MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1ß from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

Immunological and autoimmune considerations of Autism Spectrum Disorders.

Autism Spectrum Disorders (ASD) are a group of heterogeneous neurodevelopmental conditions presenting in early childhood with a prevalence ranging from 0.7% to 2.64%. Social interaction and communication skills are impaired and children often present with unusual repetitive behavior. The condition persists for life with major implications for the individual, the family and the entire health care system. While the etiology of ASD remains unknown, various clues suggest a possible association with altered immune responses and ASD. Inflammation in the brain and CNS has been reported by several groups with notable microglia activation and increased cytokine production in postmortem brain specimens of young and old individuals with ASD. Moreover several laboratories have isolated distinctive brain and CNS reactive antibodies from individuals with ASD. Large population based epidemiological studies have established a correlation between ASD and a family history of autoimmune diseases, associations with MHC complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. In addition, there is evidence that antibodies that are only present in some mothers of children with ASD bind to fetal brain proteins and may be a marker or risk factor for ASD. Studies involving the injection of these ASD specific maternal serum antibodies into pregnant mice during gestation, or gestational exposure of Rhesus monkeys to IgG subclass of these antibodies, have consistently elicited behavioral changes in offspring that have relevance to ASD. We will summarize the various types of studies associating ASD with the immune system, critically evaluate the quality of these studies, and attempt to integrate them in a way that clarifies the areas of immune and autoimmune phenomena in ASD research that will be important indicators for future research.

A short peptide protects from age-onset proteotoxicity.

Aberrant protein aggregation jeopardizes cellular functionality and underlies the development of a myriad of late-onset maladies including Alzheimer's disease (AD) and Huntington's disease (HD). Accordingly, molecules that mitigate the toxicity of hazardous protein aggregates are of great interest as potential future therapeutics. Here we asked whether a small peptide, composed of five amino acids (5MER peptide) that was derived from the human pro-inflammatory CD44 protein, could protect model nematodes from the toxicity of aggregative proteins that underlie the development of neurodegenerative disorders in humans. We found that the 5MER peptide mitigates the toxicity that stems from both; the AD-causing Aß peptide and a stretch of poly-glutamine that is accountable for the development of several disorders including HD, while minimally affecting lifespan. This protection was dependent on the activity of aging-regulating transcription factors and associated with enhanced Aß and polyQ35-YFP aggregation. A transcriptomic analysis unveiled that the peptide modifies signaling pathways, thereby modulating the expression of various genes, including these, which are known as protein homeostasis (proteostasis) regulators such as txt-13 and modifiers of proteasome activity. The knockdown of txt-13 protects worms from proteotoxicity to the same extent as the 5MER peptide, suggesting that the peptide activates the transcellular chaperone signaling to promote proteostasis. Together, our results propose that the 5MER peptide should be considered as a component of future therapeutic cocktails for the treatment of neurodegenerative maladies.

Anti-T lymphocyte globulin (ATG) induces generation of regulatory T cells, at least part of them express activated CD44.

We show here that the anti-T lymphocyte immunoglobulin (ATG) can induce Treg cells following 24-h incubation in human peripheral blood mononuclear cells (PBMCs). The ATG-induced Treg cells express known cell surface markers (e.g., CD25, FoxP3) and suppress the proliferation of autologous responder PBMCs, stimulated with allogeneic PBMCs, when added into the mixed lymphocyte culture (MLC) at zero time point or 48 h later. We expanded the characteristics of the ATG-induced human Treg cells by showing that they express a novel biomarker designated "activated CD44". ATG-induced Treg cells retain their suppressor function after freezing and thawing or irradiation. Suppression of MLC by ATG-induced Treg cells is consistently seen when the Treg cells and the responder cells were derived from the same donor, but not when they derived from different donors. Finally, patients undergoing stem cell transplantation and conditioned with ATG generate in vivo Treg cells that suppress MLC.

Involvement of CD44, a molecule with a thousand faces, in cancer dissemination.

