Hematopoietic Tissue Cells Research Articles

MANIFESTAÇÕES ORAIS DAS LEUCEMIAS EM PACIENTES PEDIÁTRICOS

Leukemias can be described as blood dyscrasias resulting from the disordered multiplication of hematopoietic tissue cells. The manifestations of the disease occur systemically and have a major impact on the oral cavity, with repercussions in response to the disease itself and to elective treatment, whether surgical, chemo- or radiotherapy. The objective of this study is to discuss the oral alterations of greatest clinical value in pediatric patients, discussing the role of the dentist in the diagnosis and treatment of these manifestations. The study was carried out from the perspective of an integrative bibliographic review, following the previously established inclusion and exclusion criteria. The results emphasize the correlation between oral health and the integrity of all systems, demonstrating that the most prevalent alterations are oral mucositis, gingival enlargement followed by spontaneous bleeding and petechiae, opportunistic infections, cavities, xerostomia and hyposalivation. It is therefore concluded that there is a need for the dentist to evaluate and monitor the treatment process of pediatric patients with leukemia who are the target of antineoplastic therapy.

Proteomic analysis of global protein acetylation in response to WSSV infection in a crustacean Cherax quadricarinatus

Protein acetylation, a crucial post-translational modification, plays a significant role in antiviral immunity of host and viral replication processes after infection. Our previous research showed that an acetyltransferase KAT2A significantly enhanced white spot syndrome virus (WSSV) replication in hematopoietic tissue (Hpt) cells from Cherax quadricarinatus, suggesting that alterations in intracellular acetylation states affect viral infection. Nevertheless, the mechanisms by which WSSV invasion regulates intracellular protein acetylation remain unexplored. To explore the connection between protein acetylation and WSSV infection, differentially acetylated proteins (DAcPs) were identified in Hpt cells during WSSV infection by proteomic analysis. The results revealed that 25 DAcPs were identified with primarily involving in transporter activity and proton ATPase activity complex during the early stage of WSSV infection, i.e., the intracellular transport of the virions. Furthermore, that 36 DAcPs associated with chromatin assembly and acetyltransferase complex were identified in the replication stage of WSSV infection, i.e., the process of extensive viral replication. Additionally, acetylation at the K27 and K36 sites of histone H3 was strongly activated during WSSV replication; similarly, the inhibition of histone acetylation by acetyltransferase inhibiter C646 significantly suppressed WSSV replication, implying that WSSV replication requires high levels of histone acetylation, while the acetylation at the K27 and K36 sites of histone H3 probably serves as critical viral regulatory targets. This study deepens the understanding of WSSV-host interaction, and lays the groundwork for future research into the relationship between protein acetylation and viral infection in crustaceans.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3–48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Characterization of the effects of Chinese peony (Radix paeoniae Alba) and its active ingredients on the hematopoietic function of the Chinese mitten crab (Eriocheir sinensis)

Hematopoietic tissue (HPT) is crucial for the proliferation and release of newly produced hemocytes during infection in crustaceans. Very few studies have reported how herbs affected the hematopoietic function of crustaceans. Radix paeoniae Alba is a candidate immunopotentiator herb with the potential to be a blood-tonifying drug. In this study, the effects of Radix paeoniae Alba and its monomers (paeoniflorin, albiflorin, and paeonol) on HPT and hemocytes of Eriocheir sinensis were evaluated. After feeding them commercial diets containing different ingredients for three weeks, the HPT development was determined via morphological observation and hematopoietic tissue body index (HTBI) calculation. The count and classification of hemocytes were evaluated using the hemocytometer and flow cytometer. The expression of hematopoietic factor astakine, transglutaminase (TGase), and marker genes for different types of hemocytes (KPI and SOD) and undifferentiated HPT cells (PCNA) were detected using real-time quantitative PCR (qRT-PCR) assay. The results indicated that the HTBI in the Radix Paeoniae Alba, paeoniflorin, and albiflorin groups increased by 114.96%, 111.42%, and 112.50%, which were higher than the control group. The Radix Paeoniae Alba, paeoniflorin, albiflorin, and paeonol groups had higher proportions of ovoid lobules than the control group, especially in the Radix Paeoniae Alba group. The total hemocyte count (THC) of the crab in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups were 8.12, 9.69, and 8.57×106 ind/mL, respectively, which were higher than the control group. Furthermore, all experimental groups showed a higher proportion of granulocytes than the control group, with increased astakine protein being simultaneously detected in the HPT. The qRT-PCR results showed a relatively higher KPI expression in all the experimental groups, and also higher SOD mRNA expression in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups, as compared with the control group. Therefore, these results implied the potential effect of Radix Paeoniae Alba and its monomers on the hematopoietic function of E. sinensis. However, due to the complex ingredient composition in Radix Paeoniae Alba and their different activities in the hematopoietic process, further research needs to be conducted in the future.

