Multi-functional 2D hybrid aerogels for gas absorption applications.

Aerogels have attracted significant attention recently due to their ultra-light weight porous structure, mechanical robustness, high electrical conductivity, facile scalability and their use as gas and oil absorbers. Herein, we examine the multi-functional properties of hybrid aerogels consisting of reduced graphene oxide (rGO) integrated with hexagonal boron nitride (hBN) platelets. Using a freeze-drying approach, hybrid aerogels are fabricated by simple mixing with various volume fractions of hBN and rGO up to 0.5/0.5 ratio. The fabrication method is simple, cost effective, scalable and can be extended to other 2D materials combinations. The hybrid rGO/hBN aerogels (HAs) are mechanically robust and highly compressible with mechanical properties similar to those of the pure rGO aerogel. We show that the presence of hBN in the HAs enhances the gas absorption capacities of formaldehyde and water vapour up to ~ 7 and > 8 times, respectively, as compared to pure rGO aerogel. Moreover, the samples show good recoverability, making them highly efficient materials for gas absorption applications and for the protection of artefacts such as paintings in storage facilities. Finally, even in the presence of large quantity of insulating hBN, the HAs are electrically conductive, extending the potential application spectrum of the proposed hybrids to the field of electro-thermal actuators. The work proposed here paves the way for the design and production of novel 2D materials combinations with tailored multi-functionalities suited for a large variety of modern applications.

Causes of double-negative T-cell lymphocytosis in children and adults.

AIMS: The causes and diagnosis of 'double-negative' (CD3+CD4-CD8-) T-cell lymphocytosis are not well studied. We aimed to define the causes of double-negative T-cell lymphocytosis in children and adults, and to identify simple clinical and laboratory features that would help to differentiate between the underlying conditions. METHODS: We collected clinical and laboratory data on 10 children and 30 adults with significantly increased peripheral-blood double-negative T-cells (>10% of total lymphocytes). We identified conditions associated with double-negative T-lymphocytosis with flow cytometry, peripheral-blood morphology and T-cell receptor-gene rearrangement studies. Patients were assigned to diagnostic categories on the basis of these test results. RESULTS AND CONCLUSIONS: The causes of double-negative T-cell lymphocytosis in children were autoimmune lymphoproliferative syndrome (ALPS) and reactive γ/δ Τ-lymphocytosis. T-cell large granular lymphocyte (T-LGL) leukaemia, reactive γ/δ T-lymphocytosis and hepatosplenic T-cell lymphoma (HSTL) were the the most common disorders underlying double-negative T-cell lymphocytosis in adults. Less common causes included hypereosinophilic syndrome, peripheral T-cell lymphoma, ALPS and monoclonal, double-negative T-lymphocytosis of uncertain significance. CD5/CD7/Vδ2 expression and absolute double-negative lymphocyte count (<1.8×109/L) were useful discriminators for distinguishing patients with reactive γ/δ T-lymphocytosis from those with γ/δ lymphoproliferative disorders. Differentiating between γ/δ T-LGL and HSTL can be difficult. Expression of CD57 and cellular morphology (pale cytoplasm with distinct granules) would support a diagnosis of γ/δ T-LGL.

Flow cytometric predictive scoring systems for common fusions ETV6/RUNX1, BCR/ABL1, TCF3/PBX1 and rearrangements of the KMT2A gene, proposed for the initial cytogenetic approach in cases of B-acute lymphoblastic leukemia.

