First phylogenetic analysis of dengue virus serotype 4 circulating in Espírito Santo state, Brazil, in 2013 and 2014.

The purpose of the present study was to reconstruct the phylogeny of dengue virus serotype 4 (DENV-4) that was circulating in Espírito Santo state, Brazil, in 2013 and 2014, and to discuss the epidemiological implications associated with this evolutionary hypothesis. Partial envelope gene of eight DENV-4 samples from Espírito Santo state were sequenced and aligned with 72 worldwide DENV-4 reference sequences from GenBank. A phylogenetic tree was reconstructed through Bayesian Inference and the Time of the Most Recent Common Ancestor was estimated. The study detected the circulation of DENV-4 genotype II in Espírito Santo state, which was closely related to strains from the states of Mato Grosso collected in 2012 and of São Paulo sampled in 2015. This cluster emerged around 2011, approximately 4 years after the entry of the genotype II in Brazil through its northern states, possibly imported from Venezuela and Colombia. This is so far the first phylogenetic study of the DENV-4 circulating in Espírito Santo state and shows the importance of an internal route of dengue viral circulation in Brazil to the introduction of the virus into this state.

Influence of demographics on clinical outcome of dengue: a cross-sectional study of 6703 confirmed cases in Vitória, Espírito Santo State, Brazil.

Dengue presents a wide clinical spectrum of signs and symptoms, with characteristics of the host potentially influencing the disease evolution. Therefore, the purpose of this study was to evaluate the influence of gender and age on dengue clinical outcomes in a recent outbreak situation in Brazil, applying a cross-sectional design and including 6703 dengue cases with laboratory confirmation, occurring in Vitória, Espírito Santo State, Brazil, between 2007 and 2013. Data were obtained from the Information System for Notifiable Diseases. Overall, 11·3% of the sample presented with severe dengue, which affected 13·0% of males, 10·0% of females, 8·8% of children, 12·5% of adolescents, 10·5% of adults and 15·5% of the elderly. Age was higher in the severe dengue group (P = 0·03). Severe dengue was associated with males and the elderly (P < 0·01); however, considering only severe cases, children presented haemorrhage and plasma leakage more frequently than older age groups. The results emphasize the importance of a differentiated protocol for management of dengue cases, taking into consideration host factors like age. These findings also suggest the elderly and children as priority groups for immunization in a future implementation of a vaccine.

CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI+2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low nonspecific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection in T-ALL, bearing the advantage of surface expression in contrast to TdT.

Selection of CD34-Positive Blood Cells for Allogeneic Transplantation: Approaches to Optimize D34-Cell Recovery, Purity, Viability, and T-cell Depletion.

BACKGROUND: Methods for clinical-scale selection of CD34-positive hematopoietic stem and progenitor cells have facilitated allogeneic transplants using HLA-mismatched healthy donors. We examined different approaches to purify mobilized CD34+ cells, focusing on yield, purity, and viability of the selected cells and T-cell depletion levels. METHODS: Sixty-seven CD34-positive selections were performed for a total of 37 allogeneic transplantations, 23 of which from HLA-haploidentical donors. The selection devices were the Isolex((R)) 300i (v. 1.12) used alone (n =13) or with the SuperMACS (n = 29); the CliniMACS (n = 3); and the Isolex 300i (v. 2.0b1). The latter was used for CD34-positive selection (n = 7) and combined CD34+/CD4 8 19-negative selections (n =15). DNAse was included to reduce cell clumping. RESULTS: With the Isolex 300i (v. 1.12), the median CD34+-cell recovery increased from 51% (without DNAse) to 61% (15 mg DNAse) and 70% (7.5 mg). DNAse (5 mg) was used for 22 selections with the Isolex (v. 2.0b1) without cell clumping. CD34-positive cell purity, yield, and viability, as well as the degree of CD3 depletion varied with the selection device and procedure used. CONCLUSION: With regard to all of the above-mentioned parameters, the best results were obtained with the Isolex 300i (v. 2.0b1). Values achieved for CD34-positive cells were 98% for purity, 50-60% for yield, and > 96% for cell viability; T-cell depletion was 4.5 to > 5 log. The automated and closed system provides target cells that are free of both magnetic particles and murine monoclonal antibody. Copyright 2000 S. Karger GmbH, Freiburg

CD99 (MIC2) expression in paediatric B-lineage leukaemia/lymphoma reflects maturation-associated patterns of normal B-lymphopoiesis.

