Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Innate immune activation in neovascular Age-related Macular Degeneration

Purpose: To compare white blood cell populations from persons with neovascular age-related macular degeneration (nAMD) with that of age-matched controls.

Methods: Immunophenotyping for white blood cell populations (including CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes, CD4 and CD8 T-lymphocytes, CD56 natural killer cells, CD19 B-lymphocytes and CD16+HLA-DR- neutrophils), chemokine receptor expression analysis (CX3CR1 and CCR2) as well as cell activation analysis (MHC-II, HLA-DR, CD62L, STAT3) was performed using samples of peripheral blood from nAMD patients and age- and gender-matched controls.

Results: The percentage of CD4+ T cells was significantly reduced while the percentage of CD11b+ cells and CD16+HLA-DR- neutrophils was significantly increased in nAMD patients compared to controls. The percentage of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes was similar between nAMD patients and controls, however there was a significant increase of CX3CR1 on the intermediate monocyte subset and on CD16+HLA-DR- neutrophils in nAMD compared to controls. HLA-DR was significantly increased in all monocyte subsets in nAMD compared to controls. Activation of Signal Transducer and Activator of Transcription 3 (STAT3) was significantly increased in nAMD patients compared to controls following stimulation with IL6.

Conclusions: Our results suggest an increased activation of the innate immune system in patients with nAMD. A better understanding of the role of the innate immune system in the pathogenesis of nAMD may help identify novel biomarkers and thus development of improved therapeutic strategies.

Mesenchymal stem cells in immunoregulation

Mesenchymal stem cells (MSCs) reside within the bone marrow cavity and serve as a reservoir for the continuous renovation of various mesenchymal tissues. Recent efforts suggest that MSCs modulate the immune reactions in vitro and escape the immune surveillance in vivo. We provide herein a discussion of the issues including the current research progress on the in vitro interactions of MSCs with multiple subsets of immune cells (dendritic cells, T cells, B cells and natural killer cells), in vivo transplantation outcomes, the possible underlying mechanisms, future research directions as well as potential clinical implications.

Visualization of the earliest steps of gammadelta T cell development in the adult thymus.

The checkpoint in cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls cell development during the penultimate CD4-CD8- stage. Passage through this checkpoint required the assembly of TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n¼56), mantle cell lymphoma (n¼54), marginal zone lymphoma (n¼41) and follicular lymphoma (n¼109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Foxp3+ T cells induce Perforin-Dependent Dendritic Cell Death in Tumor-Draining Lymph Nodes.

Regulatory T (Treg) cells limit the onset of effective antitumor immunity, through yet-ill-defined mechanisms. We showed the rejection of established ovalbumin (OVA)-expressing MCA101 tumors required both the adoptive transfer of OVA-specific CD8(+) T cell receptor transgenic T cells (OTI) and the neutralization of Foxp3(+) T cells. In tumor-draining lymph nodes, Foxp3(+) T cell neutralization induced a marked arrest in the migration of OTI T cells, increased numbers of dendritic cells (DCs), and enhanced OTI T cell priming. Using an in vitro cytotoxic assay and two-photon live microscopy after adoptive transfer of DCs, we demonstrated that Foxp3(+) T cells induced the death of DCs in tumor-draining lymph nodes, but not in the absence of tumor. DC death correlated with Foxp3(+) T cell-DC contacts, and it was tumor-antigen and perforin dependent. We conclude that Foxp3(+) T cell-dependent DC death in tumor-draining lymph nodes limits the onset of CD8(+) T cell responses.

Lymphocytic gastritis-like T cell lymphoma: molecular evidence of an unusual recurrence

This report describes a patient with a gastric biopsy specimen showing histomorphological and immunohistochemical appearances indistinguishable from those usually present in lymphocytic gastritis, a rare condition of unknown aetiology with a distinctive phenotype. The patient had a history of a biopsy confirmed T cell non-Hodgkin lymphoma at two anatomical sites ( bladder and stomach), which was subsequently treated. Molecular analysis of the T cell receptor (TCR) gamma chain gene rearrangements showed a distinct monoclonal T cell population in the bladder and gastric biopsies. The same analysis in the lymphocytic gastritis-like biopsy sample showed a monoclonal population with identical base pair size to that identified in the other specimens. This report highlights the importance of TCR gene rearrangement analysis in the diagnosis of unusual gastric inflammation, and the use of capillary electrophoresis based polymerase chain reaction in the follow up of lymphoproliferative disorders.

