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Since the majority of the current therapies lack effectiveness and efficiency in treating Multiple Sclerosis, in addition to their high cost, monitoring during usage, and the serious side effects associated with using this therapy, which in some cases may be fatal, for these reasons, there is a necessary need for effective therapy in the clinical setting and searching for an alternative therapy that is effective and safe. For this purpose, this study evaluated the impact and efficiency of Caffeic acid phenethyl ester( CAPE) in the amelioration of inflammation and demyelination in the spinal cord of experimental autoimmune encephalomyelitis(EAE) mouse model multiple sclerosis, which could be a candidate therapy for MS. Multiple sclerosis is an autoimmune T-cell mediated disease, that T- cells become active, and differentiate into Th subset. α4β1integrin increased on the surface of T- cell during inflammation, which regulates immune cell cross through the blood-brain barrier into the central nervous system, and causes inflammation in the brain and spinal cord, myelin sheath damage and neuron demyelination. The in-vivo experiment used mice. The twenty-five mice were divided into control negative, control positive, and three treatment groups. After this, EAE was induced in mice by injecting myelin oligodendrocyte glycoprotein peptide. The mice were monitored and scored daily for clinical signs. CAPE was orally administered to mice at 5 mg/kg for T1, 10 mg/kg for T2, and 20 mg/kg for T3 for 14 days. Immunofluorescence was used to assess α4integrin, Immunohistochemistry (IHC) was used to evaluate infiltration of CD3-T cell marker, and Luxol Fast Blue stain was used to evaluate demyelination. We found that CAPE treated mice model had a reduced infiltration of immune cells, demyelination in the spinal cord mice model, and decreasing α4integrin expression. These findings strongly demonstrated that CAPE could be a potential therapy for Multiple sclerosis, as it ameliorated the inflammation and demyelination in mice models. [ABSTRACT FROM AUTHOR]

Since the majority of the current therapies lack effectiveness and efficiency in treating Multiple Sclerosis, in addition to their high cost, monitoring during usage, and the serious side effects associated with using this therapy, which in some cases may be fatal, for these reasons, there is a necessary need for effective therapy in the clinical setting and searching for an alternative therapy that is effective and safe. For this purpose, this study evaluated the impact and efficiency of Caffeic acid phenethyl ester(CAPE) in the amelioration of inflammation and demyelination in the spinal cord of experimental autoimmune encephalomyelitis(EAE) mouse model multiple sclerosis, which could be a candidate therapy for MS. Multiple sclerosis is an autoimmune T-cell mediated disease, that T- cells become active, and differentiate into Th subset.α4β1integrin increased on the surface of T- cell during inflammation, which regulates immune cell cross through the blood-brain barrier into the central nervous system, and causes inflammation in the brain and spinal cord, myelin sheath damage and neuron demyelination. The in-vivo experiment used mice. The twenty-five mice were divided into control negative, control positive, and three treatment groups. After this, EAE was induced in mice by injecting myelin oligodendrocyte glycoprotein peptide. The mice were monitored and scored daily for clinical signs. CAPE was orally administered to mice at 5 mg/kg for T1, 10 mg/kg for T2, and 20 mg/kg for T3 for 14 days. Immunofluorescence was used to assess α4integrin, Immunohistochemistry (IHC) was used to evaluate infiltration of CD3-T cell marker, and Luxol Fast Blue stain was used to evaluate demyelination. We found that CAPE treated mice model had a reduced infiltration of immune cells, demyelination in the spinal cord mice model, and decreasing α4integrin expression. These findings strongly demonstrated that CAPE could be a potential therapy for Multiple sclerosis, as it ameliorated the inflammation and demyelination in mice models.

Simple Summary: Progression of chronic lymphocytic leukemia (CLL) and its response to therapies are largely dependent on the microenvironment of the bone marrow and lymph nodes, which nurtures leukemic cells and protects them from therapeutic agents. Hence, cell trafficking between the blood vessels and lymphatic tissues is critical for CLL pathophysiology. Cell adhesion molecules mediate the re-localization of CLL cells in different anatomical compartments and are involved in their survival and proliferation. Evaluation of the molecular mechanisms underlying their activation and functions has uncovered clinically relevant signaling pathways targeted by well-established and new therapeutic strategies. The aim of this review is to summarize the current knowledge regarding the microenvironment and the cell adhesion molecules that have been shown to be important in CLL and their role in transendothelial migration and cell adhesion-mediated drug resistance. We also discuss how novel therapeutic compounds modulate the functions of this important class of molecules. Chronic lymphocytic leukemia (CLL) is a B-cell malignancy whose progression largely depends on the lymph node and bone marrow microenvironment. Indeed, CLL cells actively proliferate in specific regions of these anatomical compartments, known as proliferation centers, while being quiescent in the blood stream. Hence, CLL cell adhesion and migration into these protective niches are critical for CLL pathophysiology. CLL cells are lodged in their microenvironment through a series of molecular interactions that are mediated by cellular adhesion molecules and their counter receptors. The importance of these adhesion molecules in the clinic is demonstrated by the correlation between the expression levels of some of them, in particular CD49d, and the prognostic likelihood. Furthermore, novel therapeutic agents, such as ibrutinib, impair the functions of these adhesion molecules, leading to an egress of CLL cells from the lymph nodes and bone marrow into the circulation together with an inhibition of homing into these survival niches, thereby preventing disease progression. Several adhesion molecules have been shown to participate in CLL adhesion and migration. Their importance also stems from the observation that they are involved in promoting, directly or indirectly, survival signals that sustain CLL proliferation and limit the efficacy of standard and novel chemotherapeutic drugs, a process known as cell adhesion-mediated drug resistance. In this respect, many studies have elucidated the molecular mechanisms underlying cell adhesion-mediated drug resistance, which have highlighted different signaling pathways that may represent potential therapeutic targets. Here, we review the role of the microenvironment and the adhesion molecules that have been shown to be important in CLL and their impact on transendothelial migration and cell-mediated drug resistance. We also discuss how novel therapeutic compounds modulate the function of this important class of molecules. [ABSTRACT FROM AUTHOR]