We have recently shown that CD99 (MIC2) is differentially expressed during normal early B-cell development in the bone marrow (BM). Since immature B-cell precursors (BCP) are assumed to correspond to some extent to acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) cells with respect to patterns of phenotypic differentiation, we wondered whether the particular maturation-associated expression patterns of CD99 in the normal BCP stages were conserved also in malignant cells. Therefore we compared malignant and physiological B cells from paediatric ALL/NHL and normal BM samples with respect to CD99 expression using selective gating and semi-quantitative flow cytometry. Common-ALLs (n = 45) were similar to their corresponding, very immature BCPs (stage 1) in expressing very high levels of CD99. Most pre-B ALLs (n = 16) were also CD99hi and thus differed from the patterns found in normal cytoplasmic mu-chain+ (cmu+) pre-B cells (stage 2, CD99lo). In particular, we found that those pre-B-ALL cases which were CD34+ also showed higher CD99 expression than the CD34- cases. This prompted us to investigate the levels of CD99 in those rare normal BCPs which also coexpress CD34 and cmu; these cells, which are transitory from stage 1 to stage 2, were found also CD99hi, thus precisely reflecting the patterns of CD34+ pre-B ALLs. The blasts of Burkitt-type B-cell ALL/NHL samples (n = 13) expressed considerably less CD99, similarly to the more differentiated BCP stages 2 (cmu+) and 3 (surface mu-chain+). In summary, we found that paediatric B-lineage malignancies display remarkable synchrony regarding the levels of CD99 expression compared to their putative normal counterparts.

Skin-associated lymphocytes in the peripheral blood of patients with atopic dermatitis: signs of subset expansion and stimulation.

BACKGROUND: Skin-associated T cells are defined by the cutaneous lymphocyte-associated antigen (CLA). In atopic dermatitis (AD), CLA+ T cells harbor allergen-reactive memory cells, spontaneously secrete TH2 cytokines, and display signs of increased in vivo activation, thus relating the subset to the central disease pathomechanisms. OBJECTIVES: It is not known whether the proportion of circulating CLA+ T cells might be expanded in AD. We were therefore prompted to compare the peripheral blood lymphocyte subpopulations of patients with AD with those of control subjects. METHODS: We used 3-color flow cytometry to investigate age-matched peripheral blood samples of pediatric and young adult patients with mild (n = 21) or severe (n = 15) AD, patients with allergic/atopic diseases not involving the skin (n = 9), and healthy control subjects (n = 14). RESULTS: We found no differences among the study groups with respect to the general proportions of T cells, CD4(+) T cells, CD8(+) T cells, B cells, NK cells, CD103(+) T cells, and CD25(+) T cells among total circulating lymphocytes. However, there were slightly more CD4(+) memory cells and clearly more HLA-DR+ T cells in patients with severe AD. Most remarkably, patients with severe AD had a significantly expanded proportion of CLA+ T cells (P =.024) and CLA+/CD4(+) T cells (P =.006) but similar proportions of CLA+/CD8(+) T cells compared with control subjects. Patients with severe AD also had distinctly more HLA-DR+/CLA+ T cells than control subjects (P =. 005). Similar alterations were seen in patients with mild AD, but these were not statistically significant. After correction for age, all differences were significant only in probands less than 10 years of age. CONCLUSIONS: Circulating skin-associated T cells (CLA+) show signs of subset expansion and enhanced activation in patients with AD. These alterations, compared with control values, affect CD4(+) memory T cells in particular and are prominent only in children less than 10 years of age.

Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: a study of five cases controlled by genetic methods.

We recently investigated samples of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and normal bone marrow (BM). We found that leukemic blasts, compared to their physiologic counterpart cells, frequently display aberrant phenotypes with respect to levels of expression of certain antigens. Using multiparameter flow cytometry, these differences enabled us to trace leukemic cells admixed to normal BM, which suggested that this approach might be a useful strategy for minimal residual disease detection. In the present study, we used the same multiparameter approach ("comparative phenotype mapping") to prove that such quantitative phenotypic differences really exist between malignant and normal BCP when simultaneously present in the BM. We demonstrate this by five exemplary follow-up BM samples from patients with BCP-ALL, all of which showed phenotypically aberrant cells according to levels of expression of CD10, CD11a, CD19, CD34, CD44, or CD45RA, as well as according to altered orthogonal light scattering properties. We confirmed the leukemic nature of these cells by polymerase chain reaction-based detection of bcr1/abl transcripts, and of leukemia clone-specific immunoglobulin heavy chain rearrangements in only the suspicious cells when sorted by flow cytometry, but not in normal BCP or non-B cells. Comparative phenotype mapping thus allows one to distinguish between normal and leukemic cells, and we show that it may enable rapid, specific, and quantitative detection of residual/resurgent leukemia in BCP-ALL.

Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny.

