Extranodal Lymphoproliferative Processes and Flow Cytometry

Objective: Fine-needle aspiration (FNA) cytology is a safe and cost-effective technique for the diagnosis of lymphoproliferative processes, especially when correlated with clinical and imaging studies. However, cytology alone may be unable to detect a lymphoid neoplastic process, as architectural features are less obvious than in histologic preparations and, in certain cases, reactive processes may mimic lymphoma. Flow cytometry (FC) has been recognized as an important ancillary technique in the diagnosis of lymphoid neoplasms and it can be used in conjunction with FNA in the evaluation of lymphoproliferative processes. Study Design: We performed a review of the published literature concerning FC applied to the detection of salivary glands and thyroid lymphoproliferative processes, which are frequently related to autoimmune diseases and difficult to diagnose by cytomorphology alone. Results: FC is able to detect and subtype non-Hodgkin lymphomas and may contribute to the exclusion of a neoplastic process in cytologically unclear cases. Conclusions: FC can be successfully applied in the differential diagnosis of lymphoproliferative processes in the head and neck region. The FNA-FC combined approach can reduce time to therapy and may prevent unnecessary surgical biopsies

Cooperative Induction of a Tolerogenic Dendritic Cell Phenotype by Cytokines Secreted by Pancreatic Carcinoma Cells

AbstractAg presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123− myeloid DC (MDC)) or immunosuppressive T cell development (CD11c−,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-γ. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-β, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses

Follicular Origin of a Subset of CD5+ Diffuse Large B-Cell Lymphomas

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In vivo migration of labeled autologous natural killer cells to liver metastases in patients with colon carcinoma

BACKGROUND: Besides being the effectors of native anti-tumor cytotoxicity, NK cells participate in T-lymphocyte responses by promoting the maturation of dendritic cells (DC). Adherent NK (A-NK) cells constitute a subset of IL-2-stimulated NK cells which show increased expression of integrins and the ability to adhere to solid surface and to migrate, infiltrate, and destroy cancer. A critical issue in therapy of metastatic disease is the optimization of NK cell migration to tumor tissues and their persistence therein. This study compares localization to liver metastases of autologous A-NK cells administered via the systemic (intravenous, i.v.) versus locoregional (intraarterial, i.a.) routes. PATIENTS AND METHODS: A-NK cells expanded ex-vivo with IL-2 and labeled with (111)In-oxine were injected i.a. in the liver of three colon carcinoma patients. After 30 days, each patient had a new preparation of (111)In-A-NK cells injected i.v. Migration of these cells to various organs was evaluated by SPET and their differential localization to normal and neoplastic liver was demonstrated after i.v. injection of (99m)Tc-phytate. RESULTS: A-NK cells expressed a donor-dependent CD56(+)CD16(+)CD3(- )(NK) or CD56(+)CD16(+)CD3(+ )(NKT) phenotype. When injected i.v., these cells localized to the lung before being visible in the spleen and liver. By contrast, localization of i.a. injected A-NK cells was virtually confined to the spleen and liver. Binding of A-NK cells to liver neoplastic tissues was observed only after i.a. injections. CONCLUSION: This unique study design demonstrates that A-NK cells adoptively transferred to the liver via the intraarterial route have preferential access and substantial accumulation to the tumor site

Conjugated composite containing photochromic molecule: from preparation to potential application

