natural selection and genetic differentiation. Nucleotide sequences of Pvmsp-142(N=95), Pvama-1dII (64) and PvdbpII (100), generated 27, 21 and 33 amino acid (a.a) haplotypes, respectively. Clinical isolates with amino acid haplotypes available for all three antigens (N=24) revealed uniquely different haplotype combinations in each isolate. Conversely, PvMSP-119, domain II loop of PvAMA-1 and the six a.a. in the critical binding domain of PvDBPII responsible for direct binding with the Duffy antigen on human RBC, showed 100% conservation of the amino acid sequences. In order to evaluate whether sequence differences among each antigen was indicative of strain-specific immunity, amino acid sequences of PvMSP-142, PvAMA-1DII and PvDBPII were aligned with the homologous total (IgM + IgG) antibody responses assayed by ELISA against the recombinant protein of each antigen, and the seven (P01-P07) PvAMA-1DII peptides. Though Anti-PvMSP-119 and anti-P04 antibody prevalence precludes strain-specific immune responses against the highly conserved PvMSP-119 and the P04 peptide on domain II loop of PvAMA-1, a protective immune response was clearly elicited to these two regions by the marked isotype switch from IgM to IgG with increasing exposure in areas of endemicity. Further, the presence of strain transcending (cross reactive) PvDBPII specific functionally active binding inhibitory antibodies were demonstrated among locally exposed P. vivax patients. Thus this study may support the inclusion of PvMSP-119, P04 region of PvAMA-1 domain II and region II of PvDBP as veritable vaccine components for a multi component cocktail asexual stage vaccine against P. vivax malaria in Sri Lanka.

Synthesis of glycosaminoglycan oligosaccharides. Part 5: Synthesis of a putative heparan sulfate tetrasaccharide antigen involved in prion diseases

The synthesis of the putative prion-associated heparan sulfate tetrasaccharide containing two d-glucuronic acid units is reported. Key to the synthesis were the differentiation of the N-acetylated and N-unsubstituted glucosamine units, the high-yielding glucosylation at O-4 of an N-acetyl-d-glucosamine derivative and the α-selective glycosylation of the 4′-OH group of a β-d-GlcpA-(1→4)-d-GlcpNAc disaccharide building block with a disaccharide thioglycoside donor.

A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens

BackgroundVaricella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells.ResultsWe present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera.ConclusionsThe line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.

Screening of Theileria parva apicomplexan antigen homologs for induction of MHC CD4+ and CD8+ T-cell responses.

Theileria parva, an intracellular apicomplexan pathogen transmitted byRhipicephalus appendiculatus ticks, infects and transforms lymphocytes of cattle and African buffalo causing the disease called East Coast fever (ECF). The genome of of T. parva was sequenced in order to facilitate research on parasite biology, assist the identification of schizont antigens for vaccine development and extend comparative apicomplexan genomics. In the present study, eight putative apicomplexan antigens were identified from literature and their homologs purified from a T. parva Muguga schizont cDNA library. These were immunoscreened forMHC CD4+ and CD8+ CTL response. The results showed that of the expressed proteins, only the T. parva T-complex 1 protein zeta subunit ortholog was found to elicit CD4+ response; none elicited CD8+ response. The elicitation of CD4+ T cell response by the T. parva T complex Protein-1 zeta subunit homolog indicates that it is a candidate T-helper cell target antigen. Key words: Theileria parva, apicomplexan antigen, East Coast fever (ECF), major histocompatibility complex (MHC), cytotoxic T lymphocyte.

RETRACTED: Genetic and Epigenetic Mechanisms Down-Regulate FGF Receptor 2 to Induce Melanoma-Associated Antigen A in Breast Cancer

Recent genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the gene encoding fibroblast growth factor receptor 2 (FGFR2) as a risk factor for breast cancer. We examined the relationship between these intron 2 SNPs and gene expression in breast carcinomas. Primary breast tissue showed a common occurrence of these SNPs accompanied by FGFR2 expression in normal ductal epithelium. Unexpectedly, we found that FGFR2 mRNA and protein levels were reduced in microdissected cancer cells when compared with paired normal breast epithelium. FGFR2 down-regulation was associated with DNA methylation and loss-of-heterozygosity. Where FGFR2-IIIb was expressed in tumor cells, it was accompanied by up-regulation of the RNA-binding proteins ESRP1/2, consistent with splicing of this isoform. Reduction in FGFR2 was associated with re-expression of its putative target melanoma-associated antigen (MAGE-A) in primary carcinoma cells. Conversely, forced expression or activation of FGFR2-IIIb resulted in MAGE-A silencing. These data provide the first evidence for FGFR2 down-regulation in breast carcinomas harboring intron 2 SNPs. Our findings underscore the significance of epigenetic and somatic changes that can potentially modify the effects of germline polymorphisms in determining FGFR2 gene expression.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Sia10, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10 (i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naïve fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture.