Terminal deoxynucleotidyl transferase (TdT)-positive cells in human bone marrow (BM) are a phenotypically inhomogeneous population of precursor cells. In their majority, these TdT+ cells are unambiguously committed to the B lineage, as evidenced by CD19 expression. However, TdT+ precursors that lack CD19 also exist and these may encompass a differentiation potential for the B as well as for other lineages. Because recent data suggested that CD19 expression is not the earliest differentiation event in B-cell ontogeny, we sought to reevaluate TdT+ lymphoid precursors in pediatric BM to define the phenotypic denominator of B-lineage affiliation upstream of CD19. Using four-color flow cytometry, we focused on the assessment of the CD79a antigen, which is highly B-cell specific and which may also be expressed very early in B-cell ontogeny. We found that a majority of TdT+ cells coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in all investigated samples, some TdT+ precursors lacked CD19 but expressed CD79a, which suggestively indicates also their B-lineage affiliation. In contrast to the CD19(+) precursors, which were usually CD10(hi) and CD79b+, these CD19(-)CD79a+ putative B-cell precursors preferentially expressed CD10 at low levels and were CD79b+ in only 41%. About 17% of these TdT+CD19(-)CD79a+ precursors also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or cytoplasmic CD3, which is discussed in the light of cellular activation rather than lineage promiscuity. Our data confirm that the earliest differentiation stages of B cells can be dissected upon expression of the lineage antigens CD79a and CD19 and imply that CD79a is earlier expressed than CD19. This raises the chance to follow the sequential events heralding B-cell commitment in the most immature precursors by correlating phenotypic and genetic differentiation markers.

Comparative phenotype mapping of normal vs. malignant pediatric B-lymphopoiesis unveils leukemia-associated aberrations.

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.

Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow.

We studied the differentiation profiles of B cell precursors (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molecules, the stage-specific patterns were found to differ in certain details from previously published concepts. Among the earliest BCP, a subset of CD34+ CD10(lo) precursors was repeatedly observed in addition to the well characterized CD34+ CD10(hi) CD19+ majority of cells. Only two-thirds of these CD34+ CD10(lo) cells expressed CD19. However, uniformity of phenotypic features, absence of T lineage markers, and the regeneration kinetics after chemotherapy suggest the B lineage affiliation of the CD34+ CD10(lo) precursors in general. In the more mature BCP, expression of CD10, CD20, cytoplasmic and surface mu chains (c mu and s mu) was observed to overlap more than previously recognized. We found that CD20 and c mu appear early during B cell ontogeny (already on CD34+ BCP), and that CD10 is lost late, following the onset of s mu expression. Differences between normal and post-chemotherapy BM specimens regarding the phenotypic appearance of BCP were exclusively due to differences in the subset composition, as post-chemotherapy samples showed a preponderance of immature stages. Our observations may build a framework for comparing leukemic cells with their normal counterparts to define possible leukemia-associated aberrations useful for residual disease studies.

Hepatocyte death following transforming growth factor-beta 1 addition.

Apoptosis is a morphological term which describes a sequence of events finally leading to cell death. In epithelial organs, induction of cell death is closely linked to an inhibitor of epithelial growth, transforming growth factor-beta 1 (TGF-beta 1). In this paper, we describe the morphology of TGF-beta 1-induced apoptosis in hepatocytes of the hyperplastic liver and primary cultures. Chromatin condensation, a hallmark of apoptosis, was observed in primary hepatocytes by confocal and vital UV microscopy. In addition, we have applied the morphological detection of DNA strand breaks both by in situ tailing (ISTAIL) and in situ nick translation (ISNT).

The antiandrogen cyproterone acetate induces synthesis of transforming growth factor beta 1 in the parenchymal cells of the liver accompanied by an enhanced sensitivity to undergo apoptosis and necrosis without inflammation.

