Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.

AIMS: The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD. METHODS AND RESULTS: We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signalling, facilitates autophagy, and acts as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression. CONCLUSION: Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.

Immunodeficiency-associated lymphoproliferative disorders and lymphoid neoplasms in post-COVID-19 pandemic era.

The 2017 revision of WHO Classification of tumors of hematopoietic and lymphoid tissues contains separate chapters on the immunodeficiency-associated lymphoproliferative disorders. In this mini-review, the brief description of pathological, immunophenotypical and clinical features of lymphoid neoplasms associated with primary immune disorders, HIV infection, those arising in post-transplant setting and other lymphoproliferative disorders (excluding those induced by radiation) is given. The heterogeneous spectrum of these lymphoid malignancies is specified by the nature of those factors that are capable to induce immune suppression or chronic antigenic stimulation of immune system. Taking into account the full swing of SARS-CoV-2 pandemic and our ignorance of the ability of this virus to induce the sustained stimulation of immune system, we could not exclude the high risk of autoimmune diseases and lymphoid neoplasms in the long-term post-pandemic period. In this context, the role of angiotensin-converting enzyme 2  as well as some recently reported cell receptors for SARS-CoV-2 cell entry should be considered as far as some of them (CD147, CD26) could be tumor-associated antigens.

NG2 glia regulate brain innate immunity via TGF-ß2/TGFBR2 axis.

BACKGROUND: Brain innate immunity is vital for maintaining normal brain functions. Immune homeostatic imbalances play pivotal roles in the pathogenesis of neurological diseases including Parkinson's disease (PD). However, the molecular and cellular mechanisms underlying the regulation of brain innate immunity and their significance in PD pathogenesis are still largely unknown. METHODS: Cre-inducible diphtheria toxin receptor (iDTR) and diphtheria toxin-mediated cell ablation was performed to investigate the impact of neuron-glial antigen 2 (NG2) glia on the brain innate immunity. RNA sequencing analysis was carried out to identify differentially expressed genes in mouse brain with ablated NG2 glia and lipopolysaccharide (LPS) challenge. Neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice were used to evaluate neuroinflammatory response in the presence or absence of NG2 glia. The survival of dopaminergic neurons or glial cell activation was evaluated by immunohistochemistry. Co-cultures of NG2 glia and microglia were used to examine the influence of NG2 glia to microglial activation. RESULTS: We show that NG2 glia are required for the maintenance of immune homeostasis in the brain via transforming growth factor-ß2 (TGF-ß2)-TGF-ß type II receptor (TGFBR2)-CX3C chemokine receptor 1 (CX3CR1) signaling, which suppresses the activation of microglia. We demonstrate that mice with ablated NG2 glia display a profound downregulation of the expression of microglia-specific signature genes and remarkable inflammatory response in the brain following exposure to endotoxin lipopolysaccharides. Gain- or loss-of-function studies show that NG2 glia-derived TGF-ß2 and its receptor TGFBR2 in microglia are key regulators of the CX3CR1-modulated immune response. Furthermore, deficiency of NG2 glia contributes to neuroinflammation and nigral dopaminergic neuron loss in MPTP-induced mouse PD model. CONCLUSIONS: These findings suggest that NG2 glia play a critical role in modulation of neuroinflammation and provide a compelling rationale for the development of new therapeutics for neurological disorders.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential. Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

Development and Organization of the Secondary and Tertiary Lymphoid Organs: Influence of Microbial and Food Antigens.

BACKGROUND: Secondary lymphoid organs (SLO) are distributed in many districts of the body and, especially, lymph nodes, spleen and gut-associated lymphoid tissue are the main cellular sites. On the other hand, tertiary lymphoid organs (TLO) are formed in response to inflammatory, infectious, autoimmune and neoplastic events. Developmental Studies: In the present review, emphasis will be placed on the developmental differences of SLO and TLO between small intestine and colon and on the role played by various chemokines and cell receptors. Undoubtedly, microbiota is indispensable for the formation of SLO and its absence leads to their poor formation, thus indicating its strict interaction with immune and non immune host cells. Furthermore, food antigens (for example, tryptophan derivatives, flavonoids and byphenils) bind the aryl hydrocarbon receptor on innate lymphoid cells (ILCs), thus promoting the development of postnatal lymphoid tissues. Also retinoic acid, a metabolite of vitamin A, contributes to SLO development during embryogenesis. Vitamin A deficiency seems to account for reduction of ILCs and scarce formation of solitary lymphoid tissue. Translational Studies: The role of lymphoid organs with special reference to intestinal TLO in the course of experimental and human disease will also be discussed. Future Perspectives: Finally, a new methodology, the so-called "gut-in-a dish", which has facilitated the in vitro interaction study between microbe and intestinal immune cells, will be described.