Tumor progression is substantially dependent on network of multiple factors, including adhesion and homing molecules, which support the malignant metastatic spread. CD44, one of the adhesion/homing molecules, has attracted much attention not only because it is expressed on many types of tumors, but also owing to its numerous functions, such as supporting cell migration and transmitting survival signals, thereby being pro-oncogenic by nature. We have used the mouse malignant LB lymphoma cell line as a model for comprehensive in vitro and in vivo analyses of the interaction between CD44 and hyaluronic acid (HA), and its relevance to tumor dissemination. The in vitro studies revealed that LB cells could not bind HA, either under static or dynamic (i.e., shear flow) conditions, unless their CD44 is activated by phorbol ester, deglycosylated (to increase the CD44 positive net charge) or transfected with CD44 variants. In parallel, in vivo experiments showed that LB cell dissemination could be controlled by injection of anti-CD44 monoclonal antibodies or hyaluronidase. Furthermore, LB cells transfected with CD44v4-v10 variant, rather than standard CD44, displayed enhanced invasion of the peripheral lymph nodes. This effect was completely lost if the HA binding site of CD44 were mutated. LB cell accumulation in the lymph nodes is caused by enhanced migration via the afferent lymphatics rather than by accelerated proliferation within the lymph node. This information can be exploited to tailor a "therapeutic suit" that should be maximally effective in inducing tumor resistance, while minimizing destructive side effects.

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis.

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression.

CD44 gene vaccination for insulin-dependent diabetes mellitus in non-obese diabetic mice.

BACKGROUND: Standard CD44 and its alternatively spliced variants were found to be associated with the metastatic potential of tumor cells and with cell migration of autoimmune inflammatory cells, including cells involved in experimental insulin-dependent diabetes mellitus. OBJECTIVES: To investigate whether induction of anti-CD44 immune reactivity, through cDNA vaccination, could attenuate IDDM in a transfer model of NOD mice. METHODS: Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with a 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of male NOD recipients reconstituted with diabetogenic spleen cells of female NOD donors. The VLN were implanted 20 days before and 3 days after cell transfer. RESULTS: In contrast to control groups of recipient mice, recipients vaccinated with VLN loaded with CD44v or CD44s cDNAs developed resistance to IDDM almost to the same extent. Our results suggest that the gene vaccination effect was mediated by anti-CD44 antibody rather than by cellular immunity. Histopathological examinations revealed a significant protection of pancreatic islets in the DNA-vaccinated recipients, whereas the islets of control recipients of diabetogenic cells were almost totally destroyed. CONCLUSIONS: These findings may open new opportunities for IDDM therapy in the future.

A mutation in a CD44 variant of inflammatory cells enhances the mitogenic interaction of FGF with its receptor.

Synovial fluid cells from joints of rheumatoid arthritis (RA) patients express a novel variant of CD44 (designated CD44vRA), encoding an extra trinucleotide (CAG) transcribed from intronic sequences flanking a variant exon. The CD44vRA mutant was detected in 23 out of 30 RA patients. CD44-negative Namalwa cells transfected with CD44vRA cDNA or with CD44v3-v10 (CD44vRA wild type) cDNA bound FGF-2 to an equal extent via their associated heparan sulfate chains. However, Namalwa cells, immobilizing FGF-2 via their cell surface CD44vRA, bound substantially more soluble FGF receptor-1 (FGFR-1) than did Namalwa cells immobilizing the same amount of FGF-2 via their cell surface CD44v3-v10. The former cells stimulated the proliferation of BaF-32 cells, bearing FGFR-1, more efficiently than did the latter cells. Finally, isolated primary synovial fluid cells from RA patients expressing CD44vRA bound more soluble FGFR-1 to their cell surface-associated FGF-2 than did corresponding synovial cells expressing CD44v3-v10 or synovial cells from osteoarthritis patients. The binding of soluble FGFR-1 to RA synovial cells could be specifically reduced by their preincubation with Ab's against the v3 exon product of CD44. Hence, FGF-2 attached to the heparan sulfate moiety expressed by the novel CD44 variant of RA synovium cells exhibits an augmented ability to stimulate FGFR-1-mediated activities. A similar mechanism may foster the destructive inflammatory cascade not only in RA, but also in other autoimmune diseases.

CD44 involvement in autoimmune inflammations: the lesson to be learned from CD44-targeting by antibody or from knockout mice.