INTRODUCTION: In B-acute lymphoblastic leukemia (B-ALL), the identification of cytogenetic prognostic factors is important for stratifying patients into risk groups and tailoring treatment accordingly. The purpose of this study was to propose flow cytometric (FCM) scoring systems (SSs) for predicting t(12;21)(p13;q22), t(9;22)(q34;q11), t(11q23), and t(1;19)(q23;p13.3) translocations. METHODS: We analyzed retrospectively the FCM immunophenotype of 377 patients with B-ALL with regard to the major cytogenetic findings revealed by interphase fluorescence in situ hybridization (i-FISH). Comparing descriptive data on the expression of each antigen and performing receiver operating characteristic (ROC) analysis, we identified the most reliable predictive markers for each translocation and sought to establish a specific SS for each translocation, based on specific antibody panels. RESULTS: CD27, CD9, CD66c, CD10, CD25, and CD34 were employed for the prediction of t(12;21), CD25, CD38, CD34, and CD66c for t(9;22), NG2, CD10, CD15, CD34, and CD20 for t(11q23), and CD34, cµ, CD123, and CD66c for t(1;19). The sensitivity and specificity, respectively, of each predictive score were 89.29% and 96.15% for t(12;21), 75.00% and 88.19% for t(9;22), 84.21% and 99.04% for t(11q23), and 85.71% and 92.71% for t(1;19). CONCLUSION: Four highly specific and significantly sensitive FCM-obtained SSs are proposed for the prediction of the four major translocations observed in patients with B-ALL. Prospective evaluation of the proposed SSs could lead to a better targeted cytogenetic investigation and therefore to more cost-effective laboratory practice.

B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression.

Mesenchymal derivatives of genetically unstable human embryonic stem cells are maintained unstable but undergo senescence in culture as do bone marrow-derived mesenchymal stem cells.

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here, we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages, respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations, suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs, which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells.

CD3(-)CD16(-)CD56(bright) immunoregulatory NK cells are increased in the tumor microenvironment and inversely correlate with advanced stages in patients with papillary thyroid cancer.

BACKGROUND: The innate immune system is the first line of defense and plays a key role in thyroid cancer development. The role of the tumor-infiltrating natural killer (NK) cells is becoming increasingly important in research and potential cancer therapies. NK cell subpopulations, CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright), demonstrate a significant role in the tumor immuno-surveillance process. METHODS: We investigated the distribution of CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK subpopulations in tissue and blood samples from patients with papillary thyroid cancer (PTC) and nodular goiter (NG). Twenty-eight patients with PTC, 13 patients with NG, and 50 healthy donors were included in the study. Tissue and blood samples from all patients and blood samples from healthy donors were analyzed for CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK cells by flow cytometry. RESULTS: A significant predominance of CD3(-)CD16(+)CD56(dim) cells compared to CD3(-)CD16(-)CD56(bright) NK cells was found in blood samples in all groups (p<0.0001 in PTC, NG, and healthy donors). Increased infiltration by CD3(-)CD16(-)CD56(bright) NK cells was observed in thyroid tissue of patients with PTC, as compared to CD3(-)CD16(+)CD56(dim) NK cells (p=0.046), while CD3(-)CD16(+)CD56(dim) NK cells demonstrated a higher infiltration of NG tissues. CD3(-)CD16(+)CD56(dim) NK cell tissue infiltration positively correlated with advanced stages of PTC. In contrast, the CD3(-)CD16(-)CD56(bright) NK cell population was negatively associated with tumor stage in patients with PTC. CONCLUSION: CD3(-)CD16(-)CD56(bright) NK cell infiltration seems to be associated with PTC progression. These findings contribute to a better understanding of the immune response in PTC and may lead to novel immunotherapeutic approaches in these patients.

Phenotypical analysis of lymphocytes with suppressive and regulatory properties (Tregs) and NK cells in the papillary carcinoma of thyroid.