Polímeros conjugados inseridos em filmes de biopolímeros e hidrogéis têm se mostrado de relevante importância devido às suas propriedades luminescentes, e, a introdução de moléculas fotocrômicas possibilita a formação de novos materiais. As interações intermoleculares entre estas duas espécies regem a eficiência destes materiais devido à ocorrência de processos fotofísicos. Neste sentido, compósitos contendo um polímero conjugado, o poli(3-hexiltiofeno-co-1,4-fenileno) (PTPh), e uma molécula fotocrômica, o 4-aminoazobenzeno (Azo), foram utilizadas nestes estudos. Utilizando como ferramenta a química quântica computacional (DFT), as interações e estruturas eletrônicas do sistema PTPh/Azo foram avaliadas, sugerindo que a interação C-H---π entre as moléculas de PTPh e Azo pode ser responsável pelo processo de transferência de energia que ocorre nestes materiais. Tendo estes resultados como suporte, filmes autossuportados de quitosana contendo PTPh e Azo foram estudados. A presença de fotocrômico nos filmes resultou na supressão do estado excitado singlete do polímero. Estes resultados sugerem a transferência de energia do PTPh para o Azo, e o alto valor da constante de supressão para o Azo indica a sensibilidade de PTPh à presença do fotocrômico. Além disso, os filmes se mostraram fotoestáveis, e a propriedade fotocrômica do Azo foi mantida. As interações intermoleculares e os processos fotofísicos também possuem um relevante papel na solubilidade de moléculas conjugadas. Assim, utilizando o oligômero oligo(3-hexiltiofeno-co-1,4-fenileno) (OTPh) como modelo, um estudo da solubilidade e estabilidade de moléculas conjugadas em soluções aquosas e hidrogéis foi desenvolvido envolvendo o sal biliar NaDC. A metodologia se mostrou eficiente para um polímero comercial (P3HT). Utilizando pireno como uma sonda fluorescente, foi possível caracterizar os microambientes presentes no novo sistema formado, mostrando que NaDC é capaz de solubilizar e estabilizar moléculas conjugadas em meio aquoso e hidrogel devido à formação dos agregados hidrofóbicos primários. Entretanto, nestes sistemas o Azo perdeu sua propriedade fotocrômica devido à limitada mobilidade. Desta maneira, o presente trabalho fornece estudos fundamentais e de grande relevância de materiais poliméricos luminescentes contendo molécula fotocrômica em diferentes meios e matrizes, para a formação de novos materiais para potenciais futuras aplicações em modulação de fluorescência, sensores e dispositivos optoeletrônicos.Conjugated polymers embedded in biopolymer films and hydrogels are promising materials due to their luminescent properties. The insertion of photochromic molecules enables the formation of new materials for several applications. The efficiency of these materials is due to intermolecular interactions, which lead to photophysics processes. Therefore, materials containing a conjugated polymer, the poly(3-hexylthiophene-co-1,4-phenylene) (PTPh), and a photochrome molecule, the 4-aminoazobenzene (Azo), were used in these studies. The interactions and electronic structures of the system PTPh/Azo were evaluated using computational quantum chemistry (DFT) as a tool. The results show that interaction C-H---π between PTPh and Azo might be responsible for the process of energy transfer in this material. Taking this result as support chitosan films containing PTPh and Azo was studied. The addition of the photochrome in the PTPh films resulted in quenching the polymer's excited singlet state. These results suggest the energy transfer from PTPh to Azo, and the significant value of the quenching constant for Azo indicates the sensibility of PTPh to the photochrome. In addition, a photostability of the films was observed, and the Azo molecule was able to photoisomerizate. Intermolecular interactions and photophysics also play an essential role in the solubilization of conjugated molecules. Thereby, using the oligomer oligo(3-hexylthiophene-co-1,4-phenylene) (OTPh) as a model, a study of the solubility and stability of conjugated molecules in aqueous solutions and hydrogels were developed by the use of the bile salt NaDC. The method was applied to a commercial polymer (P3HT), resulting in efficient solubilization. Using pyrene as a fluorescent probe it is possible to characterize the microenvironments in the OTPh/NaDC system. The studies showed that the capacity of NaDC to solubilize conjugated molecules in an aqueous medium is due to the formation of primary hydrophobic NaDC aggregates. However, Azo lost its photochromic property in these systems due to its limited mobility. Thus, this work provides fundamental and relevant studies of luminescent polymeric materials containing a photochromic molecule in different environments and matrices, leading to the formation of new materials for potential future applications in fluorescence modulation, sensors, and optoelectronic devices.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP: 2018/23524-9FAPESP: 2019/15498-

Sanavio F. Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colonystimulating factor

Abstract The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 Ag/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32±7 to 79±16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43±20 to 82±11%) and day 7 CFU-GM (from 41±14 to 56±20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26±14 to 41±6%, and that of erythroblasts increased from 25±12 to 30±12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B-cell chronic lymphoid leukaemias. Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/2 3 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 BCLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase. According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells