Targeting hodgkin and reed–sternberg cells with an inhibitor of heat-shock protein 90: Molecular pathways of response and potential mechanisms of resistance

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol’s toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.Fil: Segges, Priscilla. Instituto Nacional de Câncer; BrasilFil: Corrêa, Stephany. Instituto Nacional de Câncer; BrasilFil: Du Rocher, Bárbara. Fundación Oswaldo Cruz; Brasil. Instituto Nacional de Câncer; BrasilFil: Vera Lozada, Gabriela. Instituto Nacional de Câncer; BrasilFil: Krsticevic, Flavia Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas. Universidad Nacional de Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; ArgentinaFil: Arce, Debora Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Sternberg, Cinthya. Universidade Federal do Rio de Janeiro; BrasilFil: Abdelhay, Eliana. Instituto Nacional de Câncer; BrasilFil: Hassan, Rocio. Instituto Nacional de Câncer; Brasi

Revisiting the Tissue Microenvironment of Infectious Mononucleosis: Identification of EBV Infection in T Cells and Deep Characterization of Immune Profiles

To aid understanding of primary EBV infection, we have performed an in depth analysis of EBV-infected cells and of local immune cells in tonsils from infectious mononucleosis (IM) patients. We show that EBV is present in approximately 50% of B-cells showing heterogeneous patterns of latent viral gene expression probably reflecting different stages of infection. While the vast majority of EBV+ cells are B-cells, around 9% express T-cell antigens, with a predominance of CD8+ over CD4+ cells. PD-L1 was expressed by a median of 14% of EBV+ cells. The numbers of EBER+PD-L1+ cells were directly correlated with the numbers of EBER+CD3+ and EBER+CD8+ cells suggesting a possible role for PD-L1 in EBV infection of T-cells. The microenvironment of IM tonsils was characterized by a predominance of M1-polarized macrophages over M2-polarized cells. However, at the T-cell level, a heterogeneous picture emerged with numerous Th1/cytotoxic cells accompanied and sometimes outnumbered by Th2/regulatory T-cells. Further, we observed a direct correlation between the numbers of Th2-like cells and EBV– B-cells. Also, a prevalence of cytotoxic T-cells over Th2-like cells was associated with an increased viral load. These observations point to contribution of B- and Th2-like cells to the control of primary EBV infection. 35% of CD8+ cells were differentiated CD8+TBET+ cells, frequently detected in post-capillary venules. An inverse correlation was observed between the numbers of CD8+TBET+ cells and viral load suggesting a pivotal role for these cells in the control of primary EBV infection. Our results provide the basis for a better understanding of immune reactions in EBV-associated tumors

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

Macrophage polarization reflects T cell composition of tumor microenvironment in pediatric classical Hodgkin lymphoma and has impact on survival.

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients 1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL

Examples of immunostains used to identify M1-like and M2-like macrophages in classical Hodgkin lymphoma.

<p>Expression of CD68 or CD163 is indicated by blue cytoplasmic/membranous staining. The expression of transcription factors pSTAT1 and CMAF is indicated by brown nuclear staining. Examples of cases with high numbers of M2-like macrophages are shown in A, (CD68+CMAF+ macrophages) and in C, (CD163+CMAF+ macrophages). Examples of cases with large numbers of M1-like macrophages are shown in B, (CD68+pSTAT1+ macrophages) and in D (CD163+pSTAT1+ macrophages) (original magnification: 400x). The sections were not counterstained.</p

Description of macrophage subpopulations in the tumor microenvironment of pediatric classical Hodgkin lymphoma and their association with progression free survival and overall survival.

<p>PFS: progression free survival. OS: overall survival.</p><p>Description of macrophage subpopulations in the tumor microenvironment of pediatric classical Hodgkin lymphoma and their association with progression free survival and overall survival.</p

Box-plot graphs show the numerical distribution of CD68+pSTAT1+, CD68+pSTAT1-, CD68+CMAF+ and CD68+CMAF- cells/mm2 according to EBV-status (A) and Th-response group (B).

<p>The numerical distribution of CD163+pSTAT1+, CD163+pSTAT1-, CD163+CMAF+ and CD163+CMAF- cells/mm2 according to EBV-status and Th-response group is shown in C and D, respectively.</p

Kaplan-Meier curves in pediatric classical Hodgkin lymphoma, according to number of M1- or M2-like macrophages.

<p>A) Overall survival (OS) according to the numbers of CD68+CMAF+ macrophages. B) OS according to the numbers of CD163+pSTAT1+ macrophages. C) Progression-free survival (PFS) according to the numbers of CD163+CMAF+ macrophages. D) PFS according to the numbers of CD163+pSTAT1+ macrophages.</p

Pie charts showing the kind of macrophage polarization in pediatric classical Hodgkin lymphoma, considering CD68 (A) or CD163 (C) as macrophage markers.

<p>Box-plot graphs show the numerical distribution of CD68+pSTAT1+, CD68+pSTAT1-, CD68+CMAF+ and CD68+CMAF- cells/mm2 (B), as well as CD163+pSTAT1+, CD163+pSTAT1-, CD163+CMAF+ and CD163+CMAF- cells/mm2 (D).</p