Functional associations of similar MHC alleles and shared parasite species in two sympatric lemurs

Several recent studies of animals in their natural surroundings found evidence for effects of certain major histocompatibility complex (MHC) immune gene alleles on the parasite load. However, in multi-infected individuals the particular selection pressure exerted by specific parasites has rarely been explored. In this study we took advantage of the parasitological and genetic data of two previously investigated Malagasy lemur species ( Cheirogaleus medius and Microcebus murinus). We investigated whether the two sympatric and ecologically similar primates are infected by similar parasite species and explored if certain parasites are associated with particular MHC alleles. Our study revealed that most of the parasite egg morphotypes were found in both hosts. In each lemur species we identified one MHC allele which was positively associated with Ascaris-infection. Interestingly, these MHC alleles were very similar to each other but differed from all other investigated MHC alleles in an amino acid substitution in a putative functional important antigen binding site. Thus, our study gives first intriguing evidence for a direct connection between certain antigen binding sites of MHC molecules with a particular parasite in two wild primate populations. This may indicate that indeed certain parasites exert direct selective pressure on the MHC of wild living hosts.

Human immune responses in cryptosporidiosis.

Immune responses play a critical role in protection from, and resolution of, cryptosporidiosis. However, the nature of these responses, particularly in humans, is not completely understood. Both innate and adaptive immune responses are important. Innate immune responses may be mediated by Toll-like receptor pathways, antimicrobial peptides, prostaglandins, mannose-binding lectin, cytokines and chemokines. Cell-mediated responses, particularly those involving CD4(+) T cells and IFN-gamma play a dominant role. Mucosal antibody responses may also be involved. Proteins mediating attachment and invasion may serve as putative protective antigens. Further knowledge of human immune responses in cryptosporidiosis is essential in order to develop targeted prophylactic and therapeutic interventions. This review focuses on recent advances and future prospects in the understanding of human immune responses to Cryptosporidium infection.

Epidermal γδ T cells sense precancerous cellular dysregulation and initiate immune responses

Hyperplasia associated with a loss of tissue homeostasis can induce DNA replication stress, leading to precancerous dysregulation. Epidermal gammadelta T cells reside in the primary barrier that protects against diverse environmental insults; however, the functions of these T cells in tissue surveillance are not completely understood. In mice with inducible Notch1 inactivation in keratinocytes that causes epidermal hyperplasia, epidermal gammadelta T cells sensed stressed keratinocytes and migrated into the cutaneous draining lymph nodes. Simultaneous induction of beta-galactosidase (beta-Gal) as a putative antigen expressed in the process of precancerous dysregulation and Notch1 ablation in the epidermis resulted in elevated beta-Gal-specific IgG2a production. Epidermal gammadelta T cells were found to have the capacity to express chemokine (C-C motif) receptor 7 and migrate into the lymph nodes. Cutaneous draining lymph node cells in Notch1-inactivated mice expressed high levels of IFN-gamma upon anti-CD3 plus anti-CD28 stimulation. Furthermore, induced expression of beta-Gal in mice that lacked epidermal gammadelta T cells failed to induce anti-beta-Gal IgG. These results suggest that epidermal gammadelta T cells play an essential role in the initiation process of epidermal antigen-specific humoral immune responses and demonstrate the importance of epidermal gammadelta T cells in sensing precancerous dysregulation and activating adaptive immunity.

Are large wattles related to particular MHC genotypes in the male pheasant?

In sexually dimorphic species, partners can assess heritable mate quality by analyzing costly sexual ornaments in terms of their dimension and possibly of their symmetry. In vertebrates an important aspect of genetic quality is the efficiency of the immune system, and in particular the Major Histocompatibility Complex (MHC). If ornaments are honest advertisements of pathogen resistance (good genes), in line with the Hamilton-Zuk hypothesis, a correlation between ornament expression and MHC profiles should exist. We tested this hypothesis in the common pheasant Phasianus colchicus by comparing male ornament characteristics (wattle and spur size, and wattle fluctuating asymmetry) with a portion of exon 2 of the class IIB MHC genes containing 19 putative antigen recognition sites. A total of 8 new alleles was observed in the MHCPhco exon IIB. We found significant differences in the occurrence of MHC genotypes between males carrying large or small wattles. Homozygous genotypes predicted large wattle males more correctly than small wattle males. The association between the dimension of the spur and the occurrence of MHC genotypes was marginally significant, however, we did not find any significant association between MHC genotypes and asymmetry. Our results suggest that female pheasants may use the ornament size as a cue to evaluate male quality and thus choose males carrying particular MHC profiles.

