The re-emergence of the B1 cell compartment : is this a pre-lymphoma stage?

Chronic Lymphocytic Leukemia (CLL) are in some cases stereotyped for immunoglobulin variants in different populations, suggesting emergence of B cell subsets following presentation of the same antigen. CLL cells may originate from CD5+ naïve cells and from CD5 memory cells. Gene expression studies characterized a common cell of origin of the two clinical categories of CLL; the unmutated aggressive type and the mutated indolent type. The aim of this study was to investigate the presence of CD5 positive B cells in the elderly and their potential stimulation with exosomes derived from tumor cells. The findings from this study is aimed to create a model to identify instigating carcinomatous factors that may stimulate B1 cells to transform into a CLL-like model. In this study we show that CD19\textsuperscript+ cells (B cells) in cord blood have a high expression of CD5. CD19/CD5 staining of blood samples from senior citizens showed the presence of B cells which also express the CD5 marker, though at a lower expression when compared to CLL cells (CD19+/CD5 dim B cells). Measurement of clonality using λ/Κ flow cytometry staining show a monoclonal origin of the human CD19+/CD5 dim B cells. Monoclonal B cell Lymphocytosis in the elderly is a potential cell compartment that represents the origin of B cell proliferative disorders. The origin of the B cell proliferative disease requires antigen stimulation. A preliminary experiment showed that sorted lymphocytes can be stimulated by exosomes isolated from 2 cancer cells lines, A549 (lung epithelial) and PC3 (prostate cell line). In comparison with phytohaemagglutinin (PHA) and phorbolmyristate acetate (PMA), known lymphocyte stimulators, the exosomes stimulated the proliferation of monocytic-like cells. Further characterization is required to know the origin of these cells. The result shows that one can speculate that exosomes present cancer-derived antigens and stimulate cell proliferation. Further studies are required to evaluate the potential transformation capacity of cancer-derived exosomes. In addition, various cytokines were measured in the sera of senior citizens to investigate a differential release of cytokines in the presence or absence of the CD19+/CD5 dim B cells. Cytokines examined were not significantly different between the 2 groups and further evaluation of cytokine levels is required.peer-reviewe