Identification of an AnnexinB9 involve in white spot syndrome virus infection in red claw crayfish Cherax quadricarinatus

Annexin (Anx) family protein is a highly conserved protein family that plays important roles in immune defense of vertebrates and invertebrates against invading pathogens. In this study, a novel Anx was cloned and characterized from the red claw crayfish, Cherax quadricarinatus. The Open Reading Frame of CqAnxB9 consisted of 930 nucleotide bases pair and encoded 309 amino acids. The CqAnxB9 protein contained three repeat Anx domains and a typical KGLGT sequence. Tissue expression analysis showed that the expression levels of CqAnxB9 were mainly expressed in the intestine, hepatopancreas and hemocytes. After WSSV challenge, CqAnxB9 expression was up-regulated in the hematopoietic tissue (Hpt) cells. Moreover, knockdown of CqAnxB9 inhibited WSSV replication and VP28 expression, suggesting that CqAnxB9 plays a positive role in WSSV infection. Further studies revealed that recombinant CqAnxB9 protein was found to bind to the viral envelop protein VP28. All these findings indicate that new-found CqAnxB9 is likely to promote WSSV infection in crustaceans, which provides a better understanding of the pathogenesis of WSSV.

Replication of decapod iridescent virus 1 in primary cultured hematopoietic tissue cells of Cherax quadricarinatus

Decapod iridescent virus 1 (DIV1) is a newly defined iridescent virus that infects decapod crustaceans and causes significant economic loss in aquaculture. To study the mechanisms of viral infection, an in vitro infection system is required. Here, we investigated the infection and replication of DIV1 in primary cultured hematopoietic tissue (HPT) cells of crayfish Cherax quadricarinatus. RT-PCR and Western blot analysis showed that DIV1 could infect the in vitro cultured cells and express viral genes. Immunofluorescent analysis of DIV1 major capsid protein indicated an infection rate of ∼23%. As the infection progressed, viral DNA copy numbers in both the cells and culture medium increased. Transmission electron microscopy analysis and animal challenging experiments demonstrated the production and release of infectious progeny virions. In summary, the primary culture of C. quadricarinatus HPT cells supports the infection and proliferation of DIV1. Thus, it can be used as an in vitro system to study the virus.

Characterization of hemocytes and hematopoietic cells of a freshwater crayfish based on single-cell transcriptome analysis

SummaryCrustaceans constitute a species-rich and ecologically important animal group, and their circulating blood cells (hemocytes) are of critical importance in immunity as key players in pathogen recognition, phagocytosis, melanization, and antimicrobial defense. To gain a better understanding of the immune responses to different pathogens, it is crucial that we identify different hemocyte subpopulations with different functions and gain a better understanding of how these cells are formed. Here, we performed single-cell RNA sequencing of isolated hematopoietic tissue (HPT) cells and hemocytes from the crayfish Pacifastacus leniusculus to identify hitherto undescribed hemocyte types in the circulation and show that the circulating cells are more diversified than previously recognized. In addition, we discovered cell populations in the HPT with clear precursor characteristics as well as cells involved in iron homeostasis, representing a previously undiscovered cell type. These findings may improve our understanding of hematopoietic stem cell regulation in crustaceans and other animals.