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression.

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.

Distinct NG2 proteoglycan-dependent roles of resident microglia and bone marrow-derived macrophages during myelin damage and repair.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow.

Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation.

In Vivo Reprogramming of Striatal NG2 Glia into Functional Neurons that Integrate into Local Host Circuitry.

The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain.

Chondroitin sulfate proteoglycan-4 does not protect melanoma cells during inhibition of PI3K and mTOR pathways.

BACKGROUND: Chondroitin sulfate proteoglycan-4 (CSPG4) is commonly expressed in melanoma cells and induces melanoma cell proliferation and migration by enhancement of activation of the extracellular signal-regulated kinase 1, 2 (ERK1,2) pathway. The phosphoinositide 3-kinase (PI3K) -protein kinase B (AKT) and mammalian target of rapamycin (mTOR) pathways are also frequently de-regulated in melanoma. We hypothesized that CSPG4, by sustained activation of PI3K, may reduce the effect of the dual inhibition of PI3K-AKT and mTOR pathways. MATERIALS AND METHODS: CSPG4-negative melanoma cell line WM1552C was transfected with CSPG4 and CSPG4 lacking cytoplasmic domain (melanoma-associated chondroitin sulfate proteoglycan (MCSP)ΔCD). To assess the effect of CSPG4 on the mTOR pathway, PF-5212384, a dual PI3K/mTOR inhibitor was used. Cell proliferation and downstream signaling from mTOR was assayed in the presence of CSPG4. RESULTS: Forced CSPG4 expression did not provide any protection to melanoma cells from the pharmacological inhibition of mTOR pathway in vitro. In addition, we demonstrated that inhibition of signaling molecules downstream of AKT and mTOR was not diminished in the presence of CSPG4 when the cells were treated with the PI3K/mTOR inhibitor. CONCLUSION: CSPG4 expression does not have any impact on survival and signaling activity of melanoma cells during PI3K/mTOR inhibition.

Human antigen R mediated post-transcriptional regulation of epithelial-mesenchymal transition related genes in diabetic nephropathy.

BACKGROUND: Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein that modulates gene expression at the post-transcriptional level. While cytoplasmic HuR expression was identified as a marker in epithelial-mesenchymal transition (EMT) process of several types of cancer, its role in diabetic nephropathy (DN) remains unclear. METHODS: Renal biopsies from Type 2 diabetic patients and STZ-induced DN rats were stained for HuR and EMT markers. Redistribution of HuR was detected by immunostaining and western blot in high glucose stimulated cells. RNAi was used to supress HuR expression. The binding affinity for EMT-related genes was evaluated by immunoprecipitation. RESULTS: Cytoplasmic HuR expression was elevated in human and rat DN specimens along with EMT changes compared to normal controls. HuR shuttling between nucleus and cytoplasm facilitated epithelial to mesenchymal transition in renal epithelial cells. The suppression of HuR partially inhibited EMT of high glucose stimulated HK-2 cells. Furthermore, HuR bound to 3'-UTRs of critical cytokines or transcription factors mRNA involved in EMT process. CONCLUSION: Acquired phenotypic traits of EMT were partially through the enhanced HuR-binding proteins and its post-transcriptional regulation role in DN.

Molecular determinants of magnesium-dependent synaptic plasticity at electrical synapses formed by connexin36.

Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bidirectionally modulated by changes in intracellular free magnesium concentration ([Mg(2+)]i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg(2+)-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg(2+)-sensitivity. Single-channel analysis of Mg(2+)-sensitive chimeras and mutants reveals that [Mg(2+)]i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg(2+)]i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg(2+)-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg(2+)]i.

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function.

INTRODUCTION: Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT). METHODS AND RESULTS: Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast. DISCUSSION AND CONCLUSION: This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.

NG2 regulates directional migration of oligodendrocyte precursor cells via Rho GTPases and polarity complex proteins.

The transmembrane proteoglycan NG2 is expressed by oligodendrocyte precursor cells (OPC), which migrate to axons during developmental myelination and remyelinate in the adult after migration to injured sites. Highly invasive glial tumors also express NG2. Despite the fact that NG2 has been implicated in control of OPC migration, its mode of action remains unknown. Here, we show in vitro and in vivo that NG2 controls migration of OPC through the regulation of cell polarity. In stab wounds in adult mice we show that NG2 controls orientation of OPC toward the wound. NG2 stimulates RhoA activity at the cell periphery via the MUPP1/Syx1 signaling pathway, which favors the bipolar shape of migrating OPC and thus directional migration. Upon phosphorylation of Thr-2256, downstream signaling of NG2 switches from RhoA to Rac stimulation. This triggers process outgrowth through regulators of front-rear polarity and we show using a phospho-mimetic form of NG2 that indeed NG2 recruits proteins of the CRB and the PAR polarity complexes to stimulate Rac activity via the GEF Tiam1. Our findings demonstrate that NG2 is a core organizer of Rho GTPase activity and localization in the cell, which controls OPC polarity and directional migration. This work also reveals CRB and PAR polarity complexes as new effectors of NG2 signaling in the establishment of front-rear polarity.