Trypanosoma brucei effect on leukocyte differentiation and activation

Tripanossomíase Africana ou doença do sono é uma doença zoonótica causada por Trypanosoma brucei, um protozoário parasita transmitido pela mosca tsetse ou Glossina. A introdução do parasita no hospedeiro vertebrado provoca uma sucessão de eventos que envolvem a imunidade inata e adaptativa. Os macrófagos (MΦ) apresentam um papel fundamental na defesa inata por serem células apresentadoras profissionais de antigénio (APC) e células fagocíticas, importantes na eliminação dos tripanossomas. A imunidade adaptativa é assegurada pelos linfócitos, nomeadamente pelos linfócitos T. Contudo, o papel desempenhado por estas células imunitárias ainda não se encontra completamente clarificado. Assim, este estudo teve como objetivo analisar a atividade dos MΦ na infeção por T. brucei, avaliando os níveis de ureia e óxido nítrico (NO) e a expressão membranar das moléculas de classe I (MHCI) e classe II (MHCII) do complexo principal de histocompatibilidade. A diferenciação das populações linfocitárias, T helper (Th), T citotóxicas (Tc) e T reguladoras foi também avaliada. MΦ de murganho foram expostos a tripomastigotas de T. brucei e estimulados com antigénio total e exossomas de T. brucei. A caracterização da ativação macrofágica através de ensaios colorimétricos demonstrou que ambos os fenótipos M1 e M2 foram expressos e foi evidenciada correlação positiva entre a produção de ureia e de NO. Adicionalmente, os resultados da citometria de fluxo indicaram que o parasita prejudica a diferenciação das subpopulações de MΦMHCI+ e MΦMHCII+ mas induz o aumento de moléculas MHCI. Contrariamente, os exossomas demonstraram estimular as funções APC através das moléculas MHCI e MHCII. Os rácios MHCI/MHCII indicaram que o contacto com o parasita favorece a apresentação antigénica às células TCD4+, enquanto que os exossomas direcionam a apresentação antigénica para as subpopulações de linfócitos TCD4+ e TCD8+. A caraterização das populações linfocitárias através da citometria de fluxo demonstrou que T. brucei causa a diminuição das populações de linfócitos Th e Tc. Contrariamente, a estimulação com o antigénio total ou com os exossomas induziram a expansão de ambas as subpopulações linfocitárias. T. brucei parece promover a expansão das subpopulações linfocitárias CD8-CD25+FoxP3- e CD8+CD25+FoxP3-. A expansão da subpopulação celular CD25-FoxP3+ foi observada na fração celular CD8- após estimulação do antigénio e na fração celular CD8+ estimulada por exossomas. Além disso, os exossomas também promoveram a expansão da subpopulação de linfócitos T CD8+CD25+FoxP3+. Curiosamente, apesar da contração celular T. brucei induziu o aumento das moléculas FoxP3. Em conjunto, os resultados obtidos indicam que o parasita e os exossomas parecem exercer efeitos opostos nas células analisadas. Os parasitas parecem minimizar a apresentação antigénica e evitam induzir a expansão das subpopulações de linfócitos T, facilitando a sua permanência no hospedeiro. Por outro lado, os exossomas segregados por T. brucei parecem estimular a apresentação antigénica e mediar a expansão dos linfócitos Th, possivelmente deslocando o foco da atividade do sistema imunitário dos parasitas para os exossomas. Estes resultados permitiram clarificar alguns dos princípios subjacentes à resposta imunitária inata e adaptativa na fase inicial da infeção. A compreensão destes mecanismos pode vir a contribuir para o desenvolvimento de novas estratégias de controlo e eliminação da Tripanossomíase Africana.African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by tsetse fly or Glossina. Parasite introduction into mammal hosts, triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defense, since they are antigen-presenting cells (APC) and have a phagocytosis function essential for trypanosomes clearance. Adaptive immune defense is carried out by lymphocytes, in particular by T lymphocytes. However, the exact role of these immune cells remains not completely understood. Thus, this study aimed to assess the role of MΦ in T. brucei infection by measuring the urea and nitric oxide (NO) levels, and by evaluating membrane expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T helper (Th), T cytotoxic (Tc) and T regulatory cell subsets was assessed. Mouse MΦ were exposed to T. brucei trypomastigotes and stimulated by T. brucei extract and T. brucei exosomes. Characterization of MΦ activation with colorimetric assays have indicated that both M1 and M2 phenotypes were expressed, evidencing a positive correlation between urea and NO levels produced. Additionally, results of flow cytometry indicated that T. brucei impairs the expansion of both MHCI+ and MHCII+ MΦ subsets, but enhanced MHCI molecules. On the contrary, T. brucei exosomes stimulated APC functions through MHCI and MHCII molecules. MHCI/MHCII rates indicated that T. brucei shift the antigen presentation to CD4+ T cells, while exosomes directed the antigen presentation to both CD4+ and CD8+ T cells. Characterization of lymphocyte subsets by flow cytometry demonstrated that T. brucei impairs both Th and Tc lymphocytes. On the contrary, cell stimulation by extract and exosomes promote the expansion of both T cell subpopulations. T. brucei seem to promote the expansion of CD8-CD25+FoxP3- and CD8+CD25+FoxP3- T cell subsets. The expansion of CD25-FoxP3+ T cell subset was observed in CD8- cell fraction antigen stimulated and in the CD8+ cell fraction exosome stimulated. Moreover, exosomes also induced the expansion of CD8+ CD25+FoxP3+ T cell subset. Interestingly, despite cell decrease T. brucei seemed to increase FoxP3 molecules. Taken together, these findings indicate that parasite and parasite exosomes seem to have opposite effects on the evaluated cells. Parasites seem to minimize antigen presentation and avoid inducing the expansion of T cell subsets, facilitating its permanence in the host. On the other hand, T. brucei secreted exosomes seems to induce APC functions and mediate the expansion of Th lymphocytes, probably focusing the immune activity on the exosomes and not on the parasites. These findings allowed to understand some underlying principles of the innate and adaptive immune response in the early-stage of infection. Comprehension of these mechanisms can endorse the development of new strategies for control and elimination of African Trypanosomiasis

CarboxyAmido-Triazole Orotate inhibits the growth of Imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

