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.

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.

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.

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.

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.

Crotalus durissus terrificus snake venom regulates macrophage metabolism and function.

In the present study, we examined the effect of Crotalus durissus terrificus venom on rat macrophage metabolism and function. Two hours after subcutaneous injection of the venom, peritoneal resident (unstimulated), elicited (thioglycollate-stimulated), and activated Mycobacterium bovis strain bacille Calmette Guérin (BCG) macrophages were collected, and their functional and metabolic parameters were analyzed. The venom inhibited spreading and phagocytosis of macrophages. On the other hand, this treatment increased H(2)O(2) and NO production, candidacidal activity, and the activities of key enzymes of glycolysis and glutaminolysis. We also investigated whether the venom could affect macrophage activation by thioglycollate or BCG. The administration of venom 2 h before injection of thioglycollate and BCG or 2 or 3 days after injection of the thioglycollate or BCG, respectively, did not modify the previous observations. These findings suggest that crotalic venom leads the macrophage to an activated state, with high production of oxygen- and nitrogen-reactive species. This cell activation state does not include inflammatory properties of spreading and phagocytosis.

Acute Trypanosoma cruzi infection is associated with anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia: reversal by nifurtimox treatment.

In the present work we show that acute infection of C3H mice with the CL strain of Trypanosoma cruzi is characterized by an exponential growth of parasites and high mortality accompanied by anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia. Administration of nifurtimox, a trypanocydal drug currently in clinical use at different days postinfection, modulates parasitemia and prevents mortality. More importantly, none of blood and bone marrow alterations were observed in nifurtimox-treated animals when treatment was initiated early in infection, one or seven days postinoculation. The bone marrow alterations were characterized by a decrease in the total number cells as well in the number of megakaryoblasts and erythroblasts. Transfer experiments of bone marrow cells from infected mice to noninfected lethally irradiated recipients revealed a poor marrow-repopulating activity. The colony forming units-spleen assay confirmed the depression of committed clonal progenitors cells and revealed a decreased number of granulocyte/macrophage, megacariocyte and erythrocyte colonies. In summary, this is the first report showing that acute T. cruzi infection results in profound alterations of the hematopoietic system and that these alterations can be prevented by nifurtimox treatment.

Unusual horizontal and vertical root fractures of maxillary molars: an 11-year follow-up.

Intra-alveolar root fractures of posterior teeth are rare when compared with other dental injuries. This case report describes one vertical and two horizontal root fractures of teeth 3, 14, and 15. The teeth all tested normal to cold and electric pulp tests. The patient reported no history of accidental trauma, and no signs of scarring were found. These fractures were discovered during a routine full-mouth radiographic survey. All teeth were asymptomatic and in good function. During the 11 yr that followed, there was no dental treatment, except for routine periodontal maintenance. The patient had one abscess that occurred after 9 yr on tooth 3, which had to be extracted. The upper left molars are surprisingly still in function and asymptomatic. Either occlusal or lateral trauma may be the cause of these fractures. This would strongly suggest night guard appliances for patients who clench or grind. A psychological evaluation of the patient might reveal neurosis, anxiety, or stress situations affecting teeth.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice.

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Experimental study of induced inflammation in the Brazilian Boa (Boa constrictor constrictor).

The objective of this work was to identify the cellular types present in inflammatory processes in the Brazilian snake, Boa constrictor constrictor. Blood smears were first made from three normal snakes and stained by several methods to identify the cell types present, thus facilitating the identification of cells in inflammatory processes induced in 16 further snakes by the subcutaneous implantation of cotton suture threads and circular coverslips. Implanted threads induced migration of heterophils and monocytes after 4 h, more intense monocyte migration after 24 h, an intense granulocytic migration inside and around the thread after 48 h, heterophilic granulocytes, macrophages and giant cells after 7 days, and giant cells with a typical granuloma response and persistence of heterophilic cells after 15, 69 and 117 days. The cell population attached to the implanted coverslips after 4 h was composed of heterophils, thrombocytes, erythrocytes and macrophages; after 24 and 48 h heterophils predominated, and after 7 days heterophils, macrophages and giant cells predominated.

Intra-oral welding of implants for an immediate load with overdentures.

Different clinical applications of the Hruska intra-oral welding machine have been described in previous articles (Hruska, 1986, 1987, 1988, 1989a,b). These applications were related to over 100 cases of the use of non-submergible implants. This paper will describe a revised application of this machine as used with the currently popular multiform submergible implants widely used in the United States. The intra-oral welding is performed immediately after suture placement, with one or more connecting titanium wires serving as the splinting medium. This permits the patient to leave the office with a stable and retentive overdenture resting securely on the newly created splint performed the same day of surgery. This avoids the potentially troublesome problem of allowing the overdenture to be supported by the recently altered soft tissues. In addition, the patient otherwise would not have the use of a denture during the bone-healing period, which creates an alteration in psychological as well as physiological status. The disadvantages of immediate loading are counteracted by the splinting and load-sharing. This encourages the bone around the implants to reorganize and remodel according to Wolff's law, as demonstrated by De Angelis (1970) and Dalin and Olsson (1974). Different studies (Sutter et al., 1983a,b; Schroeder, 1985; Jennings, 1991) have shown that the ankylotic connection between one-stage titanium ITI implants and bone (so-called osseointegration) occurs and is maintained under load.

Reimplantation or explantation in the treatment of septic prosthetic loosening.

Indications for the surgical treatment of septic loosening of hip arthroplasty vary depending on different parameters. Explantation is indicated when patients are elderly, in generally poor condition, when there are veterate infections, particularly if these were caused by gram negative bacteria, and when there is severe skeletal injury. Reimplantation is instead indicated in patients whose general conditions are good, and who are young and motivated.

The surgical treatment of spondylolisthesis.

The authors report the results obtained in 244 patients submitted to anterior interbody fusion. The method involves preoperative reduction in plaster (when necessary) and anterior surgical stabilization, facilitated by improvement in the anatomical and biomechanical conditions obtained with reduction. This method obtained excellent results in 85% of the cases, with fusion and absence of symptoms, and good results in 10% of the cases, with occasional symptoms and/or fibrous. Results were unsatisfactory in 5% of the cases because of residual radiculopathy (4 cases), non-union of the fusion (3 cases), retrograde ejaculation (2 cases), loosening of the graft (1 case) or unthreading of a screw (1 case). It is our opinion that anterior interbody fusion is the best surgical method for the stabilization of Meyerding grades III, IV and V spondylolisthesis. Posterolateral fusion is used to treat some cases with slippage equal to less than 25% (grade I) and cases where more than one level is involved.

[Use of a latissimus dorsi myocutaneous flap in locally advanced breast cancer]. / L'uso del lembo miocutaneo di gran dorsale nel carcinoma mammario localmente avanzato.

Locally advanced breast cancer has a poor prognosis. Skin infiltration, ulceration and invasion of the chest wall, in the absence of distant metastases, worsen dramatically the quality of life. The Authors report 4 cases of one time wide resection and reconstruction using a pedicled myocutaneous flap of latissimus dorsi. This technique allowed good and rapid palliation of local symptoms.

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.

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.

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.

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.