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Abstract DUSP22, an atypical dual-specificity phosphatase enzyme, plays a significant role in regulating multiple kinase signaling pathways by dephosphorylation. Our study demonstrated that decreased DUSP22 expression is associated with shorter disease-free survival, advanced TNM (tumor, lymph nodes, and metastasis), cancer stage, and higher tumor grade in lung adenocarcinoma (LUAD) patients. Exogenous DUSP22 expression reduces the colony-forming capacity of lung cancer cells and inhibits xenograft tumor growth primarily by targeting EGFR and suppressing its activity through dephosphorylation. Knockdown of DUSP22 using shRNA enhances EGFR dependency in HCC827 lung cancer cells and increases sensitivity to gefitinib, an EGFR inhibitor. Consistently, genetic deletion of DUSP22 enhances EGFRdel (exon 19 deletion)-driven lung tumorigenesis and elevates EGFR activity. Pharmacological inhibition of DUSP22 activates EGFR, ERK1/2, and upregulates downstream PD-L1 expression. Additionally, lentiviral deletion of DUSP22 by shRNA enhances lung cancer cell migration through EGFR/c-Met and PD-L1-dependent pathways. Gefitinib, an EGFR inhibitor, mechanistically suppresses migration induced by DUSP22 deletion and inhibits c-Met activity. Furthermore, cabozantinib, a c-Met inhibitor, reduces migration and attenuates EGFR activation caused by DUSP22 deletion. Collectively, our findings support the hypothesis that loss of DUSP22 function in lung cancer cells confers a survival advantage by augmenting EGFR signaling, leading to increased activation of downstream c-Met, ERK1/2, and PD-L1 axis, ultimately contributing to the progression of advanced lung cancer.

Summary: Phosphatase and tensin homolog (PTEN) is vital for B cell development, acting as a key negative regulator in the PI3K signaling pathway. We used CD23-cre to generate PTEN-conditional knockout mice (CD23-cKO) to examine the impact of PTEN mutation on peripheral B cells. Unlike mb1-cre-mediated PTEN deletion in early B cells, CD23-cKO mutants exhibited systemic inflammation with increased IL-6 production in mature B cells upon CpG stimulation. Inflammatory B cells in CD23-cKO mice showed elevated phosphatidylinositol 3-phosphate [PI(3)P] levels and increased TLR9 endosomal localization. Pharmacological inhibition of PI(3)P synthesis markedly reduced TLR9-mediated IL-6. Single-cell RNA-sequencing (RNA-seq) revealed altered endocytosis, BANK1, and NF-κB1 expression in PTEN-deficient B cells. Ectopic B cell receptor (BCR) expression on non-inflammatory mb1-cKO B cells restored BANK1 and NF-κB1 expression, enhancing TLR9-mediated IL-6 production. Our study highlights PTEN as a crucial inflammatory checkpoint, regulating TLR9/IL-6 axis by fine-tuning PI(3)P homeostasis. Additionally, BCR downregulation prevents the differentiation of inflammatory B cells in PTEN deficiency.

ABSTRACTVaccination plays a key role in preventing morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to evaluate the safety and immunogenicity of a SARS-CoV-2 messenger ribonucleic acid (mRNA) vaccine SYS6006. In the two randomized, observer-blinded, placebo-controlled phase 1 trials, 40 adult participants aged 18–59 years and 40 elderly participants aged 60 years or more were randomized to receive two doses of SYS6006 or placebo (saline). Adverse events (AEs) were collected through 30 days post the second vaccination. Immunogenicity was assessed by live-virus neutralizing antibody (Nab), spike protein (S1) binding antibody (S1-IgG), and cellular immunity. The result showed that 7/15, 9/15 and 4/10 adult participants, and 9/15, 8/15 and 4/10 elderly participants reported at least one AE in the 20-µg, 30-µg and placebo groups, respectively. Most AEs were grade 1. Injection-site pain was the most common AE. Two adults and one elder reported fever. No vaccination-related serious AE was reported. SYS6006 elicited wild-type Nab response with a peak geometric mean titer of 232.1 and 130.6 (adults), and 48.7 and 66.7 (elders), in the 20-µg and 30-µg groups, respectively. SYS6006 induced moderate-to-robust Nab response against Delta, and slight Nab response against Omicron BA.2 and BA.5. Robust IgG response against wild type and BA.2 was observed. Cellular immune response was induced. In conclusion, two-dose primary vaccination with SYS6006 demonstrated good safety and immunogenicity during a follow-up period of 51 days in immunologically naive population aged 18 years or more. (Trial registry: Chictr.org.cn ChiCTR2200059103 and ChiCTR2200059104)