CONTEXT: The immune system seems to play a key role in preventing metastasis and recurrence of thyroid cancer. T regulatory lymphocytes (Tregs) and natural killer (NK) cells play an important role in the dysfunction of the host immune system in cancer patients. OBJECTIVE: We investigated thyroid gland infiltration by Tregs and NK cells in patients with papillary thyroid cancer (PTC) and thyroid nodular goiter (TNG). The correlation between the extent of the disease and the lymphocytic infiltration of Tregs and NK cells was examined. DESIGN, SETTING, AND PARTICIPANTS: A total of 65 patients with PTC, 25 with TNG, and 50 healthy controls were studied. Blood and tissue samples from 28 patients with PTC and 13 with TNG and blood samples from the healthy controls were analyzed for T4 (CD3(+)CD4(+)), T8 (CD3(+)CD8(+)), NK (CD3(-)CD16(+)CD56(+)), and CD4(+)CD25(+)CD127(-/low) Tregs by flow cytometry (FC). Tissue samples were also analyzed for Foxp3(+) Tregs by immunohistochemistry. RESULTS: Tregs showed greater infiltration in thyroid tissue of PTC patients compared with patients with TNG (P < 0.0009 for FC and P < 0.0001 for immunohistochemistry); FC analysis of blood samples showed no difference between the groups. Flow cytometry analysis showed significantly increased NK cells in PTC tissue compared with TNG tissue (P = 0.037), whereas blood samples showed no difference. CD4(+) and CD8(+) T cells did not differ in blood and tissue samples. Increased Tregs tissue infiltration was positively correlated with advanced disease stage (P < 0.0026), whereas NK infiltration was negatively correlated (P < 0.0041). CONCLUSION: Tregs and NK cells may be important regulators of thyroid cancer progression.

High frequency of human leukocyte antigen class II DRB1*1602 haplotype in Greek patients with myelodysplastic syndrome and of DRB1*1501 in the low-risk subgroup.

Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal hematopoietic disorders in which the immune-mediated pathogenetic mechanisms are under investigation. Overrepresentation of human leukocyte antigen (HLA)-DR2 and its serologic split HLA-DR15 has been associated with low-risk MDS in certain ethnic groups and has been proposed as a predictive factor for a favorable response to immunomodulatory treatment. Because the HLA-DRB1*15 haplotype does not predominate in the Greek population, we investigated the frequency of HLA-DRB1 alleles among 114 patients of Greek origin suffering from various types of MDS: 36 refractory anemia (RA), 24 refractory anemia with ringed sideroblasts (RARS), 19 refractory anemia with excess of blasts (RAEB), 14 refractory anemia with excess of blasts in transformation (RAEB-t), 14 chronic myelomonocytic leukemia, and 7 hypoplastic MDS patients. HLA-DRB1 molecular typing was performed with polymerase chain reaction-sequence specific oligonucleotides and results were compared with that from a previously reported control Greek population. HLA-DRB1*1602 was the only allele that was significantly overrepresented in Greek MDS patients as a whole, whereas HLA-DRB1*1501 allele frequency was significantly higher in Greek patients with low-risk myelodysplasia. Our results suggest the possible value of HLA-DR15 and HLA-DR16 as determinants for immunomodulatory interventions, at least for Greek patients with low-risk MDS.

An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions.

BACKGROUND: The aim of this study was to evaluate a flow cytometric assay for the detection of malignant effusions. METHODS: During the last 4-year period, 125 effusions suspicious for malignancy were prospectively analyzed by flow cytometry and conventional cytology. A three-step flow cytometric assay was performed, beginning with an initial informative panel of two protocols, containing SYTO-16, 7-AAD, CD71-PE, CD45-ECD, and CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PECy5, CD14-PECy7, respectively. This was followed by a basic immunophenotypic panel of seven three-color combinations, containing in the first position, EMA, Ber-EP4, CD66abce, CD56, and intracellular desmin-33, combined with CD71-PE and CD45-PeCy5 in each tube. Finally, a cytokeratin-FITC/propidium iodide DNA panel was conducted, for the detection of aneuploidy in cytokeratin positive cells. RESULTS: The sensitivity and specificity of flow cytometry were 85.1 and 97.8%, and of cytology 93.2 and 95.6%, respectively. A significant association was observed between the results of the two techniques (P < 0.001). Among eight atypical cases detected by cytology, five had been precisely characterized as malignant by flow cytometry. EMA and Ber-EP4 proved the most sensitive markers for malignancy diagnosis, while the detection of desmin-33 negative/cytokeratin positive cells had the simultaneous highest positive and negative predictive values. CD66abce was very specific, although nonsensitive, while DNA ploidy analysis was nonspecific, as hyperploidy was observed in reactive mesothelial cells. CONCLUSIONS: A flow cytometric assay of high sensitivity and specificity is proposed for the routine identification of carcinoma cells in effusions and their distinction from atypical mesothelial cells, as an ancillary to conventional cytology.

Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105+ cells.

Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long-term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM-CD105(+) cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 × 10(5) ± 0.9 × 10(5) CD105(+) cells from 10 small volume (10-25 ml) BM samples achieving an enrichment >100-fold in MSC. Seeding 2 × 10(3) immunoselected cells/cm(2) we were able to produce 2.5 × 10(8) ± 0.7 × 10(8) MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29(+), CD90(+), CD73(+), CD105(+), CD45(-); they suppressed allogeneic T-cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM-CD105(+) cells.

Spinal cord compression in an adolescent with relapsed B-precursor acute lymphoblastic leukemia and mental neuropathy.

The authors report a case of intraspinal mass associated with recurrence of B-precursor acute lymphoblastic leukemia in an adolescent male who presented with numb chin syndrome at initial diagnosis of the leukemia. The patient developed sensory changes, later on motor weakness, and eventually paraplegia. An emergent MRI scan showed an intraspinal mass at the level of T9 vertebra. Biopsy obtained during laminectomy revealed a mass composed of lymphoblasts immunophenotypically identical to the patient's known leukemia. Surgical decompression and dexamethasone were ineffective in restoration of the neurological deficits. Intraspinal extramedullary relapses should be considered in the differential diagnosis of leukemic patients with neurological symptoms.

Translocation (X;12)(p11;p13) as a sole abnormality in biphenotypic acute leukemia.

A reciprocal t(X;12)(p11;p13) was found as the sole clonal abnormality in biphenotypic leukemia with myeloid and B-lymphoid differentiation. With fluorescence in situ hybridization analysis, the ETV6 gene (previously TEL) was found to be translocated intact to the derivative X chromosome; no MLL and BCR/ABL rearrangements were found. The patient achieved complete remission after induction chemotherapy. To our knowledge, this cytogenetic aberration has not been reported previously as a sole abnormality in hematological malignancies. Its presence may suggest an important role in the pathogenesis of biphenotypic leukemia.

T-cell acute lymphoblastic leukemia relapsing as acute myelogenous leukemia.

We present the unusual case of a 16-year-old girl with T-cell acute lymphoblastic leukemia (ALL) with an early thymocyte immunophenotype without myeloid markers, who after 13 months of complete hematological remission relapsed as acute myelogenous leukemia (AML) with minimal differentiation and died of her disease. Whether the AML represented a relapse with lineage switch of the original immature T-cell clone or a new secondary malignancy, could not be proven due to the absence of molecular or clonal markers. This report suggests that a subset of CD7+ T-cell leukemias without mature T-cell antigens (CD4-, CD8-) are minimally differentiated and can relapse as AML.

Intracoronary infusion of CD133+ and CD133-CD34+ selected autologous bone marrow progenitor cells in patients with chronic ischemic cardiomyopathy: cell isolation, adherence to the infarcted area, and body distribution.

Central issues in intracoronary infusion (ICI) of bone marrow (BM)-cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM-progenitors. We could immunomagnetically isolate 0.8 +/- 0.32 x 10(7) CD133(+) cells and 0.75 +/- 0.24 x 10(7) CD133(-)CD34(+) cells from 310 +/- 40 ml BM. After labeling these cells with (99m)Tc-hexamethylpropylenamineoxime, they were infused into the infarct-related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% +/- 3.6 and 6.8% +/- 2.4 of the total infused radioactivity in the infarcted area of the heart, respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133(+) and CD133(-)CD34(+) BM-progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium.

Transferrin receptor-1 and 2 expression in chronic lymphocytic leukemia.

Transferrin receptor (TfR)-1 and 2 mRNA and CD71 (TfR1) expression was analyzed in 118 CLL patients. Ninety-five out of 109 analyzed cases expressed CD71, mostly at a high level; 60% of CD71 (+) cases were IGH-mutated. All samples were TfR1 mRNA (+); TfR2-alpha/beta mRNA was detected in, respectively, 52/102 and 100/109 cases. Competitive RT-PCR showed widely divergent levels of TfR1 mRNA in cases with high CD71 expression, alluding to post-transcriptional control of TfR1 expression in CLL. The almost uniformly high CD71 expression in CLL is in keeping with the activated status of neoplastic cells, regardless of IGH mutational load.