CqFlightless-I promotes the depolymerization of actin cytoskeleton to reduce white spot syndrome virus infection in Cherax quadricarinatus

As a member of the actin-associated protein gelsolin family, Flightless-I (FliI) is involved in promoting the depolymerization and rearrangement of the actin cytoskeleton, thus regulating a variety of biological processes, including cell proliferation, differentiation, apoptosis, and immune response. However, the molecular regulation of FliI is poorly understood in its regulation on viral infection in crustaceans. Previously, we found that the transcript of CqFliI was up-regulated in a differentially expressed transcriptome library of the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus at 1 h post white spot syndrome virus (WSSV) infection. To reveal the role of CqFliI in WSSV infection in crustacean, the open reading frame of CqFliI gene was cloned with 3870 bp, which encoded 1289 amino acids with eleven leucine-rich repeat (LRR) domains and six gelsolin-like (GEL) domains. Tissue distribution analysis showed that CqFliI was widely expressed in all the tested tissues with the highest expression in muscle, followed by a high expression in Hpt. In addition, the mRNA expression of CqFliI was significantly up-regulated in Hpt cells at 1, 3, 6, 12 and 24 h post WSSV infection. Importantly, the internalization of WSSV was clearly increased at an early infection stage of 1 hpi in Hpt cells by CqFliI gene silencing; meanwhile, the expression of viral immediate early gene IE1 and late viral envelope protein gene VP28 were both significantly increased after gene silencing of CqFliI in Hpt cells post WSSV infection, indicating a key role of CqFliI in reducing WSSV infection. Furthermore, protein pull-down assay revealed that CqFliI could bind with cytoskeletal β-actin through its GEL domains, rather than LRR domains; and the cytoskeleton of HeLa cells was obviously depolymerized after overexpression of GEL domain of CqFliI, implying that CqFliI might reduce the internalization of WSSV probably also the intracellular transport mediated by cytoskeleton via inhibiting actin polymerization. These findings may contribute to further understanding of WSSV infection mechanism which may provide more antiviral targeting design against WSSV disease.

Umbilical Cord Blood Cells Express mRNA for Neuronal Genes MAP2 and NEFL.

Hematopoietic tissue-derived cells including stem cells have been shown to differentiate into or fuse with solid organ tissue-specific cells. Among hematopoietic tissue cells, umbilical cord blood (UCB)-derived stem cells have the advantage of a higher engraftment potential. It was the purpose of this study to gather evidence on a molecular level of UCB-derived cells and stem cells generating or fusing with solid-organ tissue specific cells such as CNS-specific cells as a model for potential treatment of injured tissue.Fresh UCB was obtained by standard UCB banking procedures. Apheresis-derived mononuclear cells (adult MNC) were obtained from normal stem cells donors who underwent a 4-day G-CSF mobilization treatment. UCB cells (4 individual UCB harvests) and adult MNC (4 individuals) were selected for CD133/1+ or CD14+ or CD3+ cells by immunomagnetobead separation (autoMACS; Miltenyi Biotec). Each specimen contained between 8 and 20 × 106 selected cells at a purity of >90%.Real-time PCR was performed on the ABI Prism 7900 using the Assays-on-Demand for various neuronal genes such as Neurofilament (NEFL), Microtubule-associated protein 2 (MAP2), βTubulin 3 (TUBB3) and Nestin (NES), and the cylophilin Pre-Developed Assay Reagent (Applied Biosystems) without multiplexing. The ddCt method was used to calculate fold-difference in neuronal gene expression in the cell specimens relative to Stratagene's normal lymph node RNA.Neuronal cell-specific NEFL and MAP2 expression was found in both, UCB and adult MNC, but not TUBB3 or NES expression. The MAP2 expression was in favor of CD133−, CD14− and CD3− selected cells, whereas NEFL expression was significantly higher in CD3+ selected cells as compared with CD3− cells in both, UCB cells and adult MNC. [Display omitted] These preliminary results indicate that UCB and mobilized adult blood contain cell subsets that express mRNA for neuronal cell specific genes. NEFL expression seems to be higher in CD3-selected cells, whereas MAP2 and NEFL gene expression in progenitor cell subsets (CD133+) was found to be less.As shown by us in a clinical sex-mismatched UCB transplant setting and by others hematopoietic tissue-derived neuronal-like cells are identified in CNS tissue. Circulating cells expressing neuronal-specific genes could contribute to homeostasis of CNS tissue as known for the hematopoietic tissue.

