Oxidant/Antioxidant Status Is Impaired in Sepsis and Is Related to Anti-Apoptotic, Inflammatory, and Innate Immunity Alterations.

Oxidative stress is considered pivotal in the pathophysiology of sepsis. Oxidants modulate heat shock proteins (Hsp), interleukins (IL), and cell death pathways, including apoptosis. This multicenter prospective observational study was designed to ascertain whether an oxidant/antioxidant imbalance is an independent sepsis discriminator and mortality predictor in intensive care unit (ICU) patients with sepsis (n = 145), compared to non-infectious critically ill patients (n = 112) and healthy individuals (n = 89). Serum total oxidative status (TOS) and total antioxidant capacity (TAC) were measured by photometric testing. IL-6, -8, -10, -27, Hsp72/90 (ELISA), and selected antioxidant biomolecules (Ζn, glutathione) were correlated with apoptotic mediators (caspase-3, capsase-9) and the central anti-apoptotic survivin protein (ELISA, real-time PCR). A wide scattering of TOS, TAC, and TOS/TAC in all three groups was demonstrated. Septic patients had an elevated TOS/TAC, compared to non-infectious critically ill patients and healthy individuals (p = 0.001). TOS/TAC was associated with severity scores, procalcitonin, IL-6, -10, -27, IFN-γ, Hsp72, Hsp90, survivin protein, and survivin isoforms -2B, -ΔΕx3, -WT (p < 0.001). In a propensity probability (age-sex-adjusted) logistic regression model, only sepsis was independently associated with TOS/TAC (Exp(B) 25.4, p < 0.001). The AUCTOS/TAC (0.96 (95% CI = 0.93-0.99)) was higher than AUCTAC (z = 20, p < 0.001) or AUCTOS (z = 3.1, p = 0.002) in distinguishing sepsis. TOS/TAC, TOS, survivin isoforms -WT and -2B, Hsp90, IL-6, survivin protein, and repressed TAC were strong predictors of mortality (p < 0.01). Oxidant/antioxidant status is impaired in septic compared to critically ill patients with trauma or surgery and is related to anti-apoptotic, inflammatory, and innate immunity alterations. The unpredicted TOS/TAC imbalance might be related to undefined phenotypes in patients and healthy individuals.

Vaccine coverage in children, adolescents and adults with type 1 diabetes and their close contacts in Crete.

BACKGROUND: Individuals with type 1 diabetes (T1D) are at increased risk of infections from vaccine-preventable diseases. This study focuses on compliance of T1D patients to the recommended vaccination schedule, vaccination of their close contacts for influenza and on factors potentially contributing to vaccination program deviations. METHODS: The study population comprised children, adolescents and adults with T1D under follow-up at the Department of Pediatrics University Hospital and the Diabetic Center General Hospital, Heraklion, Crete-Greece. Data were extracted, following informed consent, from individual's vaccination booklet, medical files and telephone interview. Vaccination records, demographic parameters, glycemic control and influenza vaccination of close contacts were assessed. RESULTS: The study included 258 participants (111 children/adolescents, 147 adults). Vaccination coverage for influenza was 76.7% for children, 64.4% for adults, for PCV 90.9% for children, but only 10.8% for the 23-valent, for hepatitis B 99% for children and 78.2% for adults. Youngsters were vaccinated against Hib 91.9%, meningococcus C 98.2%, measles-mumps-rubella 90.3%, chickenpox 86.4%, hepatitis A 76.5% and HPV 42.5%. Less than 65% of all individuals were fully vaccinated for diphtheria-tetanus-pertussis and meningococcus ACWY. Approximately 50% of the 605 close contacts were not vaccinated against influenza. Individuals with better glycemic status seemed to adhere to the recommended schedule and had a better vaccinated family environment. CONCLUSIONS: Vaccination coverage for T1D individuals was sufficient regarding the majority of routine childhood vaccines, but less for adolescence and group-specific vaccines. Their family contacts were not sufficiently vaccinated for influenza. Targeted interventions are required in order to increase vaccination rates.

Longitudinal Profiles of Metabolism and Bioenergetics Associated with Innate Immune Hormonal Inflammatory Responses and Amino-Acid Kinetics in Severe Sepsis and Systemic Inflammatory Response Syndrome in Children.