Introduction Chronic lymphocytic leukemia (CLL) is the commonest hematological malignancy in the West but is relatively uncommon in India. The prognosis of CLL is determined by well-established prognostic markers. CD49d has been emerging as a promising prognostic marker in CLL. CD49d expression in CLL has been found to have an aggressive clinical course, shorter time to first treatment, and poorer prognosis. The aim of this study was to analyze the flow cytometric expression of CD49d in newly diagnosed CLL and to correlate its expression with clinico-hematological parameters. Materials and Methods Twenty-five consecutive patients of CLL, diagnosed on flow cytometry, were included in the study. Patients on treatment or those with relapse were excluded. The panel for flow cytometry included the routine markers used for CLL diagnosis along with CD49d. The expression of CD49d was correlated with clinico-hematological parameters in all patients. "R" software was used for the statistical analysis. Fisher's exact test and Wilcox test were used to assess the correlation of CD49d to categorical and continuous data, respectively. Results The mean age of the patients was 62.6 ± 12.5 years, and 80% were symptomatic at diagnosis. CD49d expression was found in 44% cases, with a higher proportion being male patients. CD49d and prolymphocyte percentage showed a statistically significant correlation (p = 0.0007). We found a statistically significant correlation between CD49d expression and lymphadenopathy and splenomegaly with p -values of 0.033 and 0.0472, respectively. CD49d positivity correlated significantly with a higher Rai stage (p = 0.0196) and intermediate and high-risk cases according to Binet staging (p = 0.033). Conclusion CD49d expression in the present study correlated with a higher prolymphocyte percentage, lymphadenopathy, splenomegaly, and higher Rai and Binet stages. CD49d expression on flow cytometry was reproducible and easy to interpret. [ABSTRACT FROM AUTHOR]

This study was carried out to assess the prognostic power of low CD49d expression (≥10%) in newly diagnosed CLL patients using a previously described cohort. Eighty‐five patients were included. Median age at diagnosis; 70 years (43–88); CD49d was expressed in 33/85 (38.8%); 23/33 (69.7%) at ≥30% referred to as 'HiCD49d' and 10/33 (30.3%) between 10 and 30% with a bimodal pattern on scatterplot analysis referred to as 'LoCD49d'. Eleven patients (12.9%) presented as Binet stage B, of whom 8 (72.7%) were CD49d+ (HiCD49d 7/8; LoCD49d 1/8). Seven of 81 patients (8.6%) were NOTCH1 mutated and all were CD49d+ (p ≤.01). IgVH analysis was performed on 29 (87.8%) of the CD49d+ cases, of whom 21 (72.4%) were unmutated and 8 (27.6%) were mutated. CD38+/CD49d+ accounted for 11/20 (55%) (CD38+/HiCD49D: 9/11; CD38+/LoCD49D: 2/11). At 42 months, treatment had been initiated in 18/85 (21%) patients, of these 10/33 (30.3%) were CD49d+ versus 8/52 (15.4%) of the CD49d− group. The median treatment free interval for the CD49d+ group was 11 months (HiCD49d; 14.5 months, LoCD49d; 11 months) compared to 21.5 months for the CD49d− group. These findings suggest that the predictive value of CD49d expression is retained at expression levels down to 10%. [ABSTRACT FROM AUTHOR]

Integrins are adhesion molecules that function as anchors in retaining tumor cells in supportive tissues and facilitating metastasis. Beta1 integrins are known to contribute to cell adhesion-mediated drug resistance in cancer. Very late antigen-4 (VLA-4), a CD49d/CD29 heterodimer, is a beta1 integrin implicated in therapy resistance in both solid tumors and haematological malignancies such as chronic lymphocytic leukemia (CLL). A complex inside-out signaling mechanism activates VLA-4, which might include several therapeutic targets for CLL. Treatment regimens for this disease have recently shifted towards novel agents targeting BCR signaling. Bruton's tyrosine kinase (BTK) is a component of B cell receptor signaling and BTK inhibitors such as ibrutinib are highly successful; however, their limitations include indefinite drug administration, the development of therapy resistance, and toxicities. VLA-4 might be activated independently of BTK, resulting in an ongoing interaction of CD49d-expressing leukemic cells with their surrounding tissue, which may reduce the success of therapy with BTK inhibitors and increases the need for alternative therapies. In this context, we discuss the inside-out signaling cascade culminating in VLA-4 activation, consider the advantages and disadvantages of BTK inhibitors in CLL and elucidate the mechanisms behind cell adhesion-mediated drug resistance. [ABSTRACT FROM AUTHOR]

Purpose: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4β1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression—BPR (VLA-4−) and BPRα (VLA-4+). Procedures: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. Results: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. Conclusions: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4−) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care. [ABSTRACT FROM AUTHOR]