Abstract Multipotent human mesenchymal stromal cells (MSCs) from multiple organs including the bone marrow (BM) and placenta harbor clinically relevant immunomodulation best demonstrated toward T lymphocytes. Surprisingly, there is limited knowledge on interactions with B lymphocytes, which originate from the BM where there is a resident MSC. With increasing data demonstrating MSC tissue‐specific propensities impacting therapeutic outcome, we therefore investigated the interactions of BM‐MSCs—its resident and “niche” MSC—and placental MSCs (P‐MSCs), another source of MSCs with well‐characterized immunomodulatory properties, on the global functional outcomes of pan‐peripheral B cell populations. We found that P‐MSCs but not BM‐MSCs significantly inhibit proliferation and further differentiation of stimulated human peripheral B populations in vitro. Moreover, although BM‐MSCs preserve multiple IL‐10‐producing regulatory B cell (Breg) subsets, P‐MSCs significantly increase all subsets. To corroborate these in vitro findings in vivo, we used a mouse model of B‐cell activation and found that adoptive transfer of P‐MSCs but not BM‐MSCs significantly decreased activated B220+ B cells. Moreover, adoptive transfer of P‐MSCs but not BM‐MSCs significantly decreased the overall B220+ B‐cell proliferation and further differentiation, similar to the in vitro findings. P‐MSCs also increased two populations of IL‐10‐producing murine Bregs more strongly than BM‐MSCs. Transcriptome analyses demonstrated multifactorial differences between BM‐ and P‐MSCs in the profile of relevant factors involved in B lymphocyte proliferation and differentiation. Our results highlight the divergent outcomes of tissue‐specific MSCs interactions with peripheral B cells, and demonstrate the importance of understanding tissue‐specific differences to achieve more efficacious outcome with MSC therapy.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Abstract Aims We collected the different prescription patterns of diabetes medications in a cohort of patients with heart failure with reduced ejection fraction (HFrEF) and analysed the impact of different prescription patterns on clinical outcomes. Methods and results Consecutive diabetic patients with HFrEF from a heart failure referral centre were retrospectively analysed between 2015 and 2016. Exclusion criteria include being lost to follow‐up, not receiving diabetes medications, and having severe renal impairment with a glomerular filtration rate < 30 mL/min/1.73 m2. Prescription of diabetes medications and the respective clinical outcomes were collected between 2016 and 2018. Among 381 patients (mean age, 64.8 ± 12.8 years; 71.9% male; mean left ventricular ejection fraction, 27.6 ± 7.0%; mean body mass index, 26.1 ± 4.7 kg/m2), the prescription rates of sodium‐glucose co‐transporter 2 inhibitor (SGLT2i) increased from 10.3% in 2016 to 17.6% in 2017 and 26.5% in 2018 (P < 0.001); the prescription rates of metformin, sulfonylurea, insulin, and dipeptidyl peptidase‐4 inhibitors did not change significantly over time. The prescription rates of metformin and SGLT2i were significantly higher in patients managed by cardiologists than non‐cardiologists (in 2018, 71.1% vs. 44.2% for metformin, 45.4% vs. 9.9% for SGLT2i, both P < 0.001). During the study period, annualized event rates of cardiovascular death or first unplanned HF hospitalization were 19.0 per 100 patient‐years. After a multivariate analysis, prescriptions of metformin {odds ratio (OR): 0.49 [95% confidence interval (CI) 0.27–0.51], P < 0.001} and SGLT2i [OR: 0.52 (95% CI 0.28–0.98), P = 0.042] were independently associated with lower annualized event rates of cardiovascular death or unplanned HF hospitalization. Conclusions Prescription patterns of diabetes medications in diabetics with HFrEF were diverse among different specialists. Prescriptions of metformin and SGLT2i were associated with favourable clinical outcomes. Our finding indicates the importance of awareness of beneficial effect of different classes of diabetes medications and collaboration between specialists in the management of diabetic HFrEF patients.

