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Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.

Abstract Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii. [ABSTRACT FROM AUTHOR]

The erythroid lineage was studied in the flounder-gloss (Platichthys flesus Linnaeus, 1758) during the annual cycle. The erythrocyte count in the blood was determined along with the contents of immature erythroid forms (basophilic and polychromatophilic normoblasts) in the head kidney (pronephros) and the blood. Cell proliferative activity was inferred from the [3H]thymidine inclusion in circulating immature erythrocytes. Irregularity was observed in erythropoiesis occurring in flounder-gloss hematopoietic tissue. Intense production of erythroid mass was mainly associated with a post-spawning period. This was evident from an increase in the contents of immature erythroid forms in the pronephros and circulating blood and an increase in their proliferative activity. The changes were associated with peculiarities of the erythroid system organization, which precludes regular production of erythropoietin in the kidney in teleost fish. [ABSTRACT FROM AUTHOR]

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Simple Summary: Cancer is one of the leading causes of death worldwide. Besides genetic risk factors and non-communicable diseases, chronic infections, including Epstein–Barr virus (EBV) infection, have been identified as promoters of cancer. The aim of our study was to investigate the association between infectious mononucleosis, the clinical manifestation of EBV infection, and cancer development in a real cohort of outpatients in Germany. Patients diagnosed with infectious mononucleosis were found to have an increased incidence of tumors of the hematopoietic and lymphoid tissues and a strong tendency to have a positive association with prostate cancer. Together, our results support the evidence for a role of EBV in the development of various malignancies and may stimulate further research efforts to elucidate the precise involvement of EBV in the carcinogenic process. Background: Cancer represents one of the leading causes of death worldwide. Besides genetic risk factors and non-communicable diseases, chronic infections including Epstein–Barr virus (EBV) infection have been identified as promotors of cancer. In the present manuscript, we evaluated the association between infectious mononucleosis, the clinical manifestation of EBV infection, and cancer development in a real-word cohort of outpatients in Germany. Methods: We used the Disease Analyzer database (IQVIA) and matched a total of 12,095 patients with infectious mononucleosis to a cohort of individuals without infectious mononucleosis based on age, sex, index year, and annual patient consultation frequency between 2000 and 2018. Results: Patients diagnosed with infectious mononucleosis had a cancer incidence of 5.3 cases per 1000 person years versus 4.4 cases per 1000 person years for patients without infectious mononucleosis. In multivariable regression models, infectious mononucleosis showed a trend towards a higher incidence of cancer in general in the age group > 50 years (incidence rate ratio (IRR): 1.32; 95% CI: 1.04–1.67) and among men (IRR: 1.36; 95% CI: 1.07–1.72). Infectious mononucleosis was significantly associated with an increased incidence of tumors of the hematopoietic and lymphoid tissues (IRR: 1.75; 95% CI: 1.22–2.50) and showed a strong trend towards an association with prostate cancer (IRR: 3.09; 95% CI: 1.23–7.76). Conclusion: Infectious mononucleosis is associated with an increased incidence of certain cancer types. The present data from a large real-world cohort support the evidence on a role of EBV in the development of different malignancies and could trigger research efforts to further elucidate its precise involvement in the carcinogenic process. [ABSTRACT FROM AUTHOR]

The crayfish Procambarus clarkii could achieve a high cumulative mortality after WSSV infections. To better understand the immune response to WSSV in hematopoietic tissue, the present study investigated the immunological response of P. clarkii and analyzed the expression of some hematopoietic cytokines. After assembly, there was an average of 47,712,411 clean reads were obtained in control and treatment groups. A total of 35,945 unigenes were discovered with N50 length of 1554 bp. Under functional classification, enrichment, and pathway analysis using different database, there were about 257 differentially expressed genes (DEGs) identified, of which 139 were up-regulated and 118 were down-regulated. The GO function analysis of these DEGs were mostly participated in activation of immune response, complement activation, complement binding, negative regulation of humoral immune response and secretory granule membrane. Under KEGG analysis, these DEGs were involved in ECM-receptor interaction, HIF-1 signaling pathway, Glycolysis/Gluconeogenesis, Thyroid hormone signaling pathway and Glucagon signaling pathway. The real-time quantitative PCR (RT-qPCR) analysis of 9 selected genes confirmed the reliability of RNA-Seq results. The present research provide for the first time the transcriptomic profile of P. clarkii hematopoietic tissue in response to WSSV infection and reveals the astakines may play important roles in antiviral immune response. The results of the present study will further enrich the theoretical basis of the crayfish immune system and provide new ideas for disease prevention and control. • The hematopoietic tissue transcriptomes of P. clarkii in response to WSSV infection were constructed. • Totally 35,945 unigenes were obtained from the transcriptome. • Totally 257 DEGs were identified including 139 up- and 118 down-regulated unigenes. • The RT-qPCR results of the transcriptome validation candidate gene are consistent with the transcriptome. • Astakines may play important roles in antiviral immune response to WSSV infections in hematopoietic tissue. [ABSTRACT FROM AUTHOR]