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of Cg CD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in Cg CD49d. Cg CD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After Cg CD49d was blocked by using its antibody, the phosphorylation level of Cg JNK in the MAPK signaling pathway and Cg TNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of Cg JNK was inhibited by using its inhibitor, the nuclear translocation of Cg Rel was restrained and Cg TNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, Cg CD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated Cg CD49d antibody labeled agranulocytes with a circle of green fluorescence signals on Cg CD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that Cg CD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes. [Display omitted] • Cg CD49d was highly expressed in agranulocytes of oyster haemocytes. • Cg CD49d was involved in regulating Cg TNF expression via MAPK signaling pathway. • Cg CD49d+ agranulocytes accounted for 24.9 ± 4.53 % of haemocytes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia among adults in Western countries; however, it is relatively rare in Asia. It is characterized by abnormal proliferation of lymphocytes in the blood, bone marrow, and lymphatic tissue. The measurement of serum beta-2 microglobulin (B2M) is essential for baseline workup of multiple myeloma and follicular lymphoma patients. CD49d, an adhesion molecule mediating cell-to-cell and cell-to-extracellular matrix interactions, represents a novel and the most reliable immunophenotypic marker regarding prognosis and independent of other markers. OBJECTIVES: This study aimed to assess the level of B2M and CD49d in serum CLL patients and correlates them with treatment response. PATIENTS AND METHODS: this is a prospective cohort study conducted on 70 patients with CLL and 40 healthy people as a control group. Patient groups were divided into two groups: The first group included 38 patients before receiving treatment and the second group included 32 patients posttreatment. Diagnosis was based on lymphocyte count of >5 × 109/L and immunophenotyping. The measurement of level B2M and CD49d in serum patients was done using enzyme-linked immunosorbent assay. RESULTS: there were 53 males and 17 females, the mean age was 59.12 ± 14.23, and the most clinical presentation was lymphadenopathy. Regarding the mean of B2M, it was 2.19 ± 0.86, 1.86 ± 0.58, and 1.41 ± 0.44 in the pre, post, and control groups, respectively, with P = 0.0001. Regarding the mean of CD49d, it was 0.22 ± 0.15, 0.30 ± 0.44, and 0.19 ± 0.13 in the pre, post, and control groups, respectively, with P = 0.211. CONCLUSION: this study showed that CD49d has no clinical impact on the treatment outcome, yet B2M has an important prognostic factor in deciding patients in advance stage.

Under conditions of lymphopenia, T lymphocytes proliferate and acquire a surface activation phenotype, which in many respects is similar to the phenotype of true memory T cells. We investigated the phenotypic features of the CD8+ T-cell population formed from donor lymphocytes after adoptive transfer of syngeneic splenocytes to sublethally irradiated mice. This population expresses markers CD44, CD122, CD5, CD49d and the chemokine receptor CXCR3. Thus, for the first time, the phenomenon of the formation of a population of T cells with signs of suppressive CD8+ T lymphocytes and true memory cells was demonstrated. [ABSTRACT FROM AUTHOR]

Flow cytometric quantification of CD154+ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154+ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154+ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154+ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations. [ABSTRACT FROM AUTHOR]

Objective: To find out the frequency of ZAP-70, CD38 and CD49d in patients diagnosed with CLL in our population. Methods: This is a cross sectional study conducted in Army Medical College in collaboration with Armed Forces Institute of Pathology and Military Hospital Rawalpindi from 1st January 2018 to 30th November 2018. Permission from Institutional Ethical Committee was obtained. Blood samples were collected by non-probability consecutive sampling technique and analyzed for blood counts and flow cytometry was done for ZAP-70, CD38 and CD49d. Manufacturer's instructions for the kits were strictly followed. Results: Fifty-one newly diagnosed patients with CLL were studied for the prognostic markers in CLL. CD 38 was expressed in 25(49%) and CD49d in 21(41.2%). ZAP-70 expression was not detected in our series of patients. Conclusion: We conclude that CD38 and CD49d expression was detected in almost half of the patients of CLL in our series. CD49d showed statistically positive correlation with CD38, showing that it is a more pragmatic choice for reliable prognostication of CLL along with CD38. [ABSTRACT FROM AUTHOR]

Purpose: Mesenchymal Stem Cells (MSCs) are the most important members of Bone Marrow (BM) milieu. MSCs affect different kinds of cells, particularly malignant cells of hematologic malignancies, but the effects of MSCs are unclear exactly. Here we analyzed the effects of derived Umbilical Cord Blood-MSCs on proliferation, cell death and some surface markers of U937 cell line in a Co-culture system with MSCs. Methods: Here we designed Co-culture systems as a model of BM milieu. We cultured U937 cells on UCB-MSCs and MSCs Conditioned Medium (C.M) driven and then treated U937 cells with optimum concentration of chloride cobalt (CoCl2) as a hypoxia-mimetic agent. In addition, we applied suitable concentrations of H2O2 to induce cell death. Proliferation rate, cell death rate and some surface markers of hypoxic U937 cells were analyzed by MTT assay, flow cytometry and Real Time-PCR were flown respectively. Results: UCB-MSCs showed supportive effects on U937 proliferation rate in normoxia and hypoxia. Lethal effect of H2O2 suppressed in the presence of UCB-MSCs in hypoxia and normoxia. Among CD11a, CD14, CD49d, CD54 and CD116 markers, CD49d was down regulated in presence of UCB-MSCs and CD116 was up regulated in hypoxia. Other markers didn’t show any significant changes. Conclusion: This work provides evidences that MSCs play critical roles in U937 cells biology. These observations shed new light on MSCs roles and demonstrated that MSCs should be regarded as an important member of BM milieu in several clinical applications such as BM transplantation prognosis and treatment of hematologic malignancies.