CD44 is a multistructural and multifunctional glycoprotein, the diversity of which is generated by alternative splicing. In this communication we review some aspects related to CD44 structure and function in experimental autoimmune inflammation, focusing on research performed in our own laboratory. We have found that CD44 targeting by antibody, passively injected into DBA/1 mice with collagen-induced arthritis (CIA) and NOD mice with type I diabetes or actively generated by CD44 cDNA vaccination of SJL/j mice with autoimmune encephalomyelitis, markedly reduced the pathological manifestations of these diseases by attenuating cell migration of the inflammatory cells and/or by their apoptotic killing. However, genetic deletion of CD44 by knockout technology enhanced the development of CIA because of molecular redundancy mediated by RHAMM (a receptor of hyaluronan-mediated motility). The mechanisms that stand behind these findings are discussed.

CD44 variant DNA vaccination with virtual lymph node ameliorates experimental autoimmune encephalomyelitis through the induction of apoptosis.

Standard CD44 (CD44s) and its alternatively spliced variants (CD44v) were found to be associated with the metastatic potential of tumor cells, and with cell migration of autoimmune inflammatory cells, including cells involved in experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to evaluate whether induction of anti-CD44 immune reactivity, through cDNA vaccination could down-regulate EAE. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of SJL/J mice immunized with myelin antigens for EAE induction. Animals vaccinated with CD44v cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. The in vitro proliferation of lymphocytes was preserved regarding myelin antigens and mitogens. Histopathological examinations revealed a significant reduction of EAE lesions and enhanced apoptosis in central nervous system (CNS)-infiltrating cells of the successfully vaccinated animals. Such methods of cDNA vaccination with CD44 could be applicable in inflammatory CNS diseases, like multiple sclerosis.

CD44 Splice Variants as Potential Players in Alzheimer's Disease Pathology.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, deposition of amyloid-ß (Aß) plaques, intracellular neurofibrillary tangles, and neuronal cell death. Neuroinflammation is commonly believed to participate in AD pathogenesis. CD44 is an inflammation-related gene encoding a widely-distributed family of alternatively spliced cell surface glycoproteins that have been implicated in inflammation, metastases, and inflammation-linked neuronal injuries. Here we investigated the expression patterns of CD44S (which does not contain any alternative exon) and CD44 splice variants in postmortem hippocampal samples from AD patients and matched non-AD controls. The expression of CD44S and CD44 splice variants CD44V3, CD44V6, and CD44V10 was significantly higher in AD patients compared to non-AD controls. Immunohistochemistry of human hippocampal sections revealed that CD44S differentially localized to neuritic plaques and astrocytes, whereas CD44V3, CD44V6, and CD44V10 expression was mostly neuronal. Consistent with these findings, we found that the expression of CD44V6 and CD44V10 was induced by Aß peptide in neuroblastoma cells and primary neurons. Furthermore, in loss of function studies we found that both CD44V10-specific siRNA and CD44V10 antibody protected neuronal cells from Aß-induced toxicity, suggesting a causal relationship between CD44V10 and neuronal cell death. These data indicate that certain CD44 splice variants contribute to AD pathology and that CD44V10 inhibition may serve as a new neuroprotective treatment strategy for this disease.

Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease.

Polyclonal anti-human thymocyte globulins (ATG) have been recently shown to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells has been suggested as one of the possible mechanisms. The aim of current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance. Phenotypic characterization revealed a significant increase in the expression of FoxP3, GITR, CD95, PD-1 and ICOS as well as the complement inhibitory molecules CD55, CD58 and CD59 on CD4+CD25+ T cells upon ATG treatment. Addition of ATG-treated cells to autologous and allogeneic peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/anti-CD28 antibodies resulted in significant inhibition of proliferation. Moreover, T-cell activation and IFNγ secretion were reduced in the presence of ATG-induced Treg cells. The CD4+CD25+CD127-low Treg fraction sorted from ATG-treated culture demonstrated greater suppressive potency than negative fraction. Conditioned medium produced by ATG-treated but not IgG-treated cells contained TGFß and suppressed T cell proliferation and activation in a TGFß receptor-dependent manner. TGFß receptor kinase inhibitor SB431542 interfered with the suppressive activity of ATG-primed cells, enabling partial rescue of proliferation and IFNγ secretion. Moreover, SB431542 prevented Treg phenotype induction upon ATG treatment. Altogether, our data reveal the role of TGFß signaling in ATG-mediated immunosuppression and further support the use of ATG, a potent inducer of regulatory T cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.