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 24 h. 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). [ABSTRACT FROM AUTHOR]

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VP AHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC 50 dose of VP AHPND ; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VP AHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VP AHPND -infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6–12 h and declined by 24 h. This evident related to the almost VP AHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VP AHPND -infected prawn was caused by an acute self-destructive immune response by hematopoietic cells. • Hematopoietic tissues respond to VP AHPND infection by increasing cellular proliferation. • A moribund prawn with VP AHPND infection showed a significant reduction of circulating hemocytes. • Damage of hematopoietic tissue is correlated with a highly potent immune-related genetic response. [ABSTRACT FROM AUTHOR]

The quality of bone marrow preparation and sections of teeth depends on the preparation method. We investigated the posterior mandibles of male rats that were processed using a modified Bouin's solution (MBS) and stained with Harris' hematoxylin and eosin, and the results were compared to a routine decalcification process using 5% formal nitric acid. We found that MBS was applicable to both soft and hard components in bone tissues and sections of teeth. MBS provided good decalcification, which facilitated sectioning. The bone marrow specimens treated with MBS exhibited clearly distinguishable hematopoietic cells, clear tissue integrity and good cell preservation. Treatment with 5% formal nitric acid for 5 days caused degeneration, poor differentiation and poorer staining of hematopoietic components, and deteriorated soft and solid tissues in bone and sections of teeth compared to MBS treatment. MBS preserved cell and tissue integrity and good staining quality. MBS can be used for rapid preparation and diagnosis for pathology and toxicity studies. [ABSTRACT FROM AUTHOR]

Melano-macrophage centers (MMCs) are nodular clusters of pigmented macrophages, implicated in homeostasis and destruction and recycling of endogenous and exogenous material. They can increase in size and/or frequency under environmental stress resulting in immunohistological biomarkers of water quality. Fluoxetine (FLX), a commonly prescribed antidepressant, can cause neuroendocrine, behavioral and reproductive alterations in teleost fish. In the present study, we analyzed the effects of a 2-week 50 µg/L FLX exposure on MMCs in histological sections of spleen and head-kidney (HK) of the cichlid fish Cichlasoma dimerus. In the spleen, FLX caused an increase in the area and a decrease in the number of MMCs. An increase in the proportion of the HK occupied by MMCs was observed in FLX-exposed fish, due to an increase in their number but not their area. The deposition rate of MMCs varies according to the hemolymphopoietic organ and would be the result of a differential response to FLX on homeostatic functions (elimination of cellular debris, iron processing and immune response). [ABSTRACT FROM AUTHOR]

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. • Radix paeoniae Albam paeoniflorin, and albiflorin obviously increased the hematopoietic tissue body index (HTBI). • Radix paeoniae Alba boosted the astakine protein in hematopoietic tissue. • Radix paeoniae Alba, paeoniflorin, and paeonol increased the hemocytes counts and the proportion of granulocytes. [ABSTRACT FROM AUTHOR]