Background: The spontaneously diabetic “non-obese diabetic” (NOD) mouse is a faithful model of human type-1 diabetes (T1D). Methods: Given the pivotal role of α4 integrin (CD49d) in other autoimmune diseases, we generated NOD mice with α4-deficient hematopoiesis (NOD.α4-/-) to study the role of α4 integrin in T1D. Results: NOD.α4-/- mice developed islet-specific T-cells and antibodies, albeit quantitatively less than α4+ counterparts. Nevertheless, NOD.α4-/- mice were completely and life-long protected from diabetes and insulitis. Moreover, transplantation with isogeneic α4-/- bone marrow prevented progression to T1D of pre-diabetic NOD.α4+ mice despite significant pre-existing islet cell injury. Transfer of α4+/CD3+, but not α4+/CD4+ splenocytes from diabetic to NOD.α4-/- mice induced diabetes with short latency. Despite an only modest contribution of adoptively transferred α4+/CD3+ cells to peripheral blood, pancreas-infiltrating T-cells were exclusively graft derived, i.e., α4+. Microbiota of diabetes-resistant NOD.α4-/- and pre-diabetic NOD.α4+ mice were identical. Co- housed diabetic NOD.α4+ mice showed the characteristic diabetic dysbiosis, implying causality of diabetes for dysbiosis. Incidentally, NOD.α4-/- mice were protected from autoimmune sialitis. Conclusion: α4 is a potential target for primary or secondary prevention of T1D.

Human B-1 cells have been proposed to be CD20+CD27+CD43+CD1c− B cells found in the umbilical cord and adult peripheral blood, but their regulatory mechanisms have not been well elucidated. Previously, we reported that mouse CD49dhigh CD4+ T cells could enhance the secretion of natural antibodies by B-1 cells. In this study, we aimed to investigate the presence and helper functions of the human equivalents of murine CD49dhigh CD4+ T cells. Here, we showed that human CD49dhigh CD4+ T cells found in the peritoneal cavity (PEC), spleen, and peripheral blood can enhance the production of IgM antibodies by B-1 cells. As revealed in mouse, CD49dhigh CD4+ T cells were more abundant in the PEC and showed a higher tendency to form conjugates with B cells than CD49dlow CD4+ T cells. Moreover, CD49dhigh CD4+ T cells showed a Th1-like memory phenotype, characterized by high expression of CD44 and CXCR3; low expression of CD62L and CCR7; rapid production of IFN-γ, tumor necrosis factor-α, and IL-2 upon stimulation with phorbol myristate acetate and ionomycin; and rapid proliferation upon stimulation with anti-CD3 and anti-CD28 antibodies. These cells also expressed high levels of PD-1, ICOS, and CD5, suggesting that they are undergoing chronic stimulation. Remarkably, CD49dhigh CD4+ T cells specifically helped B-1 cells, but not follicular memory B cells (CD27+ CD43−CD1c−) or marginal zone B cells (CD27+CD43−CD1c+), produce IgM and IgG antibodies. In parallel, the titer of human anti-blood group A IgM was positively correlated with the frequency of CD49dhigh CD4+ T cells. In conclusion, we identified human CD49dhigh CD4+ T cells with a Th1-like memory phenotype that secrete Th1 proinflammatory cytokines and help B-1 cells secrete antibodies, thereby aiding in primary defense. We suggest that these CD49dhigh CD4+ T cells are a unique type of B-cell helper T cells distinct from follicular helper T cells.

Objective: To evaluate the behavior of adhesion molecules ICAM-1 and ICAM-2 in dendritic cell (DC) immunotherapy. Materials & methods: 88 female Balb/c mice were divided into experimental groups. Tumors and lymph nodes were evaluated 7 and 14 days after immunotherapy. Results: Higher mean fluorescence intensity of ICAM-1 in the lymph nodes and tumors in the tumor group at 14 days was observed. Higher mean fluorescence intensity of ICAM-2 in the tumor DC vaccine group was observed after 14 days. A positive correlation was observed in the lymph nodes with ICAM-1 against tumoral volume in the tumor group. A negative correlation was found between ICAM-2 and tumoral volume in the lymph nodes of the tumor group. Conclusion: An increase in ICAM-2 in tumor DC vaccine and a decrease in ICAM-1 suggests the DC vaccine positively influences the immune system and that ICAM-2 could be a marker of good prognosis.