The mechanism of molecular redundancy in autoimmune inflammation in the context of CD44 deficiency.

Molecular redundancy refers to the ability of genes to back up damaged genes or gene loss. Although this term is widely discussed in many scientific circles, the process is still ill-defined, as shown by reviewing examples from the literature. Exploring the collagen-induced arthritis model in the context of CD44 knockout mice, we suggest a mechanistic explanation for molecular redundancy that depends neither on upregulation of the compensating molecule nor on structural similarity between the original molecule and the replacement molecule. The backup process is dependent, however, on two key properties shared by the two molecules: ligand binding and support of cell trafficking.

IRS-4 mediated mitogenic signalling by insulin and growth hormone in LB cells, a murine T-cell lymphoma devoid of IGF-I receptors.

Insulin and growth hormone (GH) induce mitogenic and metabolic signals in cells, GH either directly or indirectly via IGF-I production. We have studied a spontaneous murine T-cell lymphoma (LB cells) devoid of IGF-1 receptors in which proliferation is maintained by insulin [Int. J. Cancer 50 (1992) 80], and show that GH is more potent than insulin, with both GH and insulin dose-response curves for thymidine incorporation being bell-shaped. Binding showed somatogenic rather than lactogenic GH receptors. Insulin stimulated phosphorylation of the insulin receptor and of a 160-kDa protein, identified as the IRS-4 protein. This phosphorylated IRS-4 associated with PI3-kinase, which was activated along with the downstream p70(S6) kinase, whereas the Ras-MAPK pathway was not. Using selective inhibitors, the PI3-kinase, but not p70(S6) kinase or MEK, was found to be involved in insulin-stimulated DNA synthesis. GH induced tyrosine phosphorylation of IRS-4 and nuclear translocation of STAT5. The LB cells constitute a new model for studying GH and insulin signalling without interference of IGF-1 receptors.

Therapeutic properties of mesenchymal stem cells for autism spectrum disorders.

Recent studies of autism spectrum disorders (ASD) highlight hyperactivity of the immune system, irregular neuronal growth and increased size and number of microglia. Though the small sample size in many of these studies limits extrapolation to all individuals with ASD, there is mounting evidence of both immune and nervous system related pathogenesis in at least a subset of patients with ASD. Given the disturbing rise in incidence rates for ASD, and the fact that no pharmacological therapy for ASD has been approved by the Food and Drug Administration (FDA), there is an urgent need for new therapeutic options. Research in the therapeutic effects of mesenchymal stem cells (MSC) for other immunological and neurological conditions has shown promising results in preclinical and even clinical studies. MSC have demonstrated the ability to suppress the immune system and to promote neurogenesis with a promising safety profile. The working hypothesis of this paper is that the potentially synergistic ability of MSC to modulate a hyperactive immune system and its ability to promote neurogenesis make it an attractive potential therapeutic option specifically for ASD. Theoretical mechanisms of action will be suggested, but further research is necessary to support these hypothetical pathways. The choice of tissue source, type of cell, and most appropriate ages for therapeutic intervention remain open questions for further consideration. Concern over poor regulatory control of stem cell studies or treatment, and the unique ethical challenges that each child with ASD presents, demands that future research be conducted with particular caution before widespread use of the proposed therapeutic intervention is implemented.

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting ß cells renders them susceptible to the autoimmune attack, and is associated with a diminution in ß-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on ß cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in ß cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing ß cells is implicated in ß-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.

CD40-mediated activation of chronic lymphocytic leukemia cells promotes their CD44-dependent adhesion to hyaluronan and restricts CCL21-induced motility.

Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.

RHAMM and CD44 peptides-analytic tools and potential drugs.

CD44 and RHAMM are two extracellar matrix receptors whose principle ligand is the polysaccharide hyaluronan (HA). Both proteins are involved in wound repair and their aberrant regulation contributes to a variety of diseases including arthritis and cancer. Over the past decade, a number of peptide-based therapeutics that block the binding of CD44 or RHAMM-specific ligands have been developed and tested in experimental models of disease. Here, we review the structure of each of these proteins, the functions they control and the mechanisms, including their interactions with each other, responsible for these functions. We also review the development of peptide mimics that block the key functions of CD44 and RHAMM and their use in experimental models of disease.