BACKGROUND: Experimental data indicate that sepsis influences the mitochondrial function and metabolism. We aim to investigate longitudinal bioenergetic, metabolic, hormonal, amino-acid, and innate immunity changes in children with sepsis. METHODS: Sixty-eight children (sepsis, 18; systemic inflammatory response syndrome [SIRS], 23; healthy controls, 27) were enrolled. Plasma amino acids were determined by high-performance liquid chromatography (HPLC); flow-cytometry expressed as mean fluorescence intensity (MFI) of heat shock protein (HSP) levels from monocytes (m) and neutrophils (n); resistin, adiponectin, and extracellular (e) HSPs evaluated by ELISA; ATP levels in white blood cells by luciferase luminescent assay; lipid peroxidation products (TBARS) by colorimetric test; nitrite and nitrate levels by chemiluminescent assay; biliverdin reductase (BVR) activity by enzymatic assay; and energy-expenditure (EE) by E-COVX. RESULTS: Resistin, eHSP72, eHSP90α, and nitrate were longitudinally higher in sepsis compared with SIRS (p<0.05); mHSP72, nHSP72, VO2 , VCO2 , EE, and metabolic pattern were repressed in sepsis compared with SIRS (p<0.05). Septic patients had lower ATP and TBARS compared with controls on day 1, lower ATP compared with SIRS on day 3 (p<0.05), but higher levels of BVR activity. Sepsis exhibited higher phenylalanine levels on day 1, serine on day 3; lower glutamine concentrations on days 3 and 5 (p<0.05). Resistin, inversely related to ATP, was independently associated with sepsis, along with mHSP72 and eHSP90α (p<0.05); TBARS and VO2 were independently associated with organ failure (p<0.05)). Septic nonsurvivors had malnutrition, persistently repressed metabolism, mHSP72, and induced resistin and adiponectin (p<0.05). CONCLUSIONS: A pattern of early longitudinal induction of metabolic-hormones and eHSP72/HSP90α, repression of bioenergetics and innate immunity, hypo-metabolism, and amino-acid kinetics changes discriminate sepsis from SIRS; malnutrition, hypo-metabolism, and persistently increased resistin and adiponectin are associated with poor outcome.

Biological Features of Bone Marrow Mesenchymal Stromal Cells in Childhood Acute Lymphoblastic Leukemia.

OBJECTIVE: Mesenchymal stromal cells (MSCs) have a supportive role in hematopoiesis and as components of the bone marrow (BM) microenvironment may present alterations during acute lymphoblastic leukemia (ALL) and be affected by chemotherapeutic agents. We examined the biological and functional characteristics of MSCs in ALL diagnosis and treatment and their effect on MSC qualitative properties. MATERIALS AND METHODS: Immunophenotypic characterization, evaluation of clonogenicity, and proliferative capacity were measured. Apoptotic features, cell-cycle analysis, and stromal cell-derived factor 1α and angiopoietin-1 levels in MSC supernatant at diagnosis and in different phases of treatment were assessed. Chemotherapy was administered according to the Berlin-Frankfurt-Munster-2000 protocol. BM samples from children with solid tumors without BM involvement were used as the control group. RESULTS: The morphology, the immunophenotypic profile, and the apoptotic characteristics of the MSCs were not affected by leukemia. The secretion of factors involved in the trafficking of hematopoietic cells in the BM seems to be upregulated at diagnosis in comparison to the treatment phases. MSCs are influenced by the disease in terms of their functional characteristics such as clonogenicity and proliferation rate. These effects cease as soon as treatment is initiated. Chemotherapy does not seem to exert any effect on any of the MSC features examined. CONCLUSION: MSCs from children with ALL are affected by their interaction with the leukemic environment, but this phenomenon ceases upon treatment initiation, while no effect is observed by chemotherapy itself.

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

Expression levels of ASNS in mesenchymal stromal cells in childhood acute lymphoblastic leukemia.

Increased levels of asparagine synthetase (ASNS), an enzyme producing intracellular asparagine, have been implicated in the development of asparaginase resistance. The aim of this study was to assess ASNS mRNA and protein expression in bone marrow cell populations of children with acute lymphoblastic leukemia (ALL). Bone marrow mononuclear cells at diagnosis, day 33 of treatment, and after completion of chemotherapy were isolated and studied. ASNS mRNA expression was assessed by real-time PCR, and protein levels by Western blot. Our results indicate that MSC ASNS mRNA expression is upregulated in ALL samples compared to controls. ASNS expression of mesenchymal stromal cells (MSC) was found to be 2.3 times higher than that of blasts at diagnosis of ALL. We also observed that the values of the ASNS mRNA of MSC seem to reach a peak at diagnosis, and tend to decline with treatment. No correlation was found between the ASNS mRNA and protein levels. Chemotherapy does not exert any effect on the protein expression. Variability of asparaginase-induced effect may be attributable to factors involved in the interaction of hematopoietic cells with their microenvironment.

