Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

CD24 for Cardiovascular Researchers: A Key Molecule in Cardiac Immunology, Marker of Stem Cells and Target for Drug Development

The study of the membrane protein, CD24, and its emerging role in major disease processes, has made a huge leap forward in the past two decades. It appears to have various key roles in oncogenesis, tumor progression and metastasis, stem cell maintenance and immune modulation. First described in the 1980s as the homologous human protein to the mouse HSA (Heat Stable Antigen), it was reported as a surface marker in developing hematopoietic cell lines. The later discovery of its overexpression in a large number of human neoplasms, lead cancer researchers to discover its various active roles in critical checkpoints during cancer development and progression. Targeting CD24 in directed drug development showed promising results in cancer treatment. More recently, the chimeric CD24-Fc protein has shown exciting results in clinical trials as a specific modulator of auto-inflammatory syndromes. This report is aimed to summarize the relevant literature on CD24 and tie it together with recent advancements in cardiovascular research. We hypothesize that CD24 is a promising focus of research in the understanding of cardiovascular disease processes and the development of novel biological therapies.

Inhibitor of apoptosis, IAP, genes play a critical role in the survival of HIV-infected macrophages

Latent viral reservoirs of HIV-1 that persist despite antiretroviral therapy (ART) are major barriers for a successful cure. Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC)-mimetics (SM) specifically induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, and GFP-expressing HIV, chronically infected U1 cells, and ex-vivo derived MDMs from naïve and ART-treated HIV patients. SM-induced cell death was found to be mediated by IAPs using IAP siRNAs, was independent of endogenously produced TNFα and was attributed to the concomitant downregulation of IAP-1/2 and the receptor interacting protein kinase-1 degradation following HIV infection. Altogether, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Significance of CD44 and CD24 as Cancer Stem Cell Markers: An Enduring Ambiguity

Cancer stem cell population is a subset of cells capable of dictating invasion, metastasis, heterogeneity, and therapeutic resistance in tumours. Eradication of this rare population is a new insight in cancer treatment. However, prospective identification, characterization, and isolation of these CSCs have been a major challenge. Many studies were performed on surface markers for potential identification and isolation of CSCs. Lack of universal expression of surface markers limits their usage and no best combination of markers has yet been confirmed to identify CSCs capable of initiating and metastasizing tumours. CD44, a hyaluronic acid receptor, is one of the most commonly studied surface markers, which is expressed by almost every tumour cell. CD24, a heat stable antigen, is another surface marker expressed in many tumour types. However, their expression and prognostic value in isolating CSCs are still an enduring ambiguity. In this critical review, we assess the role of CD44 and CD24 in tumour initiation, development, and metastasis. We mainly focus on analysing the significance of CD44 and CD24 as CSC surface markers in combination or with other putative markers in different types of cancer.

Characterisation of antibodies for the immunochemical detection of Enterobacter sakazakii

An indirect competitive enzyme immunoassay of Enterobacter sakazakii has been developed. The rabbit polyclonal antibodies to heat-labile or heat-stable antigen of the type strain E. sakazakii CNCTC 5739^T were prepared for these purposes. The detection limits of enzyme immunoassays were within the range 0.6–14.4 × 10⁵ cells/ml. Antibodies raised to heat-labile antigen were serotype-specific. Although they contain non-specific IgG fractions binding periplasmatic and cytosol proteins, the interactions of these immunoglobulins are not manifested under conditions of ELISA developed.

Characterization of the early stages of thymic NKT cell development

Upon reaching the mature heat stable antigen (HSA)low thymic developmental stage, CD1d-restricted Vα14-Jα18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-γ–producing NKT phenotype. However, their more immature HSAhigh precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4+CD8+ double-positive (DP) stage, and when the CD4+ and CD4−CD8− double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSAhigh precursors in thymuses of newborn mice, including an elusive DPlow stage and a CD4+ stage, which were present at a frequency of ∼10−6. These HSAhigh DP and CD4+ stages appeared to be nondividing, and already exhibited the same Vβ8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN γδ T cells, the DN NKT cells did not originate from a pTα-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSAlow NK1.1neg CD4 cells. These findings identify the HSAhigh CD4+ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.

Characterization and Functional Analysis of a Murine Model for Chronic Myeloid Leukemia Stem Cells.

The purpose of this study was to characterize the phenotype and analyze the functional characteristics of CML stem cells (CML-SCs) in a mouse model system. A retrovirus transduction model was employed using a vector expressing p210/BCR/ABL and the green fluorescent protein (GFP). Transplantation studies of BCR/ABL infected cells in recipient mice demonstrated an aggressive myeloproliferative disease within approximately 3 weeks, as has previously been observed with this approach. Phenotypic analysis of bone marrow from BCR/ABL mice showed contribution by the transduced cells to multiple lineages, suggesting a primitive stem/progenitor cell gives rise to the disease. Subsequent analysis of primary cells that arise in vivo showed the leukemia-initiating activity resides in cells that are Sca-1+/c-kit+/Lin-. Cell cycle analyses (as detected by BrdU incorporation) of the Lin−/c-kit+/Sca-1+ compartment of BCR/ABL mice demonstrated an enhanced cycle rate (approximately 2-fold) in the leukemic population, signifying a possible increase in the self-renewal or proliferative properties of this primitive compartment that could lead to development/progression of the disease. Further, comparison of the BCR/ABL mice and GFP control mice revealed preferential over-expression of CD24 (heat-stable antigen) in the primitive leukemic cells (90% vs. 52% in normal counterparts) demonstrating a possible BCR/ABL induced activation of the antigen. Sorting and serial transplantation studies of CD24+ vs CD24− cells from BCR/ABL mice showed that the leukemia-initiating activity resides in the CD24+/Lin− population. Collectively, these data indicate that BCR/ABL expression increases proliferation of primitive CML cells and up-regulates the CD24 antigen. We suggest that the CD24 antigen represents a potential marker to distinguish normal from CML stem cells and that it may confer specific functional properties on the CML population.

CD24 Controls Expansion and Persistence of Autoreactive T Cells in the Central Nervous System during Experimental Autoimmune Encephalomyelitis

In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.