Natural killer cells 56bnght16- have higher counts in the umbilical cord blood than in the adult peripheral blood

ABSTRACT Introduction and hypothesis: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation in the absence of a compatible donor. The UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD), but is associated with slower engraftment and slower immune reconstitution, compared to other sources. Dendritic cells (DCs) and Natural Killer cells (NKs) play a central role in the development of GvHD and the graft versus leukemia (GvL) effect, as well as in the control of infectious complications. Method: We quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DCs, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. Results: In the UCB samples, there were higher counts of NK cells 56bright16- (median 0.024 × 109/L), compared to the PB samples (0.012 × 109/L, p < 0.0001), NK 56dim16bright (median 0.446 × 109/L vs. 0.259 × 109/L for PB samples, p = 0.001) and plasmacytoid dendritic cells (pDCs, median 0.008 × 109/L for UCB samples vs. 0.006 × 109/L for PB samples, p = 0.03). Moreover, non-classic monocyte counts were lower in UCB than in PB (median 0.024 × 109/L vs. 0.051 × 109/L, respectively, p < 0.0001). Conclusion: In conclusion, there were higher counts of NK cells and pDCs and lower counts of non-classic monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GvHD, although maintaining the GvL effect, in UCB transplant recipients, compared to other stem cell sources.

MAPK/ERK and PI3K/AKT signaling pathways are activated in adolescent and adult acute lymphoblastic leukemia.

BACKGROUND: The mitogen-activated protein kinase (MAPK)/ERK signaling cascade and the phosphoinosytol-3 phosphate/Akt (PI3K/Akt) pathways are involved in proliferation and differentiation of hematopoietic cells. The frequency of PI3K/Akt and MAPK pathway activation in adult acute lymphoblastic leukemia (ALL) still need to be elucidated. AIMS: To assess the activity and prognostic implications of MAPK/ERK and PI3K/Akt pathways in adult (ALL). METHODS: We examined 28 precursor-B-cell ALL and 6 T-cell primary ALL samples. Flow cytometry was employed to analyze the expression levels of phosphorylated ERK and phosphorylated Akt. RESULTS: Ten out of 15 (67%) ALL fresh samples (7 B-cell, 3 T-cell) showed constitutive p-ERK expression. The p-ERK mean fluorescent index ratio (MFI (R)) showed a tendency to be higher in ALL than in normal T lymphocytes (1.26 [0.74-3.10] vs. 1.08 [1.02-1.21], respectively [p = .069]) and was significantly lower than in leukemic cell lines (median MFI (R) 3.83 [3.71-5.97] [p < .001]). Expression of p-Akt was found in 35% (12/34) (10 B-cell, 2 T-cell). The median MFI (R) expression for p-Akt in primary blast cell was 1.13 (0.48-9.90) compared to 1.01 (1.00-1.20) in normal T lymphocytes (p = ns) and lower than in leukemic cell lines (median MFI (R) 2.10 [1.77-3.40] [p = .037]). Moreover, expression of p-ERK was negatively associated with the expression of CD34 (1.22 [0.74-1.33] vs. 1.52 [1.15-3.10] for CD34(+) and CD34(-) group, respectively, p = .009). CONCLUSION: Our findings suggest that both MAPK/ERK and PI3K/Akt are constitutively activated in adult ALL, indicating a targeted therapy potential for ALL by using inhibitors of these pathways.

Natural killer cells 56bright16- have higher counts in the umbilical cord blood than in the adult peripheral blood.