Chronic myelogenous leukemia is a myeloproliferative disorder characterized by the t(9:22) (q34:q11) reciprocal chromosomal translocation, resulting in the expression of the chimeric Bcr–Abl oncoprotein with constitutive tyrosine kinase activity. Deregulated Bcr–Abl induces the hyperactivation of various signalling pathways that promote cell growth, suppress apoptosis and alter cell adhesion. Bcr-Abl has also been involved in VEGF-mediated angiogenesis in CML and evidence indicates that the formation of new vessels plays an important role in the development and progression of CML. Imatinib mesylate (IM) is a selective well tolerated inhibitor of the Bcr–Abl tyrosine kinase that has significantly improved the prognosis of patients with chronic phase CML. Despite this remarkable progress, a major problem associated with the administration of imatinib is acquired resistance. Bcr-Abl gene amplification, increased expression of Bcr–Abl protein, point mutations in the Bcr–Abl tyrosine kinase domain have been reported as mechanisms of resistance to imatinib. Therefore, there is an urgent need for new anticancer agents and combinations that could improve responses and survival rates for CML. Recent studies from our laboratory have shown that addition of carboxyamidotriazole (CAI), an inhibitor of calcium-mediated signal transduction, to imatinib resistant human CML cells induces a marked decrease in cell viability and augmented apoptosis, events associated with downregulation of Bcr–Abl protein and inhibition of tyrosine phosphorylation of Bcr–Abl, STAT5, CrkL. Carboxyamidotriazole Orotate (CTO), is a derivate of CAI that has been developed at Tactical Therapeutics. CTO has a higher bioavailability and efficacy with respect to the parental compound. Exosomes are small vesicles of 40-100 nm diameter that are initially formed within the endosomal compartment and are secreted when a multivesicular body (MVB) fuses with the plasma membrane. These vesicles are released by many cell types including cancer cells and are considered messengers in intercellular communication. The exact function of exosomes in malignant cells has yet to be elucidated, but investigation has suggested roles in cell-to-cell communication, tumor-stroma interaction, and antigen presentation, thus potentially affecting cancer progression at different steps. Recent studies from our laboratory suggest that exosomes released from IM-sensitive CML cells directly affect endothelial cells modulating the process of neovascularization. Our data show that CTO is able to inhibit both in vitro and in vivo the growth of imatinib-resistant CML cells and to affect tumor microenvironment by modulating exosome-stimulated angiogenesis. CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility in the treatment of leukemia patients

LPS 자극 공여 엑소좀 전신 투여에 의한 수여 마우스 뇌의 신경염증 유도

학위논문(석사) -- 서울대학교대학원 : 융합과학기술대학원 분자의학 및 바이오제약학과, 2021.8. 오진혜.Lipopolysaccharide (LPS) is known to activate innate immune cells that promotes release of pro-inflammatory factors, which causes systemic inflammation. LPS-primed immune cells may release a huge number of exosomes to convey specific molecular information to the distant tissue as an efficient communicator to regulate physiological and biological function of target cells. From this regard, it is important to understand a biological role of exosomes for communication between periphery and brain by finding out whether exosomes peripherally stimulated by LPS may affect the brain tissue and which contents of exosomes are closely involved. In this study, I investigated whether intraperitoneally injected LPS changes the exosomes contents and LPS-stimulated exosomes may eventually cause neuroinflammation via systemic administration of those exosomes. After intraperitoneal administration of LPS to C57BL/6 donor mice, exosomes were isolated from the plasma of the donor mice, and the LPS-primed exosomes were exposed with BV2 microglia cell line or C57BL/6 recipient mice. Optimal dose and time point of LPS (5 mg, 4 hr after LPS treatment i.p.) to induce neuroinflammation was determined. Cytokine array data showed that CXCL1, CXCL10 and CCL2 chemokine levels were significantly increased in LPS-exosomes, compared to PBS-exosomes group. The expressions of proinflammatory mRNAs such as Tnfa, Il1b and Il6 were increased after treatment of LPS-exosomes in BV2 microglia cell line until 24 hr. When LPS-primed exosomes were intravenously treated to C57BL/6 recipient mice, levels of Tnfa, Ifng, Il1b, Il6 mRNA were increased in the brain, compared to PBS-exosomes-treated group. Nlrp3 and Casp1 mRNA which are known as downstream genes for CXCL1-CXCR2 signaling was increased after systemic injection of LPS-exo in recipient mouse brain. This study demonstrated that LPS-primed exosomes contain a higher amount of chemokine, which may affect the brain by inducing neuroinflammation with increased proinflammatory cytokines in the brain.지방 다당류 (LPS)는 선천 면역세포를 활성화시켜 전신 염증을 유발하는 전 염증 인자의 분비를 촉진하는 것으로 알려져 있다. LPS 프라이밍 된 면역 세포는 특정 분자 정보를 먼 조직에 전달하기 위해 표적 세포의 생리적 및 생물학적 기능을 조절하는 효율적인 매개체인 엑소좀을 다량 분비할 수 있다. 이러한 점에서 LPS로 자극된 엑소좀이 뇌 조직에 영향을 미칠 수 있는지, 그리고 엑소좀의 어떤 내용물이 이와 밀접하게 관련되어 있는지를 알아냄으로써 말초와 뇌 사이의 소통을 위한 엑소좀의 생물학적 역할을 이해하는 것이 중요하다. 이번 실험을 통해 복강 투여된 LPS가 엑소좀 내용물을 변화시키고 LPS로 자극된 엑소좀을 전신 투여하여 이러한 엑소좀이 결국 신경 염증을 유발할 수 있는지 확인하였다. C57BL/6 공여자 마우스에 LPS를 복강 내 투여한 후, 공여자 마우스의 혈장으로부터 엑소좀을 분리하고, LPS- 프라이밍 된 엑소좀을 BV2 미세아교세포주 또는 C57BL/6 수용자 마우스에 노출시켰다. 신경 염증을 유도하기위한 LPS의 최적 용량 및 시점 (5 mg, LPS 주입 4 시간 후)을 결정하였다. 사이토카인 어레이 결과는 CXCL1, CXCL10 및 CCL2 케모카인이 PBS-엑소좀 그룹에 비해 LPS-엑소좀에서 유의하게 증가했음을 보여주었다. Tnfa, Il1b 및 Il6과 같은 전 염증성 mRNA의 발현은 BV2 미세아교세포주에서 LPS-엑소좀 처리 후 24 시간까지 증가하였다. LPS-엑소좀을 C57BL/6 수용자 마우스에 정맥 주사한 경우, PBS-엑소좀 처리 군에 비해 뇌에서 Tnfa, Ifng, Il1b, Il6, Nlrp3 및 Casp1 mRNA의 발현이 증가하였다. 본 연구는 LPS 프라이밍된 엑소좀이 더 많은 양의 케모카인을 함유하고 있으며, 이는 뇌에서 증가된 전 염증성 사이토 카인과 함께 신경 염증을 유도함으로써 뇌에 영향을 미칠 수 있음을 입증하였다.Introduction 1 Materials and Methods 3 Results 7 Discussion 11 References 14 Figures 19 Abstract in Korean 30석