Could mesenchymal stromal cells have a role in childhood autoimmune diseases?

Mesenchymal stromal cells (MSCs) comprise a promising source for cellular therapy due to their ability to be readily isolated from various tissues and expand ex vivo. A unique property of these cells is the modulation of immune responses, making them attractive candidates for the treatment of autoimmune diseases. Recently, several clinical trials, mainly in adults, suggest the use of MSCs for therapy of refractory autoimmune diseases. There are a very limited number of reports in the literature addressing the cellular therapy options for pediatric patients with autoimmune diseases refractory to standard therapy. This review discusses the possible mechanisms underlying the immunosuppressive effects of MSCs on almost all cell types, and also the recent advances in cellular therapy of autoimmune diseases using MSCs as modulators of immune response, especially in children.

CD64-Neutrophil expression and stress metabolic patterns in early sepsis and severe traumatic brain injury in children.

BACKGROUND: Critical illness constitutes a serious derangement of metabolism. The aim of our study was to compare acute phase metabolic patterns in children with sepsis (S) or severe sepsis/septic shock (SS) to those with severe traumatic brain injury (TBI) and healthy controls (C) and to evaluate their relations to neutrophil, lymphocyte and monocyte expressions of CD64 and CD11b. METHODS: Sixty children were enrolled in the study. Forty-five children with systemic inflammatory response syndrome (SIRS) were classified into three groups: TBI (n = 15), S (n = 15), and SS (n = 15). C consisted of 15 non- SIRS patients undergoing screening tests for minor elective surgery. Blood samples were collected within 6 hours after admission for flow cytometry of neutrophil, lymphocyte and monocyte expression of CD64 and CD11b (n = 60). Procalcitonin (PCT), C-reactive protein (CRP), glucose, triglycerides (TG), total cholesterol (TC), high (HDL) or low-density-lipoproteins (LDL) were also determined in all groups, and repeated on day 2 and 3 in the 3 SIRS groups (n = 150). RESULTS: CRP, PCT and TG (p < 0.01) were significantly increased in S and SS compared to TBI and C; glucose did not differ among critically ill groups. Significantly lower were the levels of TC, LDL, and HDL in septic groups compared to C and to moderate changes in TBI (p < 0.0001) but only LDL differed between S and SS (p < 0.02). Among septic patients, PCT levels declined significantly (p < 0.02) with time, followed by parallel decrease of HDL (p < 0.03) and increase of TG (p < 0.02) in the SS group. Neutrophil CD64 (nCD64) expression was higher in patients with SS (81.2%) and S (78.8%) as compared to those with TBI (5.5%) or C (0.9%, p < 0.0001). nCD64 was positively related with CRP, PCT, glucose, and TG (p < 0.01) and negatively with TC, LDL, and HDL (p < 0.0001), but not with severity of illness, hematologic indices, length of stay or mechanical ventilation duration. CONCLUSIONS: In sepsis, the early stress-metabolic pattern is characterized by a high (nCD64, glucose, TG) - low (TC, HDL, LDL) combination in contrast to the moderate pattern of TBI in which only glucose increases combined with a moderate cholesterol - lipoprotein decrease. These early metabolic patterns persist the first 3 days of acute illness and are associated with the acute phase CD64 expression on neutrophils.

Genetic variants associated with susceptibility to mother-to-child transmission of hepatitis B virus.

OBJECTIVE: The mechanisms of immune tolerance to hepatitis B virus (HBV) in children infected perinatally or early in infancy still remain unclarified. We aimed to study the genetic variants of immune factors implicated in viral-host interaction in children who were born to HBV-positive mothers and who had different clinical outcome. METHODS: Mannose-binding lectin gene (mbl2) codon 54, codon 57, and promoter 221 variants, tumor necrosis factor α (TNF-α) 308G/A, and vitamin D receptor (VDR) ApaI and TaqI genotypes were analyzed in three groups of children born to HBV-positive mothers: children with chronic infection (n=33), those with resolved infection (n=36), and those naive for HBV (n=33). RESULTS: TNF-α -308G allele frequency was found to be increased in children with chronic infection compared with children who were not affected by HBV [risk ratio (RR) 1.12, 95% confidence interval (CI) 1.0-1.25; P=0.050]. The VDR ApaI A allele tended to be more frequent in children with chronic infection than in those with resolved HBV infection (RR 1.27, 95% CI 0.95-1.67; P=0.071). The VDR ApaI α allele in ApaI and TaqI joint haplotype αT was more frequent in children with resolved infection than in those with chronic infection (RR 1.74, 95% CI 0.97-3.13; P=0.049). CONCLUSION: Our results suggest that TNF-α and vitamin D pathways may be involved in the susceptibility to and outcome of HBV infection acquired early in life.