Background: The location of coelomocyte proliferation in adult sea urchins is unknown and speculations since the early 1800s have been based on microanatomy and tracer uptake studies. In adult sea urchins (Strongylocentrotus purpuratus) with down-regulated immune systems, coelomocyte numbers increase in response to immune challenge, and whether some or all of these cells are newly proliferated is not known. The gene regulatory network that encodes transcription factors that control hematopoiesis in embryonic and larval sea urchins has not been investigated in adults. Hence, to identify the hematopoietic tissue in adult sea urchins, cell proliferation, expression of phagocyte specific genes, and expression of genes encoding transcription factors that function in the conserved regulatory network that controls hematopoiesis in embryonic and larval sea urchins were investigated for several tissues.Results: Cell proliferation was induced in adult sea urchins either by immune challenge through injection of heat-killed Vibrio diazotrophicus or by cell depletion through aspiration of coelomic fluid. In response to either of these stimuli, newly proliferated coelomocytes constitute only about 10% of the cells in the coelomic fluid. In tissues, newly proliferated cells and cells that express SpTransformer proteins (formerly Sp185/333) that are markers for phagocytes are present in the axial organ, gonad, pharynx, esophagus, and gut with no differences among tissues. The expression level of genes encoding transcription factors that regulate hematopoiesis show that both the axial organ and the pharynx have elevated expression compared to coelomocytes, esophagus, gut, and gonad. Similarly, an RNAseq dataset shows similar results for the axial organ and pharynx, but also suggests that the axial organ may be a site for removal and recycling of cells in the coelomic cavity.Conclusions: Results presented here are consistent with previous speculations that the axial organ may be a site of coelomocyte proliferation and that it may also be a center for cellular removal and recycling. A second site, the pharynx, may also have hematopoietic activity, a tissue that has been assumed to function only as part of the intestinal tract.

Abstract The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 μm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph. Highlights • First detailed description of the hematopoietic organ of M. rosenbergii. • The hematopoietic organ is under the rostrum base, posterior to the brain and closely associated with the cor frontale. • The cor frontale sinus is an effective trap for foreign particles in hemolymph. • The HO can recognize and eliminate foreign particles. [ABSTRACT FROM AUTHOR]

Background: The location of coelomocyte proliferation in adult sea urchins is unknown and speculations since the early 1800s have been based on microanatomy and tracer uptake studies. In adult sea urchins (Strongylocentrotus purpuratus) with down-regulated immune systems, coelomocyte numbers increase in response to immune challenge, and whether some or all of these cells are newly proliferated is not known. The gene regulatory network that encodes transcription factors that control hematopoiesis in embryonic and larval sea urchins has not been investigated in adults. Hence, to identify the hematopoietic tissue in adult sea urchins, cell proliferation, expression of phagocyte specific genes, and expression of genes encoding transcription factors that function in the conserved regulatory network that controls hematopoiesis in embryonic and larval sea urchins were investigated for several tissues. Results: Cell proliferation was induced in adult sea urchins either by immune challenge through injection of heat-killed Vibrio diazotrophicus or by cell depletion through aspiration of coelomic fluid. In response to either of these stimuli, newly proliferated coelomocytes constitute only about 10% of the cells in the coelomic fluid. In tissues, newly proliferated cells and cells that express SpTransformer proteins (formerly Sp185/333) that are markers for phagocytes are present in the axial organ, gonad, pharynx, esophagus, and gut with no differences among tissues. The expression level of genes encoding transcription factors that regulate hematopoiesis show that both the axial organ and the pharynx have elevated expression compared to coelomocytes, esophagus, gut, and gonad. Similarly, an RNAseq dataset shows similar results for the axial organ and pharynx, but also suggests that the axial organ may be a site for removal and recycling of cells in the coelomic cavity. Conclusions: Results presented here are consistent with previous speculations that the axial organ may be a site of coelomocyte proliferation and that it may also be a center for cellular removal and recycling. A second site, the pharynx, may also have hematopoietic activity, a tissue that has been assumed to function only as part of the intestinal tract. [ABSTRACT FROM AUTHOR]

Abstract White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar) , are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii. Highlights • Hematopoietic tissue responded to viral infection by upregulating immune-related genes. • Novel immune related genes in M. rosenbergii are found during their viral response. • Cell-growth regulatory genes were highly expressed to produce more hemocytes for fighting against MrNV. [ABSTRACT FROM AUTHOR]

Abstract Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus. Highlights • Rete mirabile, respiratory tree, and polian vesicle were suggested as HPTs. • Two homologs of transcription factor GATA1 were detected in the HPTs. • EdU signals were strong in coelomocytes and HPTs. [ABSTRACT FROM AUTHOR]