CD49d and CXCR4 are key determinants of interactions between chronic lymphocytic leukemia (CLL) tumor cells and their microenvironment. In this study, we investigated the effect of CD49d and CXCR4 expressions on survival of CLL cells. Primary CLL cells were cultured with CD49d ligand, VCAM-1, or bone marrow stromal cells (BMSCs); then, apoptosis and immunophenotype analyses were performed. VCAM-1 treatment could not induce direct apoptosis protection or immunophenotype change on the CD49d-expressing CLL cells, but resulted in actin reorganization. The BMSC-induced apoptosis protection was independent from the presence of CD49d expression of CLL cells, but showed an inverse correlation with their CXCR4 expression level. We suppose that CD49d contributes to enhanced survival of leukemic cells by mediating migration to the protective microenvironment, not by direct prevention of apoptosis. Moreover, CLL cells with low CXCR4 expression represent a subpopulation that is more dependent on the microenvironmental stimuli for survival, and show increased "death by neglect" when separated from the supportive niche. [ABSTRACT FROM AUTHOR]

Integrins are cell surface receptors with bidirectional signalling capabilities that can bind to adhesion molecules in order to mediate homing of leukocytes to peripheral tissues. Gut-selective leukocyte homing is facilitated by interactions between α4β7 and its ligand, mucosal addressin cellular adhesion molecule-1 [MAdCAM-1], while retention of lymphocytes in mucosal tissues is mediated by αEβ7 binding to its ligand E-cadherin. Therapies targeting gut-selective trafficking have shown efficacy in inflammatory bowel disease [IBD], confirming the importance of leukocyte trafficking in disease pathobiology. This review will provide an overview of integrin structure, function and signalling, and highlight the role that these molecules play in leukocyte homing and retention. Anti-integrin therapeutics, including gut-selective antibodies against the β7 integrin subunit [etrolizumab] and the α4β7 integrin heterodimer [vedolizumab and abrilumab], and the non-gut selective anti-α4 integrin [natalizumab], will be discussed, as well as novel targeting approaches using small molecules. [ABSTRACT FROM AUTHOR]

Human B-1 cells have been proposed to be CD20+CD27+CD43+CD1c− B cells found in the umbilical cord and adult peripheral blood, but their regulatory mechanisms have not been well elucidated. Previously, we reported that mouse CD49dhigh CD4+ T cells could enhance the secretion of natural antibodies by B-1 cells. In this study, we aimed to investigate the presence and helper functions of the human equivalents of murine CD49dhigh CD4+ T cells. Here, we showed that human CD49dhigh CD4+ T cells found in the peritoneal cavity (PEC), spleen, and peripheral blood can enhance the production of IgM antibodies by B-1 cells. As revealed in mouse, CD49dhigh CD4+ T cells were more abundant in the PEC and showed a higher tendency to form conjugates with B cells than CD49dlow CD4+ T cells. Moreover, CD49dhigh CD4+ T cells showed a Th1-like memory phenotype, characterized by high expression of CD44 and CXCR3; low expression of CD62L and CCR7; rapid production of IFN-γ, tumor necrosis factor-α, and IL-2 upon stimulation with phorbol myristate acetate and ionomycin; and rapid proliferation upon stimulation with anti-CD3 and anti-CD28 antibodies. These cells also expressed high levels of PD-1, ICOS, and CD5, suggesting that they are undergoing chronic stimulation. Remarkably, CD49dhigh CD4+ T cells specifically helped B-1 cells, but not follicular memory B cells (CD27+ CD43−CD1c−) or marginal zone B cells (CD27+CD43−CD1c+), produce IgM and IgG antibodies. In parallel, the titer of human anti-blood group A IgM was positively correlated with the frequency of CD49dhigh CD4+ T cells. In conclusion, we identified human CD49dhigh CD4+ T cells with a Th1-like memory phenotype that secrete Th1 proinflammatory cytokines and help B-1 cells secrete antibodies, thereby aiding in primary defense. We suggest that these CD49dhigh CD4+ T cells are a unique type of B-cell helper T cells distinct from follicular helper T cells. [ABSTRACT FROM AUTHOR]

In the translational process of developing innovative therapies for DMD (Duchenne muscular dystrophy), the last preclinical validation step is often carried out in the most relevant animal model of this human disease, namely the GRMD (Golden Retriever muscular dystrophy) dog. The disease in GRMD dogs mimics human DMD in many aspects, including the inter-individual heterogeneity. This last point can be seen as a drawback for an animal model but is inherently related to the disease in GRMD dogs closely resembling that of individuals with DMD. In order to improve the management of this inter-individual heterogeneity, we have screened a combination of biomarkers in sixty-one 2-month-old GRMD dogs at the onset of the disease and a posteriori we addressed their predictive value on the severity of the disease. Three non-invasive biomarkers obtained at early stages of the disease were found to be highly predictive for the loss of ambulation before 6 months of age. An elevation in the number of circulating CD4+CD49dhi T cells and a decreased stride frequency resulting in a reduced spontaneous speed were found to be strongly associated with the severe clinical form of the disease. These factors can be used as predictive tests to screen dogs to separate them into groups with slow or fast disease progression before their inclusion into a therapeutic preclinical trial, and therefore improve the reliability and translational value of the trials carried out on this invaluable large animal model. These same biomarkers have also been described to be predictive for the time to loss of ambulation in boys with DMD, strengthening the relevance of GRMD dogs as preclinical models of this devastating muscle disease.

Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease characterised by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. Stimulation through the B cell receptor (BCR) plays a prominent role in the selection and expansion of the malignant clone in CLL. On the other hand, other external signals delivered by several cell types including T lymphocytes, macrophages, stromal cells, endothelial cells, and follicular dendritic cells, operating through either direct BCR-independent cell-cell contact or indirect production of paracrine soluble factors, synergistically cooperate in regulating proliferation and survival of CLL cells. In this context, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues, eventually delivering pro-survival signals and protecting CLL cells from drug-induced damages. In the present review, we focused on functional and physical interactions of CD49d with other microenvironmental receptors, including CD38 and BCR, and other specific CD49d-dependent interactions in lymph node and bone marrow microenvironments responsible for growth and survival-supporting signals, eventually influencing CLL prognosis and therapeutic options.

