Innovations, challenges, and minimal information for standardization of humanized mice

Mice xenotransplanted with human cells and/or expressing human gene products (also known as humanized mice ) recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called Minimal Information for Standardization of Humanized Mice (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice

CREB is a critical regulator of normal hematopoiesis and leukemogenesis

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

Figure S1. Feasibility of cryopreservation. (A) Tricistronic IDLV encoding for hGM-CSF, hIFN-α and CMV-pp65 protein used to generate SmyleDCpp65. (B) Scheme of SmyleDCpp65 generation. Monocytes were isolated by MACS selection, pre-conditioned with cytokines for 8 h, and transduced with IDLV-G2α2pp65 for 16 h. After transduction, cells were harvested and cryopreserved at 2x106 cells/mL/vial. Cells were analyzed immediately after thaw (AT) or cultured in medium without exogenous cytokines for 7 days. (C) Viability (7AADneg) and identity (CD14 + expression level) of cell product (AT). (D) Total IDLV copy numbers detected by RT-q-PCR in the transduced cell groups AT and after 7 days in culture. (E) pp65 expression in SmyleDCpp65 (CD14neg, CD11cbright) after 7 days of in vitro culture. (F) Viability, down regulation of monocyte marker (CD14), identity (CD11cbright and HLA-DR) and functional markers (CD86 and CD80) expressed in SmyleDCpp65 7 days after in vitro culture

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice

Lentivirus-Induced Dendritic Cells (iDC) for Immune-Regenerative Therapies in Cancer and Stem Cell Transplantation

Conventional dendritic cells (cDC) are ex vivo differentiated professional antigen presenting cells capable of potently stimulating naïve T cells and with vast potential for immunotherapeutic applications. The manufacture of clinical-grade cDC is relatively complex and requires several days for completion. Clinical trials showed poor trafficking of cDC from subcutaneous injection sites to lymph nodes (LN), where DC can optimally stimulate naïve lymphocytes for long-lasting memory responses. We demonstrated in mouse and human systems that a single overnight ex vivo lentiviral (LV) gene transfer into DC precursors for production of combination of cytokines and antigens was capable to induce autonomous self-differentiation of antigen-loaded DC in vitro and in vivo. These highly viable induced DC (iDC) effectively migrated from the injected skin to LN, where they effectively activated de novo antigen-specific effector memory T cells. Two iDC modalities were validated in relevant animal models and are now in clinical development: Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors co-expressing GM-CSF/IL-4/TRP2 for melanoma immunotherapy in the autologous setting (SmartDCtrp2), and Self-differentiated Myeloid-derived Lentivirus-induced against human cytomegalovirus as an allogeneic matched adoptive cell after stem cell transplantation (SmyleDCpp65). The lentiviral vector design and packaging methodology has “evolved” continuously in order to simplify and optimize function and biosafety of in vitro and in vivo genetic reprogramming of iDC. Here, we address the challenges seeking for new creations of genetically programmed iDC and integrase-defective LV vaccines for immune regeneration