The Role of Exosomes Derived From Mesenchymal Stromal Cells in Dermatology

This study has been funded by the Carlos III Health Institute of Spain through the PI13/02576 and PI17/02083 projects [cofunded by European Regional Development Fund "A way to make Europe" and Andalusian Regional Government Finance (SAS PI-0458-2016)]. The work of MQ-V was supported by a predoctoral fellowship (BOE 22/10/2019) from the Spanish Ministry of Science, Innovation and Universities. This study is part of her doctoral research in the Biomedicine program at the University of Granada.The skin is the largest organ of the human body and its main functions include providing protection from external harmful agents, regulating body temperature, and homeostatic maintenance. Skin injuries can damage this important barrier and its functions so research focuses on approaches to accelerate wound healing and treat inflammatory skin diseases. Due to their regenerative and immunomodulatory properties, mesenchymal stromal cells (MSCs) have been reported to play a significant role in skin repair and regeneration. However, it seems that the secretome of these cells and exosomes in particular may be responsible for their functions in skin regeneration and the immunomodulation field. The present review aims to gather the available information about the role of MSC-derived exosomes for both in vitro and in vivo models of different skin conditions and to highlight the need for further research in order to overcome any limitations for clinical translation.Carlos III Health Institute of Spain [European Regional Development Fund "A way to make Europe"] PI13/02576 PI17/02083Carlos III Health Institute of Spain [Andalusian Regional Government Finance] PI13/02576 PI17/02083 SAS PI-0458-2016Spanish Ministry of Science, Innovation and Universities BOE 22/10/201

Antibody light chains dictate the specificity of contact hypersensitivity effector cell suppression mediated by exosomes

Antibody light chains (LCs), formerly considered a waste product of immunoglobulin synthesis, are currently recognized as important players in the activation of the immune response. However, very little is known about the possible immune regulatory functions of LCs. Recently, we reported that hapten-specific LCs coat miRNA-150-carrying exosomes produced by CD8+ suppressor T cells downregulating the contact hypersensitivity (CHS) reaction in an antigen-specific manner, in mice tolerized by intravenous administration of a high dose of hapten-coupled syngeneic erythrocytes. Thus, the current studies aimed at investigating the role of hapten-specific LCs in antigen-specific, exosome-mediated suppression of CHS effector cells. Suppressor T cell-derived exosomes from tolerized B-cell-deficient µMT−/−, NKT-cell-deficient Jα18−/−, and immunoglobulin-deficient JH−/− mice were nonsuppressive, unless supplemented with LCs of specificity strictly respective to the hapten used for sensitization and CHS elicitation in mice. Thus, these observations demonstrate that B1-cell-derived LCs, coating exosomes in vivo and in vitro, actually ensure the specificity of CHS suppression. Our research findings substantially expand current understanding of the newly discovered, suppressor T cell-dependent tolerance mechanism by uncovering the function of antigen-specific LCs in exosome-mediated, cell–cell communication. This express great translational potential in designing nanocarriers for specific targeting of desired cells