A novel multivalent vaccine based on secretary antigen-delivery induces protective immunity against Vibrio anguillarum and Aeromonas hydrophila

In our previous work, four secretory antigen-delivery systems based on different signal peptides (SPs) (SP hlyA , SP rtxA , SP vah3 and SP empA ) have been successfully established in attenuated Vibrio anguillarum. To investigate the potential application of the antigen-delivery systems in V. anguillarum multivalent vector vaccine, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila, as a putative protective antigen, was fused to the four delivery systems, respectively, and introduced into attenuated V. anguillarum strain MVAV6203 to get four GAPDH-delivery strains in this work. Immunodetection of GAPDH indicated that among the four constructs, the SP empA -mediated GAPDH-delivery strain AV(pGap-empA) showed the highest level of GAPDH expression and secretion, and was determined as the multivalent vaccine candidate. Further immune protection evaluation of AV(pGap-empA) in turbot ( Scophtalmus maximus) demonstrated that the vaccine candidate could induce efficient protection against V. anguillarum and A. hydrophila, suggesting that this vector vaccine had great potential in serving as a valuable multivalent live vaccine.

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

Members of the Burkholderia cepacia complex (Bcc) are respiratory pathogens in patients with cystic fibrosis (CF). Close repetitive DNA sequences often associate with surface antigens to promote genetic variability in pathogenic bacteria. The genome of Burkholderia cenocepacia J2315, a CF isolate belonging to the epidemic lineage Edinburgh-Toronto (ET-12), was analysed for the presence of close repetitive DNA sequences. Among the 422 DNA close repeats, 45 genes potentially involved in virulence were identified and grouped into 12 classes; of these, 13 genes were included in the antigens class. Two trimeric autotransporter adhesins (TAA) among the 13 putative antigens are absent from the other Burkholderia genomes and are clustered downstream of the cci island that is a marker for transmissible B. cenocepacia strains. This cluster contains four adhesins, one outer-membrane protein, one sensor histidine kinase and two transcriptional regulators. By using PCR, we analysed three genes among 47 Bcc isolates to determine whether the cluster was conserved. These three genes were present in the isolates of the ET-12 lineage but absent in all the other members. Furthermore, the BCAM0224 gene was exclusively detected in this epidemic lineage and may serve as a valuable new addition to the field of Bcc diagnostics. The BCAM0224 gene encodes a putative TAA that demonstrates adhesive properties to the extracellular matrix protein collagen type I. Quantitative real-time PCR analysis indicated that BCAM0224 gene expression occurred preferentially for cells grown under high osmolarity, oxygen-limited conditions and oxidative stress. Inactivation of BCAM0224 in B. cenocepacia attenuates the ability of the mutant to promote cell adherence in vitro and impairs the overall bacterial virulence against Galleria mellonella as a model of infection. Together, our data show that BCAM0224 from B. cenocepacia J2315 represents a new collagen-binding TAA with no bacterial orthologues which has an important role in cellular adhesion and virulence.

Progress in understanding the human immune responses to Mycobacterium tuberculosis

Development of an effective vaccine against tuberculosis hinges on an improved understanding of the human immune response to Mycobacterium tuberculosis. Work in this area at the University of Texas Health Science Center at Tyler has led to advances in four areas: (1) natural killer cells contribute to innate immunity by lysing M. tuberculosis-infected mononuclear phagocytes, and to adaptive immunity by enhancing the CD8+ T-cell effector function and inhibiting expansion of T regulatory cells; (2) Interferon-gamma plays a central role in resistance to many intracellular pathogens, including M. tuberculosis, and we have identified three transcription factors that bind to the Interferon-gamma proximal promoter and increase Interferon-gamma transcription in live T-cells that are activated by M. tuberculosis antigens; (3) A DNA vaccine that encodes the M. tuberculosis 10fts;kDa culture filtrate protein and the lysosomal integral membrane protein-2 was produced to direct vaccine antigens to the MHC class II processing and presentation pathway. When this vaccine was coated with polyethylenimine and administered to mice, it yielded a remarkably potent pulmonary immune response that reduced the bacillary burden by 90% after M. tuberculosis challenge; (4) The early secreted antigenic target of 6fts;kDa (ESAT-6) is a putative vaccine antigen. We found that high concentrations of this antigen markedly inhibit Interferon-gamma production by T-cells and are working to understand the molecular mechanisms underlying this effect. Developing methods to enhance NK cell functions that favor protective immunity, increase interferon-gamma transcription, elicit protective pulmonary immune responses and prevent ESAT-6 from inhibiting T-cell function will contribute significantly to development of antituberculosis vaccines.