BATF regulates innate lymphoid cell hematopoiesis and homeostasis

Abstract Early hematopoietic progenitors undergo sophisticated developmental processes to become committed progenitors for innate lymphoid cells (ILC) in hematopoietic tissues, which ultimately become mature ILC subsets in the periphery. Basic leucine zipper ATF-like transcription factor (Batf) plays important roles in lymphocyte biology. We report that Batf regulates the production of bone marrow ILC progenitors and maintenance of peripheral ILCs. Batf is most highly expressed in ILCs among all lymphocytes. Data from in vivo and in vitro ILC differentiation experiments revealed that the expression of Batf is induced in early ILC progenitors at the α-lymphoid progenitor stage in response to the cytokine IL-7. Our Chromatin immunoprecipitation and retroviral gene transfer experiments revealed that up-regulated Batf binds and activates transcription of the Nfil3 gene to promote ILC hematopoiesis. Data from bone marrow chimera and parabiosis experiments support that Batf is intrinsically necessary to maintain normal numbers of early and late ILC progenitors in the bone marrow and mature ILC1, ILC2, ILC3, and NK cells in most peripheral tissues. Batf-deficient mice have ILC3 lymphopenia and fail to control Citrobacter rodentium infection. Moreover, ILC2 responses to cytokines were defective in Batf-deficient mice. Thus, Batf is required for normal ILC responses to inflammatory cytokines and bacterial infection. These data suggest that Batf plays fairly comprehensive roles in supporting bone marrow hematopoiesis, maintenance, and effector functions of ILCs.

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).

Pathosphysiology of Chronic Myeloid Leukemia, Prognistic Factors and Emerging Treatment Options in a Low Resource Economy

Chronic myeloid leukemia (CML) is a triphasic clonal myeloproliferative disorder characterized by the presence of Philadelphi a chromosome (Ph) in over 95% of cases alongside excessive accumulation of clonal myeloid cells in hematopoietic tissues. This occurs as a result of reciprocal translocation between the long arms of chromosome 9 and 22 t (9;22) (q34; q11) creating the fusion oncogene BCR–ABL1 which exhibits constitutive tyrosine kinase activity. It is one of the commonest haematological malignancies in low economies around the world including Nigeria. The clinical features of CML are often described in 3 phases namely the chronic phase (CP), accelerated phase (AP), and blastic phase (BP) while CP is the most common stage with progression to AP and BP occurring later. Despite that, prognosis of CML is dependent on phase of disease, age, and response to therapy, the only curative approach in use currently is hematopoietic stem cell transplantation with other drugs being used for cytogenetic responses. This study focuses on the aetiopathophysiology, cytogenetics, molecular biology, clinical/laboratory features and treatment options of CML. Rigorous review of literature on the study was retrieved from relevant oncology journals and textbooks abstracted and indexed in PubMed, Google Scholar, ProQuest, CINAHL, and Science Direct. The study discovered that, resistance of CML to imatinib has been reported with research having reached the advanced stage on the use of alternative drugs (e.g., Nilotinib and Desatinib). There are also potentials for these new drugs to become the treatment choice and first line drugs for the treatment of CML.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

Extranodal Follicular Dendritic Cell Sarcoma-A Review: "What the Mind Does Not Know the Eye Does Not See".