Recently, cases of liver damage and liver tumors have been reported after treatment of prostate cancer patients with the antiandrogen cyproterone acetate (CPA). In rat liver, CPA initiates a wave of DNA synthesis that is accompanied by apoptosis. In apoptotic hepatocytes, a latent form of transforming growth factor beta 1 (TGF-beta 1) is detectable by immunohistochemistry. Injection of a single dose of TGF-beta 1 induces apoptosis in the liver of animals pretreated with CPA but has an insignificant effect in untreated animals. In this study, we show by Northern analysis that there is increased expression of TGF-beta 1 in the liver after CPA treatment. Detection of TGF-beta 1 with in situ hybridization showed that TGF-beta 1 was synthesized in the parenchymal cells. Time course and dose-response experiments performed 48 hours after the last application of CPA showed that apoptotic nuclei with chromatin condensed at the nuclear periphery (AN) were already visible 2 hours after injection (0.13%), and apoptotic bodies (ABs) increased 2 to 9 hours after the injection (from 1.28% to 6.67%) after 25 micrograms TGF-beta 1/kg. At 4.5 hours after injection, an induction of apoptosis could be detected with 0.25 microgram TGF-beta 1/kg and after the maximum dose (250 micrograms TGF-beta 1/kg) ANs (0.24%) and ABs (16.74%) were homogeneously distributed throughout the liver lobe. Irrespective of the dose or time after injection of TGF-beta 1, 82% of the ABs were localized within hepatocytes. Liver enzymes were detected in high amounts in the serum (eightfold elevation of glutamate dehydrogenase, fivefold elevation of alanine transaminase [ALT]) 7 hours after the first visible sign of apoptosis. After an additional 20 hours, the liver contained many necrotic figures. These results suggest that the combination of TGF-beta 1 expression coupled with a strikingly enhanced sensitivity to the induction of apoptosis could be responsible both for the liver damage and the development of liver tumors observed after treatment with CPA.

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.

Hepatocarcinoma-specific mutant p53-249ser induces mitotic activity but has no effect on transforming growth factor beta 1-mediated apoptosis.

Mutations affecting the p53 gene abrogate its tumor suppressor activity. It is, however, unclear whether such mutations can generate mutant p53 proteins with an intrinsic transforming ability. More importantly, the mechanism(s) by which they exert such activity is unknown. We report here that p53-deficient hepatoma cells (Hep3B) transfected with mutant p53-249ser (codon 249 Arg-->Ser) acquire a new phenotype with an increased in vitro survival and mitotic activity. However, such a phenotypic change is not sufficient to cause a major shift in the poor tumorigenic potential of these cells. This is apparently due to transforming growth factor beta 1-mediated apoptotic death of Hep3B cells which is not affected by the expression of p53-249ser.

Apoptosis is induced by transforming growth factor-beta 1 within 5 hours in regressing liver without significant fragmentation of the DNA.

In previous studies we showed that transforming growth factor-beta 1 induces apoptosis in hepatocyte cultures and regressing livers, the mature form being more potent than the transforming growth factor-beta 1 latency-associated protein. In this study we addressed the question of whether apoptosis can be induced within a short time after administration of transforming growth factor-beta 1. Five hours after a single intravenous injection of 25 micrograms mature transforming growth factor-beta 1/kg body weight, apoptosis is augmented ninefold in the regressing rat liver. A second preceding application induces no further augmentation. Transforming growth factor-beta 1 latency-associated protein shows no effect with either regimen. Morphological evaluation shows that 5 hr after injection of transforming growth factor-beta 1 nearly all apoptotic bodies are already engulfed by their neighbor cells. After homogenization of the transforming growth factor-beta 1-treated livers, the condensed apoptotic bodies are not destroyed and remain in the nuclear pellet. No DNA fragmentation into oligosomes could be detected after purification of the DNA from the nuclear pellet and application to conventional gel electrophoresis. Application of in situ nick translation, which allows detection of DNA single- and double-strand breaks in individual apoptotic bodies, also revealed no substantial fragmentation of the DNA in apoptotic bodies. These studies show that transforming growth factor-beta 1 is able to induce apoptosis within a rather short time and also suggest that in vivo digestion of the DNA does not lead to chromatin condensation.

Induction of apoptosis in cultured hepatocytes and in the regressing liver by transforming growth factor-beta 1 occurs without activation of an endonuclease.

In previous studies in vivo apoptotic liver cells were found to be positive for transforming growth factor-beta 1 (TGF-beta 1). In hepatocyte cultures TGF-beta 1 induced rounding up and fragmentation of the cells into multiple vesicles. As revealed by the DNA specific stain H33258 the chromatin of these cells condensed and segregated into masses at the nuclear membrane, followed by nuclear fragmentation. Ultrastructurally the cytoplasm was well preserved as demonstrated by the presence of intact cell organelles. These features strongly suggest that occurrence of apoptosis. Furthermore we administered TGF-beta 1 in vivo using an experimental model in which regression of the liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate (CPA). Two doses of 1 nM TGF-beta 1/kg each augmented the incidence of apoptotic hepatocytes 5-fold. These studies strongly suggest that TGF-beta 1 is involved in the initiation of apoptosis in the liver In TGF-beta 1 treated hepatocytes both from the liver and monolayer culture no DNA fragmentation into oligosomes could be detected. Comparison of nuclei in which endonuclease was activated by Ca2+ with apoptotic nuclei revealed no obvious similarities, as demonstrated by FACS analysis, H33258 staining and electron microscopy. Thus, apoptosis induced by a growth inhibitor obviously occurs without activation of an endonuclease.