Magnesium (Mg2+) is an essential mineral for the functioning and maintenance of the body. Disturbances in Mg2+ intracellular homeostasis result in cell-membrane modification, an increase in oxidative stress, alteration in the proliferation mechanism, differentiation, and apoptosis. Mg2+ deficiency often results in inflammation, with activation of inflammatory pathways and increased production of proinflammatory cytokines by immune cells. Immune cells and others that make up the blood system are from hematopoietic tissue in the bone marrow. The hematopoietic tissue is a tissue with high indices of renovation, and Mg2+ has a pivotal role in the cell replication process, as well as DNA and RNA synthesis. However, the impact of the intra- and extracellular disturbance of Mg2+ homeostasis on the hematopoietic tissue is little explored. This review deals specifically with the physiological requirements of Mg2+ on hematopoiesis, showing various studies related to the physiological requirements and the effects of deficiency or excess of this mineral on the hematopoiesis regulation, as well as on the specific process of erythropoiesis, granulopoiesis, lymphopoiesis, and thrombopoiesis. The literature selected includes studies in vitro, in animal models, and in humans, giving details about the impact that alterations of Mg2+ homeostasis can have on hematopoietic cells and hematopoietic tissue.

The repair of lesions of the central nervous system (CNS) varies widely throughout the animal kingdom. At the level of neuronal replacement lie the major differences in CNS regeneration. At one extreme are the amniote vertebrates (reptile, avian and mammalian groups), which have very limited capacity for neuronal replacement, and therefore for neural regeneration; at the other extreme, animals such as planarians (flatworms) and colonial tunicates can repair their entire CNS after major injuries. These differences can be attributed to the abundance of multipotent and/or pluripotent stem cells and/or undifferentiated precursors among the general cell population. In this review we discuss recent advancements in knowledge of regeneration of the CNS of invertebrates. We focus on ascidians, which are a sister group of vertebrates, but we also address other invertebrate groups. Because neurogenesis is central to the events that allow regeneration of the adult CNS, we address this issue focusing on crustaceans, which have provided a paradigm to study the mechanisms underlying this phenomenon. The attraction of hemocytes toward a neurogenic niche and respecification of these cells toward a neural fate has been strongly suggested. Based on recent and emerging research, we suggest that cells of the blood lineage are not only associated with the roles that are generally attributed to them, but are the cells that either signal other cell types to differentiate into neural cells, or even eventually themselves transdifferentiate into neural cells.

Reactive oxygen species (ROS) produced in vivo during various electron transfer reactions are generally kept at a certain level since they are harmful to cells. However, it can sensitize hematopoietic progenitors to differentiation, and plays a signaling role in the regulation of hematopoietic cell fate. In the present study, the transcriptomes of crab HPT and hemocytes were sequenced using the Ion Torrent Proton sequencing platform. A total of 51,229,690 single end reads were obtained from six single-end libraries, which were assembled into 31346 unireads as reference. After mapping and transcript assembling, 362 differently expressed genes were identified and 301 of them were deemed to be more abundant in HPT. GO annotation revealed that they were mostly implicated in DNA, RNA and protein synthesis, cell division, mitochondria activities and energy metabolism. The expression level of mitochondrial complexes I (mitochondrial NADH-ubiquinone oxidoreductase) which was the main natural producers of mitochondrial ROS was found to be 8.6-fold (p < 0.01) higher in HPT than that in hemocytes. In hemocytes, the proteinase genes associated with proPO activation from the 61 up-regulated genes in hemocytes were the main up-regulated genes which might be the potential markers for mature hemocytes. ROS level in HPT cells was relatively higher which was confirmed with the high expression level of mitochondria related genes identified by transcriptome sequencing. After the ROS level was depressed by N-acetyl- l -cysteine (NAC), the production of hemocytes from HPT was inhibited, and the recovery of the total hemocytes counts was delayed. These results collectively indicated that the genes in redox system were more active in HPT, and ROS could function as an important modulator in the hematopoiesis of crab and promote the production of hemocytes from HPT. [ABSTRACT FROM AUTHOR]

Crayfish haematopoietic tissue (HPT) has a thin-sheet-like structure with a thickness of 100-160 µm and a width of approximately 1-2 cm. This structure makes HPT extremely easy to fold after removal from the animal. Therefore, it is difficult to handle the tissue without folding when processing for sectioning and histological study. The degree of tissue folding reflects the size of the tissue sections obtained, how complicated it is to interpret the location of each tissue section, and the accuracy of the interpretation of the location of a specific transcript. To facilitate the interpretation of a specific transcript location in the HPT, we optimized a whole-mount in situ hybridization technique to minimize tissue folding. This optimized protocol effectively reduced the tissue folding. Therefore, the location of a specific transcript in the HPT was easily and accurately defined. This protocol will be useful for whole-mount staining of other tissues with similar structure. [ABSTRACT FROM AUTHOR]