Indoleamine 2,3-dioxygenase (IDO) is one of the initial rate-limiting enzymes of the kynurenine pathway (KP), which causes immune suppression and induction of T cell anergy. It is associated with the imbalance of immune homeostasis in numerous diseases including cancer, chronic viral infection, allergy, and autoimmune diseases. Recently, IDO has extended its role to liver field. In this review, we summarize the dysregulation and potentials of IDO in the emerging field of liver injuries, as well as current challenges for IDO targets. In particular, we discuss unexpected conclusions against previous work published. IDO is induced by pro-inflammatory cytokines in liver dysfunction and exerts an immunosuppressive effect, whereas the improvement of liver injury may require consideration of multiple factors besides IDO.

Intensive cancer chemotherapy is well known to cause bone vasculature disfunction and damage, but the mechanism is poorly understood and there is a lack of treatment. Using a rat model of methotrexate (MTX) chemotherapy (five once-daily dosses at 0.75 mg/kg), this study investigated the roles of the Notch2 signalling pathway in MTX chemotherapy-induced bone micro-vasculature impairment. Gene expression, histological and micro-computed tomography (micro-CT) analyses revealed that MTX-induced micro-vasculature dilation and regression is associated with the induction of Notch2 activity in endothelial cells and increased production of inflammatory cytokine tumour necrosis factor alpha (TNFα) from osteoblasts (bone forming cells) and bone marrow cells. Blockade of Notch2 by a neutralising antibody ameliorated MTX adverse effects on bone micro-vasculature, both directly by supressing Notch2 signalling in endothelial cells and indirectly via reducing TNFα production. Furthermore, in vitro studies using rat bone marrow-derived endothelial cell revealed that MTX treatment induces Notch2/Hey1 pathway and negatively affects their ability in migration and tube formation, and Notch2 blockade can partially protect endothelial cell functions from MTX damage.

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/β-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option.

Abstract Background Use of anti-programmed cell death-1 (anti-PD-1) has been successful in treating many types of cancers. Despite its promising efficacy, immune-related adverse events are still a major concern. Immune-related cardiotoxicity, which is rare but fatal, has recently become a focus of attention. Cardiotoxicities including myocarditis, cardiomyopathy, cardiac fibrosis, heart block and cardiac arrest have been reported. Of these toxicities, myocarditis is often accompanied by dysrhythmia. The presentation of sick sinus syndrome as an immune-related adverse event has not yet been reported. Here, we reported the first case of sick sinus syndrome, a rare toxicity induced by anti-PD-1. Case presentation A 42-year-old male patient who had metastatic hepatocellular carcinoma failed treatment with sorafenib. Pembrolizumab at a fixed dose of 100 mg every three weeks was given. His heart rate gradually slowed down and he presented sick sinus syndrome with a lowest heart rate of 38 bpm after six cycles of pembrolizumab. He denied chest tightness, cold sweating, palpitation and dyspnea. Lab data including cardiac enzyme, electrolytes and thyroid function were all within a normal range. Simultaneously, he complained of fatigue, dizziness and anorexia with hypotension. Lab data revealed low cortisol and ACTH levels. Anti-PD-1 induced adrenal insufficiency was suspected. Low-dose cortisone (12.5 mg) was prescribed, and the patient’s symptoms, hypotension and sick sinus syndrome showed rapid improvement. Cortisone was gradually titrated and discontinued three weeks later. His sick sinus syndrome did not relapse and the cortisol and ACTH level returned to normal. Conclusions Sick sinus syndrome caused by anti-PD-1 treatment is a rare adverse event. With the development of sick sinus syndrome, myocarditis should be the first differential diagnosis because of its lethality. From this case, we learned that sick sinus syndrome may be a presentation of immune- or adrenal insufficiency-mediated sinus node dysfunction, both could be reversed with a glucocorticoid supplement.