INTRODUCTION AND HYPOTHESIS: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation in the absence of a compatible donor. The UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD), but is associated with slower engraftment and slower immune reconstitution, compared to other sources. Dendritic cells (DCs) and Natural Killer cells (NKs) play a central role in the development of GvHD and the graft versus leukemia (GvL) effect, as well as in the control of infectious complications. METHOD: We quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DCs, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. RESULTS: In the UCB samples, there were higher counts of NK cells 56bright16- (median 0.024 × 109/L), compared to the PB samples (0.012 × 109/L, p < 0.0001), NK 56dim16bright (median 0.446 × 109/L vs. 0.259 × 109/L for PB samples, p = 0.001) and plasmacytoid dendritic cells (pDCs, median 0.008 × 109/L for UCB samples vs. 0.006 × 109/L for PB samples, p = 0.03). Moreover, non-classic monocyte counts were lower in UCB than in PB (median 0.024 × 109/L vs. 0.051 × 109/L, respectively, p < 0.0001). CONCLUSION: In conclusion, there were higher counts of NK cells and pDCs and lower counts of non-classic monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GvHD, although maintaining the GvL effect, in UCB transplant recipients, compared to other stem cell sources.

Lower access to risk stratification tests and drugs, and worse survival of chronic lymphocytic leukaemia patients treated in public as compared to private hospitals in Brazil: A retrospective analysis of the Brazilian registry of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) has a highly variable clinical course. In addition to biological factors, socioeconomic factors and health system characteristics may influence CLL outcome. Data from the Brazilian Registry of CLL were analyzed to compare clinical and treatment-related characteristics in patients with CLL, from public or private institutions. A total of 3326 patients from 43 centres met the eligibility criteria, of whom 81% were followed up at public hospitals and 19% at private hospitals. The majority were male (57%), with a median age of 65 years. Comparing public and private hospitals, patients in public hospitals were older, had more advanced disease at diagnosis, and more frequently had elevated creatinine levels. All investigated prognostic markers were evaluated more often in private hospitals. First-line treatment was predominantly based on chlorambucil in 41% of the cases and fludarabine in 38%. Anti-CD20 monoclonal antibody was used in only 36% of cases. In public hospitals, significantly fewer patients received fludarabine-based regimens and anti-CD20 monoclonal antibodies. Patients from public hospitals had significantly worse overall survival (71% vs. 90% for private hospitals, p < 0.0001) and treatment-free survival (32% vs. 40%, for private hospitals, p < 0.0001) at seven years. Our data indicate striking differences between patients followed in public and private hospitals in Brazil. A worse clinical condition and lack of accessibility to basic laboratory tests and adequate therapies may explain the worse outcomes of patients treated in public institutions.

Bystander effects induced by the interaction between urothelial cancer cells and irradiated adipose tissue-derived stromal cells in urothelial carcinoma.

Cell-cell interactions between cancer cells and neighboring adipose tissue-derived stromal cells (ATSCs) are known to regulate the aggressiveness of cancer cells. In addition, the radiation-induced bystander effect is an important modulator of cancer cell kinetics. Radiation therapy is often given for urinary cancer, but the biological effects of the irradiated cancer stroma, including adipose tissue, on urothelial carcinoma (UC) remain unclear. We investigated the bystander effect of irradiated ATSCs on UC using a collagen gel culture method to replicate irradiated ATSC-cancer cell interactions after a single 12-Gy dose of irradiation. Proliferative activity, invasive capacity, protein expression and nuclear translocation of p53 binding protein-1 (53BP1) were analyzed. Irradiated ATSCs significantly inhibited the growth and promoted the apoptosis of UC cells in comparison to non-irradiated controls. The invasiveness of UC cells was increased by irradiated ATSCs, but not irradiated fibroblasts. Nuclear translocation of 53BP1 protein due to the bystander effect was confirmed in the irradiated group. Irradiated ATSCs regulated the expressions of the insulin receptor, insulin-like growth factor-1 and extracellular signal-regulated kinase-1/2 in UC. In conclusion, the bystander effect of irradiated ATSCs is a critical regulator of UC, and the actions differed depending on the type of mesenchymal cell involved. Our alternative culture model is a promising tool for further investigations into radiation therapy for many types of cancer.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels

ABSTRACT Introduction: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eightto ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. Objective and Method: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. Results: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. Conclusion: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation

Abstract Introduction: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. Method: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. Results and conclusion: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

