Application of the crypt isolation technique to the assessment of genetic alterations of colorectal carcinomas

The crypt isolation technique (CIT) allows for the isolation of pure tumor crypts from colon tumor tissue. In a previous study we reported on the genetic alterations found in colorectal tumor crypts using the CIT; however, a direct comparison of the genetic alterations found in colorectal carcinomas using either conventional methods (CM) or the CIT has not previously been performed. Here, we analyzed the impact of this method on the genetic analysis of colon tumor cells by comparing the observed frequency of genetic alterations in colon tumors isolated using CM or the CIT. We used a combination of the CIT and the fluorescent polymerase chain reaction assay to accurately assess the incidence of allelic imbalances (AI) at a number of chromosomal loci (17p, 5q, 18q, 1p, 8p, 22q), microsatellite instability (MSI), and mutations of cancer-related genes (p53 and APC genes) in 48 sporadic colorectal carcinomas. In addition, genetic alterations seen in multiploid tumors (defined as tumors with both diploid and aneuploid cell populations) identified by the CIT were also examined. The incidence of AI at the chromosomal loci tested was more frequently detected in samples isolated from tumors using the CIT than in those isolated from the same tumors using CM. In contrast, we observed no differences in the frequency of MSI or cancer-related gene mutation between the two groups. Although there was no difference in the frequency of genetic alterations between tumors with evidence of multiploidy, sorting of diploid and aneuploid populations allowed detection of distinct genetic changes. The crypt isolation method thus appears to be useful in that it allows purification of tumor cells and the accurate assessment of their genetic alterations. In addition, it may also be of benefit in clarifying the genetic profile of multiploid tumor cell populations.     

Class II positive human dermal fibroblasts restimulate cloned allospecific T cells but fail to stimulate primary allogeneic lymphoproliferation

Recently, a number of laboratories have shown that gamma interferon (IFN-gamma) is a potent modulator of HLA class II antigen expression in a variety of cell types ranging from classical antigen presenting cells to those not expected to participate in physiological antigen presentation such as fibroblasts. In order to examine the role of HLA class II expressing fibroblasts in antigen presentation, we established dermal fibroblast (FIB) strains from five HLA typed donors. After optimal preculture with IFN-gamma, class II positive FIB were fully competent to restimulate proliferative responses of two DR specific T cell clones and one DP specific T cell line. However, they failed to elicit strong primary allogeneic proliferation from fully DR mismatched fresh PBMC. This failure was not due to a direct suppressive effect of FIB and could not be corrected by exogenous IL1 or by factors contained in conventional mixed leukocyte culture supernatants.     

非小细胞肺癌组织中抑癌基因APC表达的研究

目的探讨非小细胞肺癌组织中APC基因mRNA转录水平。方法采用RT-PCR技术,以SybrgreenⅠ为荧光染料,beta-actin基因为内参照,对17例临床确诊的非小细胞肺癌病例的正常肺组织和肿瘤组织的APC基因mRNA进行实时荧光定量检测。计算每份标本Ct(APC)/Ct(beta-actin)比值,比较每份病例正常肺组织和肿瘤组织比值之间的差值(d),配对t检验。结果在17例病例中,APCmRNA在肺癌组织中的表达较正常肺组织明显降低(d=-0.29,P<0.05)。结论肺癌患者正常肺组织和肿瘤组织APC基因转录存在明显差异,肺癌组织APC基因转录水平降低。     

Adoptive cell therapy in multiple Myeloma

Introduction: Recent breakthrough advances in Multiple Myeloma (MM) immunotherapy have been achieved with the approval of the first two monoclonal antibodies, elotuzumab and daratumumab. Adoptive cell therapy (ACT) represents yet another, maybe the most powerful modality of immunotherapy, in which allogeneic or autologous effector cells are expanded and activated ex vivo followed by their re-infusion back into patients. Infused effector cells belong to two categories: naturally occurring, non-engineered cells (donor lymphocyte infusion, myeloma infiltrating lymphocytes, deltagamma T cells) or genetically- engineered antigen-specific cells (chimeric antigen receptor T or NK cells, TCR-engineered cells).

Areas covered: This review article summarizes our up-to-date knowledge on ACT in MM, its promises, and upcoming strategies to both overcome its toxicity and to integrate it into future treatment paradigms.

Expert opinion: Early results of clinical studies using CAR T cells or TCR- engineered T cells in relapsed and refractory MM are particularly exciting, indicating the potential of long-term disease control or even cure. Despite several caveats including toxicity, costs and restricted availability in particular, these forms of immunotherapy are likely to once more revolutionize MM therapy.     