Expressão do gene da alfa-amilase de Bacillus subtilis em Xanthomonas campestris

Orientadores : Yoko Bomura Rosato , Spartaco Astolfi FilhoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A bactéria Xanthomonas campestris é um fitopatógeno que produz a goma xantana, de grande importância econômica, devido ao seu variado potencial de utilização na Indústria e na agricultura. Com o objetivo de se verificar a expressão de um gene de amilase exógeno em X.campestris, estabeleceu-se um sistema de clonagem para esta bactéria. Este sistema constituiu-se no plasmídio promíscuo pMFY40 ao qual se inseriu o gene da alfa-emllese de Bacillus subtilis, resultando no plesmídio pAP1. Este plesmídio possui aproximadamente 14.0 Kb, á mobilizável por plasmídios ¿helper¿ e razoavelmente estável, em condições de fermentação, em X. campestris. O pAP1 foi introduzido por transformação e por conjugação em duas linhagens de campestris, uma delas originalmente amilolítica (REF) e outra não-amilolítica (280), mas que é uma boa produtora de goma. Após se verificar a secreção da enzima em meio sólido pelos recombinantes, analisou-se o consumo de amido e a produção de açúcar redutor em meto líquido. Verificou-se que a linhagem 280/pAPl foi capaz de degradar o amido do meio e que a linhagem REF/pAPl teve seu caráter amiolítico amplificado. A produção de xantana em meios contendo diferentes com posições de substratos foi verificada através da viscosidade da goma, para se analisar as potencialidades da substituição do substrato tradicional, que é a sacarose, pelo amido. Foi observado um acréscimo de produção nas linhagens contendo o pAPl em meio com 2X de amido e em meto com 1X de amido e IX de sacarose. Apesar da substituição total da sacarose pelo amido ser Inviável para a fermentação pela linhagem 280/pAP1, verificou-se que a composição de IX de sacarose e de 1X de amido Já fornece bons resultadosAbstract: Xanthomonas campestris Is a phytopathogenlc bacteria. Which produces the xanthan-gum a biopolimer of economical Importance due to lts great poss1b111tles of ut111zat1on ln the Industry and In the agriculture. A cloning system for the analysis of expression of the extracellular amylase was established. The alfa-amylase gene from Bacillus subtilis was introduced in the promiscuous plasmid pMFY40, giving rise to the hybrid plasmid pAP1. This plasmid contains approximately 14.0 Kb, is mobilizable by helper plasmid and is reasonably stable, in fermenting conditions, in X.campetris. The plasmid pAP1 has been introduced by transformation and by conjugation into two strains of X. campestris one of them originally amylolytlc (REF), and the other not amylolytic (280) but a very good gum producer. Besides the observation of the enzyme production by the recombinants in solid media, the starch consumption and the reducing-sugar production in liquid media were also analysed. It was verified that the strain 280/pAP1 was able to degradate the starch of the media and that the strain REF/pAP1 had its amylolytic character amplified. The xanthan production in media containing different substrates compositions was analysed to evaluate the potentialities of the substitution of the traditional substrate, sucrose, by starch. The strains bearing the plasmid pAP1 showed viscosity enhancement of the media containing 2% starch and also 1% starch plus 1% sucrose. Although the total substitution of sucrose by starch was not successful for the fermentation of 280/pAP1, good results were observed in the media containing 1% starch plus 1% sucroseMestradoGeneticaMestre em Ciência

Modeling Human Cytomegalovirus in Humanized Mice for Vaccine Testing

Human cytomegalovirus (HCMV or HHV-5) is a globally spread pathogen with strictly human tropism that establishes a life-long persistence. After primary infection, high levels of long-term T and B cell responses are elicited, but the virus is not cleared. HCMV persists mainly in hematopoietic reservoirs, whereby occasional viral reactivation and spread are well controlled in immunocompetent hosts. However, when the immune system cannot control viral infections or reactivations, such as with newborns, patients with immune deficiencies, or immune-compromised patients after transplantations, the lytic outbursts can be severely debilitating or lethal. The development of vaccines for immunization of immune-compromised hosts has been challenging. Several vaccine candidates did not reach the potency expected in clinical trials and were not approved. Before anti-HCMV vaccines can be tested pre-clinically in immune-compromised hosts, reliable in vivo models recapitulating HCMV infection might accelerate their clinical translation. Therefore, immune-deficient mouse strains implanted with human cells and tissues and developing a human immune system (HIS) are being explored to test anti-HCMV vaccines. HIS-mice resemble immune-compromised hosts as they are equipped with antiviral human T and B cells, but the immune reactivity is overall low. Several groups have independently shown that HCMV infections and reactivations can be mirrored in HIS mice. However, these models and the analyses employed varied widely. The path forward is to improve human immune reconstitution and standardize the analyses of adaptive responses so that HIS models can be forthrightly used for testing novel generations of anti-HCMV vaccines in the preclinical pipeline