Transglutaminase Type 2 is Involved in the Hematopoietic Stem Cells Homeostasis.

Type 2 transglutaminase (TG2) is a multifunctional protein involved in various biological processes playing a key regulatory role in cell homeostasis such as cell death and autophagy. New evidence is emerging that support an important role of autophagy in regulating normal hematopoiesis. Prompted by these findings, in this study we investigated in vivo involvement of TG2 in mouse hematopoiesis under normal or nutrient deprivation conditions. We found that the number and rate of differentiation of bone marrow hematopoietic stem cell was decreased in the TG2 knockout mice. We present evidence showing that these effects on hematopoietic system are very likely due to the TG2-dependent impairment of autophagy. In fact, stimulation of autophagy by starvation is able to rescue the block of the differentiation of stem cells progenitors in the TG2 KO mice. It was also shown that the RhoA/ERK½ pathway, known to be essential for regulation of the bone marrow progenitor cells homeostasis, was significantly impaired in the absence of TG2. Hence, this study expanded our knowledge about TG2 discovering a role of this enzyme in regulation of hematopoiesis.

7,12-Dimethylbenz(a)anthracene-induced genotoxicity on bone marrow cells from mice phenotypically selected for low acute inflammatory response

Exposure to polycyclic aromatic hydrocarbon (PAH) environmental contaminants has been associated with the development of mutations and cancer. 7,12-Dimethylbenz(a)anthracene ( DMBA), a genotoxic agent, reacts with DNA directly, inducing p53-dependent cytotoxicity resulting in cell death by apoptosis or giving rise to cancer. DMBA metabolism largely depends on activation of the aryl hydrocarbon receptor (AhR). Mice phenotypically selected for high (AIRmax) or low (AIRmin) acute inflammatory response present a complete segregation of Ahr alleles endowed with low (Ahr(d)) or high (Ahr(b1)) affinity to PAHs, respectively. To evaluate the role of AhR genetic polymorphism on the bone marrow susceptibility to DMBA, AIRmax and AIRmin mice were treated with a single intraperitoneal injection of DMBA (50 mg/kg b.w.) in olive oil. Bone marrow cells (BMCs) were phenotyped by both flow cytometry and cytoslide preparations. Despite a significant decrease in total cell count in BM from AIRmin mice, there was an increase of blast cells and immature neutrophils at 1 and 50 days after DMBA treatment, probably due to a cell-cycle blockade at the G1/S transition leading to immature stage cell production. A panel of proteins related to cell cycle regulation was evaluated in immature BM cells (Lin(-)) by Western Blot, and DNA damage and repair were measured using an alkaline version of the Comet assay. In Lin(-) cells isolated from AIRmin mice, high levels were found in both p53 and p21 protein contents in contrast with the low levels of CDK4 and Ciclin D1. Evaluation of DNA repair in DMBA-treated BMCs, indicated long-lasting genotoxicity and cytotoxicity in BMC from AIRmin mice and a blockade of cell cycle progression. On the other hand, AIRmax mice have a high capacity of DNA damage repair and protection. These mechanisms can be associated with the differential susceptibility to the toxic and carcinogenic effects of DMBA observed in these mice. (C) 2015 Elsevier B.V. All rights reserved.

Quantification of capillary blood cell flow using reflectance confocal microscopy.

BACKGROUND/PURPOSE: In vivo reflectance confocal microscopy (IVCM) is a new tool for skin microcirculation. However, the measure of quantitative blood cell flow (QBCF) has not been standardized. We studied the inter-investigator and the intra-capillary reproducibility of the manual measure of QBCF on IVCM videos and investigated if a software program might help measure QBCF and be sensitive to vascular occlusion tests. METHODS: The inter-investigator reproducibility of the manual QBCF was evaluated on 107 videos. The intra-capillary reproducibility of QBCF measured manually and by 2 semi-automatic procedures based on Image J software analysis was evaluated on 19 capillaries. One of the semi-automatic methods (peaks of luminous intensity) was also used to measure the QBCF during vascular occlusion tests. RESULTS: The manual measure did not show a good inter-investigator reproducibility (Pearson's coefficient <0.5). The 'peaks of luminous intensity' method was found to have a good intra-capillary reproducibility and to be sensitive to vascular occlusion. CONCLUSION: Differently from the manual count, the count of peaks of luminous intensity by Image J software seems to be promising to measure QBCF. The future is to create software allowing for real-time measure of the QBCF based on the peaks of luminous intensity inside the capillaries recorded by IVCM.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Impairment of the hematological response and interleukin-1ß production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide.

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1ß (IL-1ß) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1ß in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1ß, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Splenectomy increases mortality in murine Trypanosoma cruzi infection.

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-γ and TNF-α production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4 percent) or were fed a control diet (20 percent protein) ad libitum. When the experimental group had lost about 20 percent of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates.

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4%) or were fed a control diet (20% protein) ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions.

BACKGROUND AND PURPOSE: We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells. EXPERIMENTAL APPROACH: Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA. KEY RESULTS: RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats. CONCLUSIONS AND IMPLICATIONS: Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

Cytotoxicity analysis of EDTA and citric acid applied on murine resident macrophages culture.