Methotrexate (MTX) treatment for childhood malignancies has shown decreased osteogenesis and increased adipogenesis in bone marrow stromal cells (BMSCs), leading to bone loss and bone marrow adiposity, for which the molecular mechanisms are not fully understood. Currently, microRNAs (miRNAs) are emerging as vital mediators involved in bone/bone marrow fat homeostasis and our previous studies have demonstrated that miR-6315 was upregulated in bones of MTX-treated rats, which might be associated with bone/fat imbalance by directly targeting Smad2. However, the underlying mechanisms by which miR-6315 regulates osteogenic and adipogenic differentiation require more investigations. Herein, we further explored and elucidated the regulatory roles of miR-6315 in osteogenesis and adipogenesis using in vitro cell models. We found that miR-6315 promotes osteogenic differentiation and it alleviates MTX-induced increased adipogenesis. Furthermore, our results suggest that the involvement of miR-6315 in osteogenesis/adipogenesis regulation might be partially through modulating the TGF-β/Smad2 signalling pathway. Our findings indicated that miR-6315 may be important in regulating osteogenesis and adipogenesis and might be a therapeutic target for preventing/attenuating MTX treatment-associated bone loss and marrow adiposity.

Intensive methotrexate (MTX) treatment for childhood malignancies decreases osteogenesis but increases adipogenesis from the bone marrow stromal cells (BMSCs), resulting in bone loss and bone marrow adiposity. However, the underlying mechanisms are unclear. While microRNAs (miRNAs) have emerged as bone homeostasis regulators and miR-542-3p was recently shown to regulate osteogenesis in a bone loss context, the role of miR-542-3p in regulating osteogenesis and adipogenesis balance is not clear. Herein, in a rat MTX treatment-induced bone loss model, miR-542-3p was found significantly downregulated during the period of bone loss and marrow adiposity. Following target prediction, network construction, and functional annotation/ enrichment analyses, luciferase assays confirmed sFRP-1 and Smurf2 as the direct targets of miR-542-3p. miRNA-542-3p overexpression suppressed sFRP-1 and Smurf2 expression post-transcriptionally. Using in vitro models, miR-542-3p treatment stimulated osteogenesis but attenuated adipogenesis following MTX treatment. Subsequent signalling analyses revealed that miR-542-3p influences Wnt/β-catenin and TGF-β signalling pathways in osteoblastic cells. Our findings suggest that MTX treatment-induced bone loss and marrow adiposity could be molecularly linked to miR-542-3p pathways. Our results also indicate that miR-542-3p might be a therapeutic target for preserving bone and attenuating marrow fat formation during/after MTX chemotherapy.

The E3 ligase Mule has been previously reported to be essential for B cell development and function by modulating p53 ubiquitination and degradation. Here Haoet al. identify KLF4 as a novel ubiquitination target of Mule and show it controls T cell proliferation and autoimmunity.

Fixation and permeabilization of cells and tissues are essential processes in biological techniques like immunofluorescence and immunohistochemistry for cell biology studies. In typical procedures, the biological samples are treated by paraformaldehyde and Triton X-100 to achieve cellular fixation and permeabilization, respectively, prior to the incubation with specific antibodies. While it is well-known that the integrity of cell membrane has been broken during these processes, quantitative studies on the loss of cellular mass density and the enhancement of molecular accessibility at single cell level are still rare. In this study, we employed the surface plasmon resonance (SPR) imaging technique to monitor the mass density change of single cells during sequential fixation and permeabilization processes. We further utilize the osmotic responses of single cells to sugar molecules as an indicator to evaluate the integrity of cell membranes. It was found that, while fixation initially destructed the integrity of cell membranes and increased the permeability of intra- and extra-cellular molecules, it was permeabilization process that substantially induced significant loss in cellular mass density.