Background: Impairment of regulatory T cells (Tregs) in common autoimmune diseases seems likely. However, the extent of Treg deficiency (number, function) or differential susceptibility of T effector cells (Teffs) to suppression is not completely understood. We hypothesize that even in healthy individuals both cell populations are heterogeneous and differ in their suppressive capability and their susceptibility to suppression. Material and Methods: Lymphocytes were enriched by MACS for CD4+CD25+ Tregs or CD4+CD25- Teffs. After multicolour staining (anti-CD25, anti-CD127, anti-CD49d or anti-CD45RA) highly purified Treg and Teff subpopulations were collected by FACS. Functional capacity of Tregs or suppressive susceptibility of Teffs was analyzed in an in vitro assay. Results: When CD4+CD25highCD127-/low CD49d- Tregs were tested on naive CD4+CD127+CD25-CD45RA+ Teffs (93.8 %) suppression was almost complete, while the suppressive capacity of CD4+CD25highCD127-/low CD49d+ Tregs was significantly less (71.8 %). Suppressive activity was low when CD4+CD25highCD127-/low CD49d+ Tregs were analyzed on CD4+CD127+CD25-CD45RA- Teffs (48.7%). Conclusion: Although CD49d+ Tregs are functional, the suppressive capacity is significantly lower compared to CD49d- Tregs. CD45RA+ Teffs can be completely suppressed, while CD45RA- Teffs display relative resistance. Phenotypic and functional heterogeneity of Tregs as well as Teffs has to be considered when analyzing deficiencies in immune regulation.

CD49d is a surface integrin that is expressed on chronic lymphocytic leukaemia ( CLL) cells, and strongly correlates with more aggressive disease. Given its association with cell-cell adhesion and leucocyte trafficking, we hypothesized that patients with high CD49d expression would experience a clinical course dominated by lymphadenopathy. CD49d expression was measured by flow cytometry and considered positive if expressed by ≥30% of CLL cells. The study included 797 newly diagnosed CLL/small lymphocytic leukaemia patients; 279 (35%) were CD49d positive. CD49d-positive patients were more likely to present with lymphadenopathy ( P < 0·001); a finding that persisted after adjusting for fluorescence in situ hybridisation ( FISH) and IGHV mutation status [odds ratio ( OR) 2·51; 95% confidence interval ( CI) 1·64-3·83; P < 0·001]. Among CLL Rai 0 patients, CD49d positivity was associated with shorter time to development of lymphadenopathy (3·2 years vs not reached, P < 0·01). This association was maintained after adjusting for either FISH [hazard ratio ( HR) 2·18; 95% CI 1·25-3·81; P = 0·006) or IGHV status ( HR 2·02; 95% CI 1·11-3·69; P = 0·02) individually, but was attenuated when adjusting by both ( HR 1·72; 95% CI 0·88-3·38; P = 0·11).These data demonstrate that CD49d-positive CLL patients experience a disease course dominated by lymphadenopathy. These findings could have implications for therapy selection and disease monitoring. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of this study was to assess the prognostic value of CD49d expression in Turkish chronic lymphocytic leukemia patients. METHODS: Data for 118 patients from a single center were evaluated. In all, the study included 73 patients for whom complete clinical follow-up data, and flow cytometry test results for CD5/19, CD23/43, CD38, Zap- 70, Kappa, and Lambda light chains, and CD49d were available. The effect of the level of CD49d expression on overall survival (OS) and time to treatment (TTT) was investigated retrospectively. RESULTS: Patients with high CD49d expression (≥30%) had more advanced disease at the time of diagnosis (median Rai stage 3 vs. Rai stage 1, P = 0.03). Patients resistant to treatment had higher CD49d expression than patients that responded to treatment (mean CD49d expression of 58% vs. 46%, P = 0.08). The level of CD49d expression was not associated with OS or TTT. CONCLUSION: The study's findings show that the patients with high CD49d expression at the time of diagnosis had more advanced disease and poorer response to therapy; however, their overall survival did not differ from that of the patients with advanced disease stage, but lower levels of CD49d expression.

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer. [ABSTRACT FROM AUTHOR]

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. The frequency of symptomatic central nervous system (CNS) involvement is unknown but thought to be a rare phenomenon. Currently there are no known risk factors for CNS involvement. Case presentation: We describe a clinically staged low-risk CLL case that presented with symptomatic CNS involvement and progressed rapidly to death. Evaluation of the surface adhesion molecules identified a markedly altered expression pattern of the integrin, CD49d and the tetraspanin, CD82, in the index case when compared to similar low-risk CLL cases. We found that the early Rai clinical stage CLL patients showed linear correlation for the co-expression of CD82 and CD49d. In contrast, this unique index case with CNS involvement, which has the same Rai clinical stage, had a significantly lower expression of CD82 and higher expression of CD49d. Conclusions: These data suggest that the expression profile of CD49d and CD82 may represent potential biomarkers for patients with increased propensity of CNS involvement. Moreover, this study illustrates the critical need for a better mechanistic understanding of how specific adhesion proteins regulate the interactions between CLL cells and various tissue sites. [ABSTRACT FROM AUTHOR]