Follicular dendritic cell sarcoma (FDCS) is an intermediate-grade malignancy of follicular dendritic cells, which are derived from mesenchymal stem cells. Nodal FDCS is well-recognized. However, when it occurs at an extranodal site, it may not be recognized and is often misdiagnosed. These tumors exhibit a variable spindle to epithelioid cell morphology with a lymphocytic infiltrate and a distinct immunophenotype. The World Health Organization has classified this entity under tumors of hematopoietic and lymphoid tissue, that is, histiocytic and dendritic cell neoplasms. However, its occurrence at extranodal sites and its behavior and management more closely resemble that of a soft tissue tumor. Increased awareness about the existence of FDCS at extranodal sites may aid in the reduction of diagnostic errors. We wish to draw attention to this entity by reporting our experience of 54 cases of extranodal FDCS encountered over a period of 14 years and present a review of the literature of this underrecognized entity. We also describe the ontogeny and molecular pathogenesis of this uncommon lesion.

A cytokine receptor domeless promotes white spot syndrome virus infection via JAK/STAT signaling pathway in red claw crayfish Cherax quadricarinatus

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is pivotal in immune responses for a variety of pathogens in both vertebrates and invertebrates. Domeless (Dome), as a unique cytokine receptor, involves in the upstream JAK/STAT pathway in invertebrates. In this study, the full-length cDNA sequence of a cytokine receptor Dome was identified from red claw crayfish Cherax quadricarinatus (named as CqDome), which contained an open reading frame of 4251 bp, encoding 1416 amino acids. The CqDome contained extracellular conservative domains of a signal peptide, two cytokine binding modules (CBM), three fibronectin-type–III–like (FN3) domains and a transmembrane region. Tissue distribution analysis showed that CqDome generally expressed in all the tissues selected with a high expression in hemocyte. The gene expression of both the viral immediately early gene (IE1) and a late gene envelope protein VP28 of white spot syndrome virus (WSSV) were significantly decreased after gene silencing of CqDome in crayfish haematopoietic tissue (Hpt) cells, indicating a key role of CqDome in promoting WSSV infection. Furthermore, the phosphorylation level of CqSTAT was significantly inhibited by gene silencing of CqDome in Hpt cells, indicating that CqDome participated in signal transduction of JAK/STAT pathway in red claw crayfish. These data together suggest that CqDome is likely to promote WSSV infection via JAK/STAT pathway, which sheds new light on further elucidation of the pathogenesis of WSSV.

Large–scale screening of molecules involved in virus–host interaction by specific compounds in Cherax quadricarinatus hematopoietic tissue cells

White spot syndrome virus is one of the major pathogens that seriously threaten crustacean aquaculture. However, knowledge about the molecular mechanism of virus–host interaction remains limited. In the present study, a library containing 303 compounds targeting specific molecules was used for large–scale screening for the first time. Cherax quadricarinatus hematopoietic tissue cells were used in the present study. Combining the analysis of viral infection and cell toxicity test, 30 compounds were demonstrated to be effective. Their targeted molecules were involved in the regulation of a variety of cell signaling pathways and participated in several cellular processes, such as Hedgehog and NF–κB cell signaling pathway and regulation of cellular ions. The present study provides valuable insights into the molecular mechanism of the viral pathogenesis and host immunity, and will help to advance disease control.

Quantitative In-Depth Analysis of the Mouse Mast Cell Transcriptome Reveals Organ-Specific Mast Cell Heterogeneity.