It has been well-established that malondialdehyde (MDA), which is generated during the process of lipid peroxidation, is a commonly known biomarker for oxidative stress. Therefore, the serum levels of MDA are detected by using the lipid peroxidation assay with commercially available kit to determine the induction of oxidative stress in rat models.

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host.

Psoriasis is a chronic inflammatory skin disorder that affects ~2%–3% of the worldwide population. Inappropriate and excessive activation of endosomal Toll-like receptors 7, 8, and 9 (TLRs 7–9) at the psoriatic site has been shown to play a pathogenic role in the onset of psoriasis. Macrophage is a major inflammatory cell type that can be differentiated into phenotypes M1 and M2. M1 macrophages produce proinflammatory cytokines, and M2 macrophages produce anti-inflammatory cytokines. The balance between these two types of macrophages determines the progression of various inflammatory diseases; however, whether macrophage polarization plays a role in psoriatic inflammation activated by endosomal TLRs has not been investigated. In this study, we investigated the function and mechanism of macrophages related to the pathogenic role of TLRs 7–9 in the progression of psoriasis. Analysis of clinical data in database revealed significantly increased expression of macrophage markers and inflammatory cytokines in psoriatic tissues over those in normal tissues. In animal studies, depletion of macrophages in mice ameliorated imiquimod, a TLR 7 agonist-induced psoriatic response. Imiquimod induced expression of genes and cytokines that are signature of M1 macrophage in the psoriatic lesions. In addition, treatment with this TLR 7 agonist shifted macrophages in the psoriatic lesions to a higher M1/M2 ratio. Both of the exogenous and endogenous TLR 7–9 ligands activated M1 macrophage polarization. M1 macrophages expressed higher levels of proinflammatory cytokines and TLRs 7–9 than M2 macrophages. These results suggest that by rendering macrophages into a more inflammatory status and capable of response to their ligands in the psoriatic sites, TLR 7–9 activation drives them to participate in endosomal TLR-activated psoriatic inflammation, resulting in an amplified inflammatory response. Our results also suggest that blocking M1 macrophage polarization could be a strategy which enables inhibition of psoriatic inflammation activated by these TLRs.

Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

BACKGROUND:Diabetes mellitus (DM) is associated with higher incidence and poorer prognosis of hepatocellular carcinoma (HCC). The influence of DM on patient survival in different HCC stages is not known. METHODS:A prospective dataset of 3,182 HCC patients was collected between 2002 and 2014. Patients were divided into three groups according to BCLC stages (BCLC stage 0 and stage A, BCLC stage B, BCLC stage C and stage D). We compared the cumulative survival rate of diabetic and non-diabetic patients in different BCLC groups. The correlation between DM and overall survival was also analyzed by multivariate Cox regression model within each group. RESULTS:DM is present in 25.2% of all patients. Diabetic patients had lower cumulative survival in BCLC stage 0 plus BCLC stage A group (log rank p

Pleurotus tuber-regium (PTR) is an edible and medicinal mushroom found mainly in tropical and subtropical countries. In this study, we investigated the anti-inflammatory effects of PTR mycelia on RAW 264.7 macrophages. Samples of PTR mycelia were prepared by submerged fermentation. An ethanolic extract of PTR mycelia (PTRA) inhibited the production of nitric oxide (NO), TNF-α, and IL-6 in LPS-treated RAW 264.7 cells. Two compounds were purified and identified as cerevisterol (CE) and ergosta-4,6,8(14),22-tetraen-3-one (EG). Both CE and EG inhibited the production of NO, TNF-α, and PGE2 in LPS-treated RAW 264.7 cells. The expression of the iNOS and COX2 proteins was inhibited by CE and EG in a dose-dependent manner. In addition, CE and EG repressed the expression of iNOS, COX2, TNF-α, and SOCS3 mRNAs. This study revealed that an ethanolic extract of PTR mycelia exhibited anti-inflammatory activity and that CE and EG might contribute to this activity.