Activated plasmacytoid dendritic cells regulate type 2 innate lymphoid cell–mediated airway hyperreactivity

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Glia–neuron interactions in the mammalian retina

The mammalian retina provides an excellent opportunity to study glia–neuron interactions and the interactions of glia with blood vessels. Three main types of glial cells are found in the mammalian retina that serve to maintain retinal homeostasis: astrocytes, Müller cells and resident microglia. Müller cells, astrocytes and microglia not only provide structural support but they are also involved in metabolism, the phagocytosis of neuronal debris, the release of certain transmitters and trophic factors and K⁺ uptake. Astrocytes are mostly located in the nerve fibre layer and they accompany the blood vessels in the inner nuclear layer. Indeed, like Müller cells, astrocytic processes cover the blood vessels forming the retinal blood barrier and they fulfil a significant role in ion homeostasis. Among other activities, microglia can be stimulated to fulfil a macrophage function, as well as to interact with other glial cells and neurons by secreting growth factors. This review summarizes the main functional relationships between retinal glial cells and neurons, presenting a general picture of the retina recently modified based on experimental observations. The preferential involvement of the distinct glia cells in terms of the activity in the retina is discussed, for example, while Müller cells may serve as progenitors of retinal neurons, astrocytes and microglia are responsible for synaptic pruning. Since different types of glia participate together in certain activities in the retina, it is imperative to explore the order of redundancy and to explore the heterogeneity among these cells. Recent studies revealed the association of glia cell heterogeneity with specific functions. Finally, the neuroprotective effects of glia on photoreceptors and ganglion cells under normal and adverse conditions will also be explored.     

Role of α-glucan-induced oxygen species in dendritic cells and its impact in immune response against tuberculosis

With 10 million new cases and three million deaths estimated to occur yearly ? more than any time in history ? tuberculosis (TB) remains the single most widespread and deadly infectious disease. Until recently, it was thought that both latent and active TB was primarily related to host factors. Nonetheless, the participation of bacterial factors is becoming increasingly evident. Minimal variations in genes related to Mycobacterium tuberculosis (Mtb) virulence and pathogenesis can lead to marked differences in immunogenicity. Dendritic cells (DC) are professional antigen presenting cells whose maturation can vary depending on the cell wall composition of each particular Mtb strain being critical for the onset of the immune response against Mtb. Here we evaluated the role played by α-glucan, in the endogenous production of reactive oxygen species, ROS, and the impact on DC maturation and function. Results showed that α-glucans on Mtb induce ROS production leading to DC maturation and lymphocyte proliferation. Even more, α-glucans induced Syk activation but were not essential in non-opsonized phagocytosis. In summary, α-glucans of Mtb participates in ROS production and the subsequent DC maturation and antigen presentation, suggesting a relevant role of α-glucans for the onset of the protective immune response against TB.     

PHA eludes macrophage suppression to activate CD8+ T cells

Tumors may include a high proportion of immune modulatory cells and molecules that restrain the anti-cancer response. Activation of T cells to eliminate cancer cells within the immune-suppressive tumor microenvironment remains a challenge. We have shown that C57BL/6?J peritoneal cell culture models features of macrophage-dense tumors as TCR ligation fails to activate T cells unless IFNγ is neutralized or iNOS is inhibited. We tested other forms of T cell activation and found phytohemagglutinin (PHA) distinctive in the ability to markedly expand CD8 T cells in this model. IFNγ or iNOS inhibition was not necessary for this response. PHA triggered less IFNγ production and inhibitory PD-L1 expression than TCR ligation. Macrophages and CD44^hi T cells bound PHA. Spleen T cell responses to PHA were markedly enhanced by the addition of peritoneal cells revealing that macrophages enhance T cell expansion. That PHA increases CD8 T cell responses within macrophage-dense culture suggests this mitogen might enhance anti-tumor immunity.     

Role of membrane environment and membrane-spanning protein regions in assembly and function of the Class II Major Histocompatibility complex

Class II Major Histocompatibility complex (MHC-II) is a polymorphic heterodimer that binds antigen-derived peptides and presents them on the surface of antigen presenting cells. This mechanism of antigen presentation leads to recognition by CD4 T-cells and T-cell activation, making it a critical element of adaptive immune response. For this reason, the structural determinants of MHC-II function have been of great interest for the past 30 years, resulting in a robust structural understanding of the extracellular regions of the complex. However, the membrane-localized regions have also been strongly implicated in protein-protein and protein-lipid interactions that facilitate Class II assembly, transport and function, and it is these regions that are the focus of this review. Here we describe studies that reveal the strong and selective interactions between the transmembrane domains of the MHC α, and invariant chains which, when altered, have broad reaching impacts on antigen presentation and Class II function. We also summarize work that clearly demonstrates the link between membrane lipid composition (particularly the presence of cholesterol) and MHC-II conformation, subsequent peptide binding, and downstream T-cell activation. We have integrated these studies into a comprehensive view of Class II transmembrane domain biology.