Age-related changes in non-receptor dependent generation of reactive oxygen species from phagocytes of healthy adults

Several authors have shown that neutrophil generation of reactive oxygen species (ROS) declines with advancing age. Similar changes have also been suggested in monocytes. In both cases alterations in second messenger activity have been implicated as the most likely explanation for these observations. The aim of this study was to investigate the effect of age on phagocyte ROS generation, stimulated by the direct activation of protein kinase C (PKC). Venous blood was drawn from normal healthy subjects, cells were separated on a double density gradient into mononuclear and polymorphonuclear (pmn) cells. Phorbol myristate acetate (PMA) was employed as a cell stimulus. Superoxide generation was measured by cytochrome c reduction and myeloperoxidase (MPO) products by measurement of peak luminol chemiluminescence (CL). Fifty-eight subjects, 25 males and 33 females, were studied, median age 49 years (range 26-88 years). Polymorphonuclear cell superoxide generation was significantly higher in males and there was a trend towards higher pmn MPO product generation in males. Using Spearman's ranked correlation coefficient, monocyte superoxide generation was negatively correlated with age (r = -0.473, P <0.001). No changes in the generation of MPO products was found. There were also trends towards a negative correlation of pmn cytochrome c reduction and peak luminol CL with age in males but not females. Since PMA directly activates protein kinase C, reduced monocyte superoxide generation with increasing age appears to be related to alterations in the ROS generating system downstream of the cell receptor. Impaired monocyte superoxide generation may have implications for non-specific defence against certain infections and early tumour growth in the elderly. Factors underlying these changes in monocyte function therefore require further study.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFN?(+) cells dominate disease initiation with IL-17(+) cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFN? responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFN? driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFN? cytokine production by CD4 and CD8 cells, but not IL-17 responses.

Use of SLAM and PVRL4 and Identification of Pro-HB-EGF as Cell Entry Receptors for Wild Type Phocine Distemper Virus

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Critical Role for STAT4 Activation by Type 1 Interferons in the Interferon-Y Response to Viral Infection

Interferons (IFNs) are essential for host defense. Although the antiviral effects of the type 1 IFNs IFN- and IFN- (IFN-/) have been established, their immunoregulatory functions, especially their ability to regulate IFN- production, are poorly understood. Here we show that IFN-/ activate STAT4 directly (STAT, signal transducers and activators of transcription) and that this is required for IFN- production during viral infections of mice, in concert with T cell receptor-derived signals. In contrast, STAT1 appears to negatively regulate IFN-/ induction of IFN-. Thus, type 1 IFNs, in addition to interleukin-12, provide pathways for innate regulation of adaptive immunity, and their immunoregulatory functions are controlled by modulating the activity of individual STATs.

Proteomic Analysis of Recurrent Joint Inflammation in Juvenile Idiopathic Arthritis

The synovial fluid proteome in juvenile idiopathic arthritis was investigated to isolate joint-specific biomarkers that are expressed in patients displaying recurrent joint inflammation. To identify the synovial specific proteome, matched synovial fluid and plasma samples were subjected to protein separation by 2-dimension electrophoresis (2DE). Forty-three protein spots, overexpressed in the joint, were identified. Synovial fluids from children with single-event knee joint inflammation were then compared with a group with recurrent knee disease. Nine synovial specific proteins were significantly differentially expressed in the recurrent group. Proteolytic fragments of collagen X, fibrin beta-chain, and T-cell receptor alpha-region have been identified among this protein cluster. Putative biomarkers, overexpressed in the joint and differentially expressed in children with recurrent joint inflammation, have been identified. These proteins may play a significant role determining the pathological state within the chronically inflamed joint and influence disease progression in JIA. This is the first study of the synovial proteome in children.

ZAP-70 restoration in mice by in vivo thymic electroporation.

Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.