Purpose: Mesenchymal Stem Cells (MSCs) are the most important members of Bone Marrow (BM) milieu. MSCs affect different kinds of cells, particularly malignant cells of hematologic malignancies, but the effects of MSCs are unclear exactly. Here we analyzed the effects of derived Umbilical Cord Blood-MSCs on proliferation, cell death and some surface markers of U937 cell line in a Co-culture system with MSCs. Methods: Here we designed Co-culture systems as a model of BM milieu. We cultured U937 cells on UCB-MSCs and MSCs Conditioned Medium (C.M) driven and then treated U937 cells with optimum concentration of chloride cobalt (CoCl2) as a hypoxia-mimetic agent. In addition, we applied suitable concentrations of H2O2 to induce cell death. Proliferation rate, cell death rate and some surface markers of hypoxic U937 cells were analyzed by MTT assay, flow cytometry and Real Time-PCR were flown respectively. Results: UCB-MSCs showed supportive effects on U937 proliferation rate in normoxia and hypoxia. Lethal effect of H2O2 suppressed in the presence of UCB-MSCs in hypoxia and normoxia. Among CD11a, CD14, CD49d, CD54 and CD116 markers, CD49d was down regulated in presence of UCB-MSCs and CD116 was up regulated in hypoxia. Other markers didn't show any significant changes. Conclusion: This work provides evidences that MSCs play critical roles in U937 cells biology. These observations shed new light on MSCs roles and demonstrated that MSCs should be regarded as an important member of BM milieu in several clinical applications such as BM transplantation prognosis and treatment of hematologic malignancies. [ABSTRACT FROM AUTHOR]

Summary Extensive transcriptional and ontogenetic diversity exists among normal tissue-resident macrophages, with unique transcriptional profiles endowing the cells with tissue-specific functions. However, it is unknown whether the origins of different macrophage populations affect their roles in malignancy. Given potential artifacts associated with irradiation-based lineage tracing, it remains unclear if bone-marrow-derived macrophages (BMDMs) are present in tumors of the brain, a tissue with no homeostatic involvement of BMDMs. Here, we employed multiple models of murine brain malignancy and genetic lineage tracing to demonstrate that BMDMs are abundant in primary and metastatic brain tumors. Our data indicate that distinct transcriptional networks in brain-resident microglia and recruited BMDMs are associated with tumor-mediated education yet are also influenced by chromatin landscapes established before tumor initiation. Furthermore, we demonstrate that microglia specifically repress Itga4 (CD49D), enabling its utility as a discriminatory marker between microglia and BMDMs in primary and metastatic disease in mouse and human. [ABSTRACT FROM AUTHOR]

Chemotherapeutic drug resistance in acute lymphoblastic leukemia (ALL) is a significant problem, resulting in poor responsiveness to first-line treatment or relapse after transient remission. Classical anti-leukemic drugs are non-specific cell cycle poisons; some more modern drugs target oncogenic pathways in leukemia cells, although in ALL these do not play a very significant role. By contrast, the molecular interactions between microenvironment and leukemia cells are often neglected in the design of novel therapies against drug resistant leukemia. It was shown however, that chemotherapy resistance is promoted in part through cell-cell contact of leukemia cells with bone marrow stromal cells, also called cell adhesion-mediated drug resistance (CAM-DR). Incomplete response to chemotherapy results in persistence of resistant clones with or without detectable minimal residual disease (MRD). Approaches for how to address CAM-DR and MRD remain elusive. Specifically, studies using anti-functional antibodies and genetic models have identified integrin alpha4 as a critical molecule regulating bone marrow homing and active retention of normal and leukemic cells. Pre-clinical evidence has been provided that interference with alpha4-mediated adhesion of ALL cells can sensitize them to chemotherapy and thus facilitate eradication of ALL cells in an MRD setting. To this end, Andreeff and colleagues recently provided evidence of stroma-induced and alpha4-mediated NFkappaB signaling in leukemia cells, disruption of which depletes leukemia cells of strong survival signals. We here review the available evidence supporting the targeting of alpha4 as a novel strategy for treatment of drug resistant leukemia.

We investigated CD49d (also termed ITGA4) expression and its biological and clinical correlations in 415 patients with chronic lymphocytic leukaemia. CD49d expression was stable over the course of the disease. A high expression of CD49d (>30%) was found in 142/415 (34%) patients and was associated with progressive disease (advanced clinical stage, high serum lactate dehydrogenase or β2-microglobulin levels; all p < 0·05) and aggressive disease biology (increased ZAP70 or CD38, unmutated IGHV, trisomy 12, mutations of NOTCH1 and SF3B1; all P < 0·05). A higher CD49d expression was also associated with a lower blood lymphocyte count and a higher number of lymphoid areas involved by the disease. Patients with high CD49d expression were treated more frequently (55% vs. 27%; P < 0·001) and earlier (median time to treatment [TTT] 65·4 months vs. not reached; P < 0·001) than those with low CD49d expression. However, no significant differences in response rates were observed. In the subgroup of patients with mutated IGHV, high CD49d expression was predictive of a shorter TTT while other markers, such as ZAP70 and CD38, were not. In conclusion, in this study CD49d expression correlated with high-risk CLL biomarkers and proved to be useful for separating patients with mutated IGHV into two different prognostic groups. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. The immune inflammatory response also contributes to disease progression in DMD patients. In a previous study, we demonstrated higher levels of circulating CD49dhi and CD49ehi T cells in DMD patients compared to healthy control. DMD patients are clinically heterogeneous and the functional defect cannot be correlated with genotype. Therefore, it is important to be able to define reliable noninvasive biomarkers to better define the disease progression at the beginning of clinical trials. Results: We studied 75 DMD patients at different stages of their disease and observed that increased percentages of circulating CD4+CD49dhi and CD8+CD49dhi T lymphocytes were correlated with both severity and a more rapid progression of the disease. Moreover, T+CD49d+ cells were also found in muscular inflammatory infiltrates. Functionally, T cells from severely affected patients exhibited higher transendothelial and fibronectin-driven migratory responses and increased adhesion to myotubes, when compared to control individuals. These responses could be blocked with an anti-CD49d monoclonal antibody. Conclusion: CD49d can be used as a novel biomarker to stratify DMD patients by predicting disease progression for clinical trials. Moreover, anti-CD49d peptides or antibodies can be used as a therapeutic approach to decrease inflammation-mediated tissue damage in DMD. [ABSTRACT FROM AUTHOR]