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood.

BACKGROUND AIMS: Umbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols. METHODS: We assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133(+) subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control. RESULTS: The mononuclear cell fraction expressed 0.53±0.06% CD133. The corresponding value for CD34 was 1.64±0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93±1.25% CD34 while, after the corresponding process, CD133(+) expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133(+) yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC). CONCLUSIONS: CD34 isolation by the immunomagnetic method results in highly pure CD34(+) population, while the efficient and reproducible yield of a pure CD133(+) population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133(+) cells.

TRAF6 ubiquitinates TGFß type I receptor to promote its cleavage and nuclear translocation in cancer.

Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.

Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults.

BACKGROUND AIMS: Age-related changes that could affect the biologic features of mesenchymal stromal cells (MSC), such as a decrease in proliferation and osteoblast differentiation capacity and an increase of senescence markers and apoptosis, have been reported recently. The aim of this study was the evaluation of age-related characteristics and the correlation of age with the functional properties of MSC. METHODS: The doubling time (DT), colony-forming unit­fibroblast (CFU-F) colonies and surface antigen expression of MSC isolated from bone marrow (BM) of children (C-MSC) were compared with those from adults (A-MSC). The expression of Oct-4 and Nanog transcripts and the relative telomere length were evaluated in both groups. RESULTS: DT values were lower in C-MSC compared with A-MSC, and a higher CFU-F count was observed in children. However, the expression of Oct-4 and Nanog did not differ between C-MSC and A-MSC and was not correlated with the proliferative capacity. The telomere length was significantly higher in C-MSC compared with A-MSC. CONCLUSIONS: These data suggest that children's BM-derived MSC could be a more advantageous source of these cells for tissue engineering and cell therapy.

Stem cells: promises versus limitations.

Stem cells are the self-renewing progenitors of several body tissues and are classified according to their origin and their ability to differentiate. Current research focuses on the potential uses of stem cells in medicine and how they can provide effective treatment for a range of diseases. This approach has resulted in the field of medical practice called regenerative medicine. To attain the promises of regenerative medicine, it is necessary to fully understand the biology and properties of stem cells, achieve their successful differentiation into functional tissues, overcome the barriers related to immune responses after administration, and assess any oncogenic properties that limit their use. The availability of human stem cells not only raises hope for cell replacement therapies, but also provides a system for understanding the mechanisms of embryonic development and disease progression. Nevertheless, it raises ethical concerns that need to be addressed before the use of stem cells in clinical practice.

Biologic characteristics of mesenchymal stromal cells and their clinical applications in pediatric patients.

In the past few years, intensive research in the understanding of the biologic characteristics of the mesenchymal stromal cells has already led to some early clinical applications. The aim of this review is to summarize the latest information from basic science advances and the outcome of their use in clinical practice with a particular focus in pediatric patients. The minimum criteria required to identify mesenchymal stromal cells, their immunosuppressive-nonimmunogenic properties and their attribution in the treatment of graft-versus-host disease, in the acceleration of hematopoietic recovery, in tissue repair/tissue engineering and in the treatment of selected inherited disorders are discussed. Appropriate preclinical models, completion of ongoing and development of new clinical trials will establish the role of these cells in the treatment of both adult and pediatric patients.

Opioids modulate constitutive B-lymphocyte secretion.

The opioid system plays a major role in immunomodulation, while its action on cells of the immune system may be opioid receptor-mediated or not. Opioid effects on B-lymphocytes are considered as indirect, attributed to an interplay between distinct cell populations. The aim of the present study was to investigate whether opioid agonists (morphine, alpha(S1)-casomorphin and ethylketocyclazocine) may have a direct action on the secretion of antibodies and cytokines by multiple myeloma-derived cell lines and normal CD19+ B-lymphocytes. Our results show that opioids modulate antibody and cytokine secretion by multiple myeloma cells in a cell line-dependent and opioid receptor-independent manner, while they decrease antibody secretion by normal B-lymphocytes. Furthermore, they decrease the proliferation rate of multiple myeloma cells through opioid receptor activation. Our data suggest two different mechanisms of action of opioids, mediated by different signaling pathways: an early non-opioid receptor-related effect, modulating the constitutive immunoglobulin and cytokine secretion, and a long-term receptor-mediated action on cell growth. These data suggest a further opioid implication in the control of humoral immunity.