The hematopoietic stem cell regulatory gene latexin has tumor-suppressive properties in malignant melanoma.

Despite recent advancements in therapy, melanoma remains a highly lethal skin cancer. A better understanding of the genetic and epigenetic changes responsible for melanoma formation and progression could result in the development of more effective treatments. Advanced melanomas are known to exhibit widespread promoter region CpG island methylation leading to the inactivation of key tumor suppressor genes. Meta-analyses of relevant microarray data sets revealed the hematopoietic stem cell regulator gene latexin (LXN) to be commonly downregulated in approximately 50% of melanomas. The CpG island in the promoter region of LXN was almost universally hypermethylated in melanoma cell lines and tumors, and treatment of the cell lines with the demethylating drug 5-aza-2'-deoxycytidine resulted in increased LXN expression. In this paper, we demonstrate that the exogenous expression of LXN in melanoma cell lines results in a significant inhibition of tumor cell proliferation. In addition, we show that the increased expression of LXN in these lines correlates with reduction in the expression levels of stem cell transcription factors OCT4, NANOG, SOX2, KLF4, and MYCN, indicating that LXN may exert its tumor-suppressive function by altering the stem cell-like properties of melanoma cells.

Skeletal muscle neural progenitor cells exhibit properties of NG2-glia.

Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.

CD8 T cell priming in the presence of IFN-α renders CTLs with improved responsiveness to homeostatic cytokines and recall antigens: important traits for adoptive T cell therapy.

Previous mouse and human studies have demonstrated that direct IFN-α/ß signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α-primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15(-/-) and IL-7(-/-) recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2(+) CD8 T cells with autologous dendritic cells loaded with MART1(26-35) peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.

The endothelial antigen ESAM monitors hematopoietic stem cell status between quiescence and self-renewal.

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.

What shall we do with the damaged proteins in lung disease? Ask the proteasome!

The proteasome constitutes the main protein waste disposal and recycling system of the cell. Together with endoplasmic reticulum stress and the autophagosome pathway, it takes centre stage in cellular protein quality control. In lung research, the proteasome is, first of all, a promising therapeutic target to intervene in the malignant growth of lung cancer cells. Therapeutic targeting of the proteasome has also been extended to pulmonary fibrosis and asthma using animal models. Moreover, the proteasome is involved in lung pathogenesis. In cystic fibrosis, rapid proteasomal degradation of mutant cystic fibrosis transmembrane conductance regulator contributes to loss of function of lung epithelial cells. In chronic obstructive pulmonary disease (COPD), pulmonary proteasome expression and activity are downregulated and inversely correlate with lung function. In addition, as the proteasome degrades signalling mediators that have been oxidatively modified in COPD, it contributes to further compromise cellular function. The consequences of proteasomal dysfunction are loss of protein quality control, accumulation of misfolded proteins and exacerbation of cellular stress, which are also hallmarks of protein quality diseases and premature ageing. This suggests that proteasome dysfunction can be regarded as a new pathomechanism for chronic lung diseases, awaiting further therapeutic exploration in the future.

Cancer/testis antigens are novel targets of immunotherapy for adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an intractable hematologic malignancy caused by human T-lymphotropic virus type 1 (HTLV-1), which infects approximately 20 million people worldwide. Here, we have explored the possible expression of cancer/testis (CT) antigens by ATLL cells, as CT antigens are widely recognized as ideal targets of cancer immunotherapy against solid tumors. A high percentage (87.7%) of ATLL cases (n = 57) expressed CT antigens at the mRNA level: NY-ESO-1 (61.4%), MAGE-A3 (31.6%), and MAGE-A4 (61.4%). CT antigen expression was confirmed by immunohistochemistry. This contrasts with other types of lymphoma or leukemia, which scarcely express these CT antigens. Humoral immune responses, particularly against NY-ESO-1, were detected in 11.6% (5 of 43) and NY-ESO-1-specific CD8(+) T-cell responses were observed in 55.6% (5 of 9) of ATLL patients. NY-ESO-1-specific CD8(+) T cells recognized autologous ATLL cells and produced effector cytokines. Thus, ATLL cells characteristically express CT antigens and therefore vaccination with CT antigens can be an effective immunotherapy of ATLL.