Leukemic Dendritic Cells Expand Central Memory T Lymphocytes From HCT Donors Able to React against the Original Leukemia in Vitro and In Vivo.

Abstract 4090Poster Board III-1025Allogeneic hematopoietic transplantation (allo-HCT) is the only curative option for patients affected by high-risk acute myeloid leukemia (AML). This is largely due to the ability of allogeneic immune system to eradicate leukemic stem cells (LSC). However, the fact that some patients still relapse after allo-HCT, suggests that strategies to increase LSC targeting by donor T cells are needed. For this purpose, we exploited the unique ability of myeloid blasts to differentiate into leukemic dendritic cells (LDC). We observed that a short (48h) exposure to calcium ionophore A23187 and IL-4 is able to induce LDC differentiation in 14/16 (86%) of AML that we studied, both de novo and secondary. Importantly, despite phenotypic and functional changes indicative of differentiation into DC-like cells, the process was accompanied by the maintenance of disease markers such as CD34 and CD117. Moreover, LDC maintained the expression of the oncogenic protein WT1, which is a putative LSC antigen. Thanks to these favourable characteristics, LDC proved to be superior to the original blasts in expanding leukemia-reactive T lymphocytes both in the autologous and allogeneic HCT setting (on average, 5-fold expansion of blasts-stimulated T cells vs 95-fold expansion of LDC-stimulated T cells, SEM=2,7 and 67,7 respectively, p=0,01). We observed that the level of T-cell expansion directly correlate with the percentage of LDC obtained upon treatment with A23187 and IL-4. Most importantly, LDC proved to be more potent than blasts in expanding central memory T lymphocytes (TCM), which are known to confer superior anti-tumor immunity (on average, 29% of TCM upon stimulation with blasts vs 53% TCM upon stimulation with LDC, SEM=7,2 and 5,7 respectively, p=0,01). LDC-expanded T lymphocytes were able to efficiently recognize and kill leukemic blasts in vitro (on average, 953 specific spots of IFN-g/50'000 effectors at E:T ratio of 10:1 -SEM=120- and 29% of specific killing at E:T ratio of 50:1 -SEM=7,4-). Importantly, analysis of different HLA-settings and different targets of patient origin, suggests that LDC can expand T lymphocytes with specificities against multiple antigens expressed by the original leukemia. In particular, we observed the expansion of WT-1 specific T cells upon LDC stimulation. Finally, when infused in NOD/Scid mice transplanted with the original leukaemia, LDC-stimulated T lymphocytes were able to induce long-term complete remissions (>16 weeks) in all mice analyzed, suggesting that this approach may be active against leukemic stem cells. These results show for the first time that LDC-stimulated human T cells could exert a strong GvL activity in vivo. Disclosures:Bordignon:Molmed Spa: Employment.

Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen

AbstractSeveral studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

Surface Protein EF3314 Contributes to Virulence Properties of Enterococcus faecalis

Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and biofilm associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.

The Dendritic Cell–like Functions of IFN-Producing Killer Dendritic Cells Reside in the CD11b+ Subset and Are Licensed by Tumor Cells

IFN producing killer dendritic cells (IKDC) were originally defined as CD11c(int) B220(+)NK1.1(+) (or CD49b(+)) cells that exert a potent tumoricidal activity in animals lacking B, T, and conventional natural killer effectors. MHC class II expression on tumor infiltrating IKDC prompted us to investigate their putative antigen presenting function. Here, we show that tumor cells license IKDC to acquire the properties of antigen presenting cells, i.e., expression of MHC class II and costimulatory CD86 molecules. We show that the CD11b(+) subset of IKDC are able to prime naïve CD4(+) T cells and cross-prime naïve CD8(+) T lymphocytes. Licensing of IKDC by tumor cells was mandatory for the full differentiation of T cells into polarized effectors. IKDC could engulf and process soluble Ova protein in a CD206-dependent manner. Finally, we show that CD11b(+)IKDC is selectively endowed with CTLA4Ig-inhibitable antigen presenting capacities and that targeting this subset with the detoxified adenylate cyclase toxin of Bordetella pertussis fused to antigen resulted in efficient cross-presentation of antigen by IKDC to specific TCR transgenic CD8(+)T cells in vivo. Collectively, our data indicate that upon exposure to tumor cells, IKDC subserve DC-like functions.

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.