Bioluminescence imaging of calvarial bone repair using bone marrow and adipose tissue-derived mesenchymal stem cells.

A combined strategy using bioluminescence imaging, bone densitometry and histology was used to analyze the bone regeneration capacity of human bone marrow (hBMSC) and adipose tissue (hAMSC) mesenchymal stem cells, seeded in an osteoconductive arginine-glycine-aspartate (RGD) crosslinked hydrogel scaffold, implanted in a mouse calvarial bone defect. We show that firefly luciferase labeled stem cells can be monitored in vivo through a prolonged 90 days period, during which hBMSCs survive better than hAMSCs and that the density of scaffold bearing defects increased significantly more than that of defects without scaffolds.

Serum and urine interleukin-6 and transforming growth factor-beta1 in young infants with pyelonephritis.

BACKGROUND: Cytokines are involved both in the acute response during pyelonephritis and in the progression to renal scarring. The aim of the present study was to assess the pro-inflammatory interleukin-6 (IL-6) and the anti-inflammatory pro-fibrotic transforming growth factor-beta1 (TGF-beta) in very young infants with pyelonephritis. METHODS: Serum and urine concentrations of IL-6 and TGF-beta1 were determined by enzyme immunoassay in infants with acute pyelonephritis before antibiotic treatment and in infants with non-renal fever. IL-6 was studied in 12 infants with pyelonephritis and in eight with non-renal fever (median ages, 2 months for both groups). TGF-beta1 was studied in 11 infants with pyelonephritis and in nine with non-renal fever (median ages, 2 and 4 months, respectively). RESULTS: No significant differences were documented in serum concentrations of IL-6 and TGF-beta1 between patients and controls. Urine IL-6 levels were significantly higher in infants with pyelonephritis than in controls (medians, 147 and 21.4 pg/ml, respectively; P = 0.0106). The urine levels of TGF-beta1 were lower in infants with pyelonephritis than in controls, although not significantly (medians, 6.12 and 11.0 ng/ml, respectively; P = 0.0705). CONCLUSIONS: Our findings confirm the implication of IL-6 but not of TGF-beta1 in the pathogenesis of the early stages of pyelonephritis in young infants. Tauhe role of the pro-fibrotic TGF-beta1 in the development of renal scarring deserves further investigation.

The impact of mode of delivery and gestational age on cord blood hematopoietic stem/progenitor cells.

Human cord blood has been successfully used as an alternative source of hematopoietic stem cells suitable for transplantation. The aim of this study was to assess the impact of gestational age and the mode of delivery on cord blood hematopoietic stem/progenitor cell characteristics. The mode of delivery does not seem to affect either the replating capacity of hematopoietic progenitors colony-forming unit-granulocyte-macrophage or the cord blood content in CD34(+) cells. The higher percentage of CD34(+) cells in cord blood from preterm deliveries compared to full-term ones indicates that hematopoietic progenitors from preterm cord blood may be suitable for transplantation. These findings should be taken into consideration when selection of cord blood units is required for potential use in transplantation.

Assessment of megakaryopoiesis in children with acute lymphoblastic leukemia.

The effect of the underlying disease and chemotherapy on megakaryopoiesis has not been extensively studied in children with acute lymphoblastic leukemia (ALL) during and at the end of therapy. Using a serum-free assay, we assessed the megakaryocyte (Mk) colony formation in vitro from bone marrow mononuclear cells of 25 children with ALL during chemotherapy and shortly after the cessation of it. Twelve children with solid tumors without bone marrow involvement and cord blood from 10 full-term normal vaginal deliveries were used as controls. A significant reduction in the number of Mk colonies was observed at diagnosis of ALL, and Mk colony formation remained lower than controls throughout the different phases of leukemia treatment. Our study suggests that defects in megakaryopoiesis of children with ALL in long-term remission may persist during chemotherapy and at least shortly after the end of it.

A morphometric approach for the evaluation of angiogenesis in acute lymphoblastic leukemia of childhood.

Angiogenesis was estimated in childhood acute lymphoblastic leukemia (ALL) by the use of a novel morphometric method. This was compared with the widely used one of microvessel density (MVD). Bone marrow biopsies were evaluated by immunostaining with anti-Factor VIII related antigen (FVIIIRAg). Angiogenesis was calculated as volume corrected microvessel density index (VC-MVDI), taking into account the bone marrow cellularity. According to our results both MVD and VC-MVDI were increased at diagnosis of ALL in comparison with the control group. However the VC-MVDI increment was not statistically significant. Therefore, VC-MVDI could be more representative of the true increase of angiogenesis, correlating better with the outcome of the disease.