Mast cells (MCs) are primarily resident hematopoietic tissue cells that are localized at external and internal surfaces of the body where they act in the first line of defense. MCs are found in all studied vertebrates and have also been identified in tunicates, an early chordate. To obtain a detailed insight into the biology of MCs, here we analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR-based transcriptomics. We show that MCs at different tissue locations differ substantially in their levels of transcripts coding for the most abundant MC granule proteins, even within the connective tissue type, or mucosal MC niches. We also demonstrate that transcript levels for the major granule proteins, including the various MC-restricted proteases and the heparin core protein, can be several orders of magnitude higher than those coding for various surface receptors and enzymes involved in protease activation, as well as enzymes involved in the synthesis of heparin, histamine, leukotrienes, and prostaglandins. Interestingly, our analyses revealed an almost complete absence in MCs of transcripts coding for cytokines at baseline conditions, indicating that cytokines are primarily produced by activated MCs. Bone marrow-derived MCs (BMMCs) are often used as equivalents of tissue MCs. Here, we show that these cells differ substantially from tissue MCs with regard to their transcriptome. Notably, they showed a transcriptome indicative of relatively immature cells, both with respect to the expression of granule proteases and of various enzymes involved in the processing/synthesis of granule compounds, indicating that care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. Furthermore, the latter finding indicates that the development of fully mature tissue-resident MCs requires a cytokine milieu beyond what is needed for in vitro differentiation of BMMCs. Altogether, this study provides a comprehensive quantitative view of the transcriptome profile of MCs resident at different tissue locations that builds nicely on previous studies of both the mouse and human transcriptome, and form a solid base for future evolutionary studies of the role of MCs in vertebrate immunity.

Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF).

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

Directed differentiation of granular cells from crayfish hematopoietic tissue cells

Hemocytes are the major immune cells of crustaceans. New hemocyte production is required throughout the life cycle of these animals to maintain a functional immune system. The mechanism of crustacean hematopoiesis has just begun to be understood and new methods are needed for the investigation of this process. Here we report the directed differentiation of granular cells (GCs) from the hematopoietic tissue (HPT) cells of Cherax quadricarinatus in vitro. We started by providing the cultured HPT cells with different additives to induce possible differentiation. We found that crayfish muscle extract greatly promoted the physical status of the cells and induced the formation of refractile cytoplasmic granules. The transcription of marker genes and the production of functional prophenoloxidase further confirmed the formation of mature GCs. In our experiments, young GCs usually started to develop in ∼2 weeks post induction and over 60% of the cells became mature within 3–4 weeks. This is the first time that the fully differentiation of crustacean hemocytes is accomplished in vitro. It provides a powerful tool for in-depth study of crustacean hematopoiesis.

Reduction of Delayed Homologous Recombination by Induction of Radioadaptive Response in RaDR-GFP Mice (Yonezawa Effect): An Old Player With a New Role.

Radiotherapy (RT) treats cancer effectively with high doses of ionizing radiation (IR) to killing cancer cells and shrinking tumors while bearing the risk of developing different side effects, including secondary cancer, which is most concerning for long-term health consequences. Genomic instability (GI) is a characteristic of most cancer cells, and IR-induced GI can manifest as delayed homologous recombination (HR). Radioadaptive response (RAR) is capable of reducing genotoxicity, cell transformation, mutation, and carcinogenesis, but the rational evidence describing its contributions to the reduction of radiation risk, in particular, carcinogenesis, remains fragmented. In this work, to investigate the impact of RAR on high-dose, IR-induced GI measured as delayed HR, the frequency of recombinant cells was comparatively studied under RAR-inducible and -uninducible conditions in the nucleated cells in hematopoietic tissues (bone marrow and spleen) using the Rosa26 Direct Repeat-green fluorescent protein (RaDR-GFP) homozygote mice. Results demonstrated that the frequency of recombinant cells was significantly lower in hematopoietic tissues under RAR-inducible condition. These findings suggest that reduction in delayed HR may be at least a part of the mechanisms underlying decreased carcinogenesis by RAR, and application of RAR would contribute to a more rigorous and scientifically grounded system of radiation protection in RT.