AIM: To assess the ex vivo cytotoxicity of EDTA and citric acid solutions on macrophages. METHODOLOGY: The cytotoxicity of 17% EDTA and 15% citric acid was evaluated on murine macrophage cultures using MTT-Tetrazolium method [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide]. A total of 5 x 10(5) cells were plated in medium culture with 17% EDTA or 15% citric acid. Fresh medium was used as a control. Toxicity values were analysed statistically by anova and Tukey's test (P<0.05) at short (0, 6, 12, 24 h) and medium periods (1, 3, 5, 7 days), using ELISA absorbance. RESULTS: On the short term, both EDTA (0.253 nm) and citric acid (0.260 nm) exhibited cytotoxic effects on macrophage cultures (P<0.05). On the medium term, statistical differences were observed (P<0.05) between the groups. EDTA (0.158 nm) and citric acid (0.219 nm) were cytotoxic when compared with the control group; EDTA-reduced macrophage viability significantly more than citric acid (P<0.05). CONCLUSIONS: Both EDTA and citric acid had effects on macrophages cells ex vivo, but citric acid was less toxic in periods from 1 to 7 days of use.

Protein-energy malnutrition alters histological and ultrastructural characteristics of the bone marrow and decreases haematopoiesis in adult mice.

Protein-energy malnutrition (PEM) decreases resistance to infection by impairing a number of physiological processes, including haematopoiesis. The aim of this study was to evaluate the microanatomical aspects of bone marrow (BM) in mice that were subjected to PEM, in particular, with respect to the components of the local extracellular matrix and the proliferative activity of haematopoietic cells. For this, histological, histochemical, immunohistochemical and ultrastructural techniques were used. Two-month old male Swiss mice were fed with a low-protein diet containing 4% protein and control mice fed a 20% protein diet. When the experimental group had attained a 25% loss of their original body weight, we collected the different biological samples. Malnourished mice had presented severe BM atrophy as well as a reduction in proliferating cell nuclear antigen and gelatinous degeneration. The malnourished mice had more fibronectin accretion in paratrabecular and endosteal regions and more laminin deposition in perisinusal sites than controls. Endosteal cell activation and hyperplasia were found, suggesting their participation in the process. Additionally, we have observed a decrease in the capacity of malnourished haematopoietic stroma to support the growth of haematopoietic stem cells (CD34+) in vitro. These findings point to a structural impairment of the haematopoietic microenvironments in mice with PEM, possibly hampering the interactions between cells and cellular signalling.

Effect of in vivo phenol or hydroquinone exposure on events related to neutrophil delivery during an inflammatory response.

Phenol (PHE) and hydroquinone (HQ) are metabolites of benzene that affect leukocytes after solvent intoxication. Hence, we investigated the effects of PHE or HQ exposure on neutrophil mobilization during an inflammatory response. Male Wistar rats received intraperitoneal injections of PHE, HQ or vehicle only and assays were performed 24 h after the last dose. Quantifications of bone marrow or circulating leukocytes showed that only HQ exposure induced neutrophilia, probably due to the accelerated mobilization from the bone marrow compartment, since reduced numbers of segmented cells in the last phase of maturation were detected there. Intravital microscopy showed that circulating leukocytes of HQ-exposed rats increased their rolling behavior and adherence to the mesenteric postcapillary venule wall in vivo. The enhanced leukocyte-endothelium interaction was not dependent on microvascular reactivity or perivascular mast cell degranulation. Instead, it was the result of neutrophil activation, demonstrated by a decrease in L-selectin and an increase in beta2 integrin expression on neutrophil membranes. This pattern of neutrophil activation may have contributed to the higher number of neutrophils in the subcutaneous inflammatory response of HQ-exposed rats after oyster glycogen injection. Taken together, our results indicate that HQ exposure alters neutrophil mobilization, which results in an exacerbated response after an injury. Although PHE is endogenously metabolized to HQ, PHE exposure only induced an increment in rolling behavior, which was not sufficient to alter the inflammatory response.

Chronic blockade of nitric oxide biosynthesis in rats: effect on leukocyte endothelial interaction and on leukocyte recruitment.

INTRODUCTION: Previous studies showed that animals chronically treated with NG-nitro-L-arginine methyl ester (L-NAME) have a reduced inflammatory reaction. Now the role of L-NAME treatment (20 mg/Kg/day/14 days) on leukocyte mobilisation was assessed in rats. METHODS: In vivo leukocyte recruitment evoked by Bothrops jararaca venom (BjV) and nitrite/nitrate (NO2-/NO3-; Griess reaction) were evaluated in the air pouch cavity. Haematological parameters were evaluated in the bone marrow and in the peripheral compartment. Microcirculatory blood flow, number of rolling and adhered leukocytes, vascular reactivity and mast cell activity were studied by intravital microscopy. Blood pressure was measured by the tail-cuff method. L-selectin and beta(2) integrin expressions on peripheral and bone marrow leukocytes were quantified by flow cytometry. RESULTS: When compared with control rats (D-NAME) L-NAME treated rats had reduced PMN cell infiltrate (50%) and NO2-/NO3- (27%) in the air pouch cavity. Rolling leukocytes were decreased (70%) in L-NAME-treated animals, which was reversed by topical application of NO donor (SIN-1). BjV stimulation increased the number of rolling and adhered leukocytes only in control rats. Systemic blood pressure, microcirculatory blood flow and microvascular reactivity was not altered by the treatment. Only the vessel response to acetylcholine was delayed in treated rats. Peripheral PMN cells were increased by L-NAME treatment (100%), but the number of bone marrow cells was not altered. The treatment reduced L-selectin expression on circulating leukocytes, by either with (16%) or without (26%) stimulation with BjV; PMN cells were more affected (32-37%). Impairment of L-selectin expression was also verified in bone marrow cells under stimulation with BjV. CONCLUSIONS: Results show that this schedule of L-NAME treatment promotes a decrease on L-selectin expression. This effect may promote the standstill of leukocytes in the blood compartment and may be responsible, at least in part, for the observed deficient leukocyte-endothelium interactions with subsequent impairment of leukocyte migration to the inflammatory site.