Immunoglobulin light chain repertoire in chronic lymphocytic leukemia.

Immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) light chain repertoire was analyzed in 276 chronic lymphocytic leukemia (CLL) cases and compared with the relevant repertoires from normal, autoreactive, and neoplastic cells. Twenty-one functional IGKV genes were used in IGKV-J rearrangements of 179 kappa-CLL cases; the most frequent genes were IGKV3-20(A27), IGKV1-39/1D-39(O2/O12), IGKV1-5(L12), IGKV4-1(B3), and IGKV2-30(A17); 90 (50.3%) of 179 IGK sequences were mutated (similarity < 98%). Twenty functional IGLV genes were used in IGLV-J rearrangements of 97 lambda-CLL cases; the most frequent genes were IGLV3-21(VL2-14), IGLV2-8(VL1-2), and IGLV2-14(VL1-4); 44 of 97 IGL sequences (45.4%) were mutated. Subsets with "CLL-biased" homologous complementarity-determining region 3 (CDR3) were identified: (1) IGKV2-30-IGKJ2, 7 sequences with homologous kappa CDR3 (KCDR3), 5 of 7 associated with homologous IGHV4-34 heavy chains; (2) IGKV1-39/1D-39-IGKJ1/4, 4 unmutated sequences with homologous KCDR3, 2 of 4 associated with homologous IGHV4-39 heavy chains; (3) IGKV1-5-IGKJ1/3, 4 sequences with homologous KCDR3, 2 of 4 associated with unmutated nonhomologous IGHV4-39 heavy chains; (4) IGLV1-44-IGLJ2/3, 2 sequences with homologous lambda CDR3 (LCDR3), associated with homologous IGHV4-b heavy chains; and (5) IGLV3-21-IGLJ2/3, 9 sequences with homologous LCDR3, 3 of 9 associated with homologous IGHV3-21 heavy chains. The existence of subsets that comprise given IGKV-J/IGLV-J domains associated with IGHV-D-J domains that display homologous CDR3 provides further evidence for the role of antigen in CLL pathogenesis.

Analysis of expressed and non-expressed IGK locus rearrangements in chronic lymphocytic leukemia.

Immunoglobulin kappa (IGK) locus rearrangements were analyzed in parallel on cDNA/genomic DNA in 188 kappa- and 103 lambda-chronic lymphocytic leukemia (CLL) cases. IGKV-KDE and IGKJ-C-intron-KDE rearrangements were also analyzed on genomic DNA. In kappa-CLL, only 3 of 188 cases carried double in-frame IGKV-J transcripts: in such cases, the possibility that leukemic cells expressed more than one kappa chain cannot be excluded. Twenty-eight kappa-CLL cases also carried nonexpressed (nontranscribed and/or out-of-frame) IGKV-J rearrangements. Taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 38% of kappa-CLL cases carried biallelic IGK locus rearrangements. In lambda-CLL, 69 IGKV-J rearrangements were detected in 64 of 103 cases (62%); 24 rearrangements (38.2%) were in-frame. Four cases carried in-frame IGKV-J transcripts but retained monotypic light-chain expression, suggesting posttranscriptional regulation of allelic exclusion. In all, taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 97% of lambda-CLL cases had at least 1 rearranged IGK allele, in keeping with normal cells. IG repertoire comparisons in kappa- versus lambda-CLL revealed that CLL precursor cells tried many rearrangements on the same IGK allele before they became lambda producers. Thirteen of 28 and 26 of 69 non-expressed sequences in, respectively, kappa- or lambda-CLL had < 100% homology to germline. This finding might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in some cases. The inactivation of potentially functional IGKV-J joints by secondary rearrangements indicates active receptor editing in CLL and provides further evidence for the role of antigen in CLL immunopathogenesis.