INTRODUCTION: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. OBJECTIVE AND METHOD: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. RESULTS: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. CONCLUSION: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Cellular and physical microenvironments regulate the aggressiveness and sunitinib chemosensitivity of clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is the most predominant type of kidney cancer in adults and is responsible for approximately 85% of clinical cases. The tumor-specific microenvironment includes both cellular and physical factors, and it regulates the homeostasis and function of cancer cells. Perirenal adipose tissue and tumor-associated macrophages are the major cellular components of the RCC microenvironment. The RCC microvasculature network generates interstitial fluid flow, which is the movement of fluid through the extracellular compartments of tissues. This fluid flow is a specific physical characteristic of the microenvironment of RCC. We hypothesized that there may be an interaction between the cellular and physical microenvironments and that these two factors may play an important role in regulating the behavior of RCC. To elucidate the effects of adipose tissue, macrophages, and fluid flow stimulation on RCC and to investigate the relationships between these factors, we used a collagen gel culture method to generate cancer-stroma interactions and a gyratory shaker to create fluid flow stimulation. Adipose-related cells, monocytes, and fluid flow influenced the proliferative potential and invasive capacity of RCC cells. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interactions between RCC and adipose tissue fragments or macrophages. Fluid flow stimulation synergistically enhanced the anti-proliferative effect of sunitinib on RCC cells, but macrophages abolished the synergistic anti-proliferative effect related to fluid flow stimulation. In conclusion, we established a reconstructed model to investigate the cellular and physical microenvironments of RCC in vitro. Our alternative culture model may provide a promising tool for further therapeutic investigations into many types of cancer. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation.

INTRODUCTION: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. METHOD: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. RESULTS AND CONCLUSION: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Dental and oropharyngeal lesions in rats with chronic acid reflux esophagitis.

OBJECTIVE: In this study, we evaluated pathological changes in the tooth and pharynx of GERD rats to elucidate the association between gastric acid reflux and oral and pharyngeal diseases. METHODS: An experimental rat model of chronic acid reflux esophagitis was surgically created. The oral cavities were observed histologically every 2 weeks until 20 weeks after surgery. RESULTS: At 10 weeks after surgery, molar crown heights in GERD rats were shorter than that in control rats, and inflammatory cell infiltration by gastric acid reflux was found in the periodontal mucosa of GERD rats. Furthermore, dental erosion progressed in GERD rats at 20 weeks after surgery, and enamel erosion and dentin exposure were observed. During the same period, inflammatory cell infiltration was observed in the mucosa of the posterior part of the tongue. These findings suggest that gastric acid reflux may be one of the exacerbating factors of dental erosion, periodontitis and glossitis. CONCLUSION: We investigated oral changes in an experimental rat model of GERD and observed development of dental erosion, periodontitis and glossitis. Our findings suggested chronic gastric acid reflux may be involved in the pathogenesis of oral disease.

Decreased alertness due to sleep loss increases pain sensitivity in mice.

Extended daytime and nighttime activities are major contributors to the growing sleep deficiency epidemic, as is the high prevalence of sleep disorders like insomnia. The consequences of chronic insufficient sleep for health remain uncertain. Sleep quality and duration predict presence of pain the next day in healthy subjects, suggesting that sleep disturbances alone may worsen pain, and experimental sleep deprivation in humans supports this claim. We demonstrate that sleep loss, but not sleep fragmentation, in healthy mice increases sensitivity to noxious stimuli (referred to as 'pain') without general sensory hyper-responsiveness. Moderate daily repeated sleep loss leads to a progressive accumulation of sleep debt and also to exaggerated pain responses, both of which are rescued after restoration of normal sleep. Caffeine and modafinil, two wake-promoting agents that have no analgesic activity in rested mice, immediately normalize pain sensitivity in sleep-deprived animals, without affecting sleep debt. The reversibility of mild sleep-loss-induced pain by wake-promoting agents reveals an unsuspected role for alertness in setting pain sensitivity. Clinically, insufficient or poor-quality sleep may worsen pain and this enhanced pain may be reduced not by analgesics, whose effectiveness is reduced, but by increasing alertness or providing better sleep.

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.