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease ( Pl cSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of Pl cSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells. [ABSTRACT FROM AUTHOR]

Emergency granulopoiesis (EG) is a response to severe inflammation in which increased neutrophils are generated in the hematopoietic tissue. Photolabeling is utilized to distinguish newly developed neutrophils from existing neutrophils. However, this technique requires a strong laser line and labels subsets of the existing neutrophils. Here we create a transgenic zebrafish line that expresses a time-dependent switch from green fluorescent protein (GFP) to red fluorescent protein (RFP) in neutrophils, which allows quantification of EG using simple GFP/RFP ratiometric imaging.

The Litopenaeus vannamei is a prime aquaculture species and has a worth market value all over the world. In this study, we investigated the genesis and morphology of hematopoietic tissue (HPT) and types of hemocyte based on its morphology and cytochemical feature using histological and histochemical methods. The results revealed that HPT in L. vannamei is not visible histologically in mysis and early two post larvae (P1 & P2), and emerges in P3 which locates in epigastric region. The HPT showed a continuous propagation of cells in successive post larval stages. Four types of cells can be identified in the HPT from adult specimen and mitotic activity is visible in the HPT. The hemocytes have been differentiated into five types and some of these types are co-related with the HPT cells. The cytochemical studies suggested that type I and II cells of hemocytes are PAS positive while scanty presence of prophenoloxidase was observed in type I cells. Similarly the type I cells are Sudan Black B positive and rest of the cells showed weak activity against lipid detective stain. This is the first effort towards genesis of HPT and its relation with circulating hemocytes in L. vannamei.

El mielolipoma adrenal es un tumor benigno poco frecuente, compuesto de tejido adiposo maduro y elementos hematopoyéticos diversos. Estos tumores pueden localizarse en las glándulas adrenales aunque también pueden encontrarse en sitios extradrenales. Las manifestaciones clínicas son variadas tales como sangramientos digestivos, dolor abdominal en los flancos, masa abdominal palpable, hematuria, hemorragia retroperitoneal y pérdida de peso. Entre los métodos diagnósticos figuran el ultrasonido, la tomografía axial computarizada y la resonancia magnética. La resección quirúrgica es el tratamiento de elección, sobre todo cuando haya sintomatología o en tumores mayores de 4 cm, por el riesgo de ruptura espontánea con hemorragia retroperitoneal. Presentamos la historia clínica de una paciente de 42 años de edad con antecedentes de dolores en hipocondrio derecho de aproximadamente un año de evolución, la cual sufrió un cólico nefrítico que cedió con tratamiento médico. Posteriormente en la consulta externa, en el examen físico, se constata una masa tumoral en hipocondrio derecho, la cual fue diagnosticada por ultrasonido y tomografía axial computarizada como un tumor adrenal. Se intervino la paciente y se confirma el diagnóstico. La evolución posoperatoria fue buena y la paciente continúa asintomática. El objetivo de este trabajo es presentar un nuevo caso de esta enfermedad, poco frecuente, la cual fue diagnosticada en una paciente de 42 años de edad y realizar una breve revisión de la literatura.Mielolipoma is a rare benign tumor of the adrenal gland, which is formed by mature adipose tissue and several hematopoietic elements. They are generally located in the adrenal glands, although they can occur in other sites. The clinical manifestations are varied such as gastrointestinal bleeding, abdominal pain in the flanks, abdominal mass, retroperitoneal hemorrhage, hematuria and loss of weight. Among the diagnosing methods are ultrasound, computerized axial tomography and magnetic imaging resonance. The surgical excision is the treatment of choice, mainly when symptoms are present or in case of over 4 cm tumors, because of the spontaneous rupture risk with retroperitoneal hemorrhage. The objective of this paper was to present a new case of this rare disease together with a brief literature review. A 42 years-old female with a history of abdominal pain located in the right upper quadrant of the abdomen, who suffered nephritic colic that was successfully treated. The physical exam at the outpatient service revealed a tumorous mass diagnosed as adrenal tumor by means of ultrasound and axial computerized tomography. She was operated on and the diagnosis was confirmed. Her postoperative recovery was good and the patient is still asymptomatic.