Aims Natalizumab is a humanized monoclonal antibody specific for CD49d receptors of integrins. It inhibits the entry of inflammatory cells into the central nervous system and is approved for the treatment of relapsing-remitting multiple sclerosis ( MS). Several lines of evidence indicate an involvement of B cells and plasma cells in MS pathogenesis. However, treatment with the natalizumab analogon PS/2 immunoglobulin G ( IgG) has so far only been investigated in T cell-mediated animal models of MS. Due to the importance of B lineage cells in the pathogenesis of MS, the objective of the present study has thus been to analyse the effects of PS/2 IgG in a mouse model of MS with T and B cell cooperation ( OSE mice). Methods OSE mice were treated with the natalizumab analogon PS/2 IgG either at disease onset or after peak of disease. Treatment was also performed with PS/2 F(ab′)2 fragments. Results PS/2 IgG treatment improved the clinical outcome and decreased spinal cord demyelination and immune cell infiltration if given early in the disease course. Treatment increased blood leukocytes and resulted in a partial internalization of CD49d in T and B cells. The therapeutic effects of PS/2 IgG injections were independent of the Fc fragment as F(ab′)2 injections were equally beneficial. In contrast, PS/2 IgG was not effective when given late in the disease course. Conclusions Results indicate that natalizumab may also be beneficial in MS with B cell-driven immunopathogenesis. [ABSTRACT FROM AUTHOR]

Background: Impairment of regulatory T cells (Tregs) in common autoimmune diseases seems likely. However, the extent of Treg deficiency (number, function) or differential susceptibility of T effector cells (Teffs) to suppression is not completely understood. We hypothesize that even in healthy individuals both cell populations are heterogeneous and differ in their suppressive capability and their susceptibility to suppression. Material and Methods: Lymphocytes were enriched by MACS for CD4+CD25+ Tregs or CD4+CD25- Teffs. After multicolour staining (anti-CD25, anti-CD127, anti-CD49d or anti-CD45RA) highly purified Treg and Teff subpopulations were collected by FACS. Functional capacity of Tregs or suppressive susceptibility of Teffs was analyzed in an in vitro assay. Results: When CD4+CD25highCD127-/low CD49d- Tregs were tested on naive CD4+CD127+CD25-CD45RA+ Teffs (93.8 %) suppression was almost complete, while the suppressive capacity of CD4+CD25highCD127-/low CD49d+ Tregs was significantly less (71.8 %). Suppressive activity was low when CD4+CD25highCD127-/low CD49d+ Tregs were analyzed on CD4+CD127+CD25-CD45RA- Teffs (48.7%). Conclusion: Although CD49d+ Tregs are functional, the suppressive capacity is significantly lower compared to CD49d- Tregs. CD45RA+ Teffs can be completely suppressed, while CD45RA- Teffs display relative resistance. Phenotypic and functional heterogeneity of Tregs as well as Teffs has to be considered when analyzing deficiencies in immune regulation. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chemotherapeutic drug resistance in acute lymphoblastic leukemia (ALL) is a significant problem, resulting in poor responsiveness to first-line treatment or relapse after transient remission. Classical anti-leukemic drugs are non-specific cell cycle poisons; some more modern drugs target oncogenic pathways in leukemia cells, although in ALL these do not play a very significant role. By contrast, the molecular interactions between microenvironment and leukemia cells are often neglected in the design of novel therapies against drug resistant leukemia. It was shown however, that chemotherapy resistance is promoted in part through cell-cell contact of leukemia cells with bone marrow (BM) stromal cells, also called cell adhesion-mediated drug resistance (CAM-DR). Incomplete response to chemotherapy results in persistence of resistant clones with or without detectable minimal residual disease (MRD). Approaches for how to address CAM-DR and MRD remain elusive. Specifically, studies using anti-functional antibodies and genetic models have identified integrin alpha4 as a critical molecule regulating BM homing and active retention of normal and leukemic cells. Pre-clinical evidence has been provided that interference with alpha4-mediated adhesion of ALL cells can sensitize them to chemotherapy and thus facilitate eradication of ALL cells in an MRD setting.To this end, Andreeff and colleagues recently provided evidence of stroma-induced and alpha4-mediated nuclear factor-κB signaling in leukemia cells, disruption of which depletes leukemia cells of strong survival signals. We here review the available evidence supporting the targeting of alpha4 as a novel strategy for treatment of drug resistant leukemia. [ABSTRACT FROM AUTHOR]