Although bone marrow and bone toxicities have been reported in breast cancer survivors, preventative strategies are yet to be developed. Clinical studies suggest consumption of long chain omega-3 polyunsaturated fatty acids (LCn3PUFA) can attenuate age-related bone loss, and recent animal studies also revealed benefits of LCn3PUFA in alleviating bone marrow and bone toxicities associated with methotrexate chemotherapy. Using a female rat model for one of the most commonly used anthracycline-containing breast cancer chemotherapy regimens (adriamycin + cyclophosphamide) (AC) chemotherapy, this study investigated potential effects of daily LCn3PUFA consumption in preserving bone marrow and bone microenvironment during chemotherapy. AC treatment for four cycles significantly reduced bone marrow cellularity and increased marrow adipocyte contents. It increased trabecular bone separation but no obvious changes in bone volume or bone cell densities. LCn3PUFA supplementation (375 mg/100 g/day) attenuated AC-induced bone marrow cell depletion and marrow adiposity. It also partially attenuated AC-induced increases in trabecular bone separation and the cell sizes and nuclear numbers of osteoclasts formed ex vivo from bone marrow cells isolated from AC-treated rats. This study suggests that LCn3PUFA supplementation may have beneficial effects in preventing bone marrow damage and partially protecting the bone during AC cancer chemotherapy.

Intensive cancer chemotherapy is known to cause bone defects, which currently lack treatments. This study investigated the effects of polyphenol resveratrol (RES) in preventing bone defects in rats caused by methotrexate (MTX), a commonly used antimetabolite in childhood oncology. Young rats received five daily MTX injections at 0.75 mg/kg/day. RES was orally gavaged daily for seven days prior to, and during, five‐day MTX administration. MTX reduced growth plate thickness, primary spongiosa height, trabecular bone volume, increased marrow adipocyte density, and increased mRNA expression of the osteogenic, adipogenic, and osteoclastogenic factors in the tibial bone. RES at 10 mg/kg was found not to affect bone health in normal rats, but to aggravate the bone damage in MTX‐treated rats. However, RES supplementation at 1 mg/kg preserved the growth plate, primary spongiosa, bone volume, and lowered the adipocyte density. It maintained expression of genes involved in osteogenesis and decreased expression of adipogenic and osteoclastogenic factors. RES suppressed osteoclast formation ex vivo of bone marrow cells from the treated rats. These data suggest that MTX can enhance osteoclast and adipocyte formation and cause bone loss, and that RES supplementation at 1 mg/kg may potentially prevent these bone defects.

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(cre)PP4(F/F) mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(cre)PP4(F/F) mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(cre)PP4(F/F) mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJ(H) recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM(+) B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of V(H)DJ(H) recombination.

Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss.

Drug-induced liver injury (DILI) has been a public, economic and pharmaceutical issue for many years. Enormous effort has been made for discovering and developing novel biomarkers for diagnosing and monitoring both clinical and preclinical DILI at an early stage, though progress has been relatively slow. Additionally, herb-induced liver injury is an emerging cause of liver disease because herbal medicines are increasingly being used worldwide. Recently, circulating microRNAs (miRNAs) have shown potential to serve as novel, minimally invasive biomarkers to diagnose and monitor human cancers and other diseases at early stages.In order to identify candidate miRNAs as diagnostic biomarkers for DILI, miRNA expression profiles of serum and liver tissue from two parallel liver injury Sprague-Dawley rat models induced by a compound (acetaminophen, APAP) or an herb (Dioscorea bulbifera, DB) were screened in this study. The initial screens were performed on serum using a MicroRNA TaqMan low-density qPCR array and on liver tissue using a miRCURY LNA hybridization array and were followed by a TaqMan probe-based quantitative reverse transcription-PCR (qRT-PCR) assay to validate comparison with serum biochemical parameters and histopathological examination. Two sets of dysregulated miRNA candidates in serum and liver tissue were selected in the screening phase. After qRT-PCR validation, a panel of compound- and herb- related serum miRNAs was identified.We have demonstrated that this panel of serum miRNAs provides potential biomarkers for diagnosis of DILI with high sensitivity and specificity.