Oral mucosa keratinocytes and their exosomes for epithelial tissue regeneration

Early tumors, including high grade dysplasia and intramucosal invasive cancer, of the esophagus can today be removed using endoscopic resection, often using a technique called endoscopic submucosal dissection (ESD). This treatment is better tolerated and has considerably less mortality and morbidity compared to conventional, more invasive surgery, which usually entails esophagectomy. However, after larger endoscopic dissections, stricture formation is a common complication. Such strictures are usually treated with balloon dilatations, but the procedure often has to be repeated several times and is associated with risks such as perforations. In recent years, Japanese researchers developed a new method to reduce the risk of strictures. About two weeks before the treatment, an oral mucosa biopsy is taken from the patient from which epithelial cells are isolated and grown on special temperature-responsive polymer-coated surfaces. The polymer changes morphology and wettability properties depending on temperature, which enables non-enzymatic cell harvesting – the cells can be detached as contiguous sheets and with a large amount of extracellular matrix (ECM) maintained. After the ESD, cell sheets can be transplanted to the wound bed without the need for suture or other fixative, it is thought that the remaining ECM acts as a glue. Nine patients were treated in Tokyo and we subsequently transferred the technology to Stockholm where five additional patients were treated. Although one of nine patients in the Japanese cohort and three of five patients in the Swedish cohort still developed strictures, they appeared to be milder and easier to treat than expected. However, the aim of these projects was to evaluate safety and feasibility, further studies are needed to evaluate efficacy of the treatment. Since some patients still developed strictures despite the cell sheet transplantation, we next aimed to evaluate if exosomes from the cell culture media could be used as a pro-regenerative agent, perhaps in combination with cell sheet therapy. Media was collected from clinical- grade production of cell sheets from eight healthy donors. The media was concentrated by ultra-filtration and exosomes were isolated by size-exclusion chromatography. The exosomes were characterized by western blot (CD9+, Flotillin-1+, GRP94-), electron microscopy and nanoparticle tracking analysis (~125 nm). They reduced the proliferation of skin fibroblasts and stimulated upregulation of gene expression of growth factors relevant for wound healing. We studied the exosomes’ adhesion to esophageal wound bed by topical application to porcine esophageal wounds ex vivo and could detect signal after as little as one minute adhesion time. We also found that the exosomes stimulated wound healing of full-thickness skin wounds in immunocompetent rats, both at the 6th day and 17th day time point. In conclusion we found that exosomes could be isolated from cell sheet media and that they exhibited pro-regenerative properties even in a xenogeneic setting. Further studies are necessary to evaluate their potential to stimulate mucosal wound healing and reduce stricture formation of the esophagus

Immunogenicity of Exosomes from Dendritic Cells Stimulated with Toxoplasma gondii Lysates in Ocularly Immunized Mice

Immunogenicity of dendritic cell-derived exosomes stimulated with Toxoplasma gondii lysates (TLA exo), mixed with cholera toxin as an adjuvant, was investigated in mice immunized via 2 mucosal routes (ocular vs intranasal). BALB/c mice were injected 3 times with TLA exo vaccine at 2 week interval, and the levels of IgG in serum and IgA in tear, saliva, feces, and vaginal wash were measured. To observe the expression of T. gondii-specific B1 gene, mice infected with ME49 T. gondii cysts were immunized with TLA exo or PBS exo (not stimulated with TLA), and their brain tissues were examined. The mice vaccinated via intranasal route elicited significantly higher humoral and mucosal immune responses compared with mice treated with PBS alone. Also, mice immunized via ocular route (by eyedrop) induced significantly higher T. gondii-specific IgG in serum and IgA in tear and feces in comparison with PBS controls. B1 gene expression was significantly lower in TLA exo vaccinated mice than in PBS or PBS exo vaccinated mice. These results demonstrated that ocular immunization of mice with TLA exo vaccine has the potential to stimulate systemic or local antibody responses. This study also highlighted an advantage of an eyedrop vaccine as an alternative for T. gondii intranasal vaccines.ope