Microvascular lung injury and endoplasmic reticulum stress in systemic lupus erythematosus-associated alveolar hemorrhage and pulmonary vasculitis.

Human COPA mutations affecting retrograde Golgi-to-endoplasmic reticulum (ER) protein transport cause diffuse alveolar hemorrhage (DAH) and ER stress ("COPA syndrome"). Patients with SLE also can develop DAH. C57BL/6 (B6) mice with pristane-induced lupus develop monocyte-dependent DAH indistinguishable from human DAH, whereas BALB/c mice are resistant. We examined Copa and ER stress in pristane-induced lupus. Copa expression, ER stress, vascular injury, and apoptosis were assessed in mice and COPA was quantified in blood from patients with SLE. Copa mRNA and protein expression were impaired in B6 mice with pristane-induced DAH, but not in pristane-treated BALB/c mice. An ER stress response (increased Hsp5a/BiP, Ddit3/CHOP, Eif2a, and spliced Xbp1) was seen in lungs from pristane-treated B6, but not BALB/c, mice. Resistance of BALB/c mice to DAH was overcome by treating them with low-dose thapsigargin plus pristane. CB6F1 mice did not develop DAH or ER stress, suggesting that susceptibility was recessive. Increased pulmonary expression of von Willebrand factor (Vwf), a marker of endothelial injury, and the chemokine Ccl2 in DAH suggested that pristane promotes lung microvascular injury and monocyte recruitment. Consistent with that possibility, lung endothelial cells and infiltrating bone marrow-derived cells from pristane-treated B6 mice expressed BiP and showed evidence of apoptosis (annexin-V and activated caspase-3 staining). COPA expression also was low in patients with SLE with lung involvement. Pristane-induced DAH may be initiated by endothelial injury, resulting in ER stress, apoptosis of lung endothelial cells, and recruitment of myeloid cells that propagate lung injury. The pathogenesis of DAH in SLE and COPA syndrome may overlap.

Myxomavirus serpin alters macrophage function and prevents diffuse alveolar hemorrhage in pristane-induced lupus.

C57BL/6 mice with pristane-induced lupus develop macrophage-dependent diffuse alveolar hemorrhage (DAH), which is blocked by treatment with liver X receptor (LXR) agonists and is exacerbated by low IL-10 levels. Serp-1, a myxomavirus-encoded serpin that impairs macrophage activation and plasminogen activation, blocks DAH caused by MHV68 infection. We investigated whether Serp-1 also could block DAH in pristane-induced lupus. Pristane-induced DAH was prevented by treatment with recombinant Serp-1 and macrophages from Serp1-treated mice exhibited an anti-inflammatory M2-like phenotype. Therapy activated LXR, promoting M2 polarization and expression of Kruppel-like factor-4 (KLH4), which upregulates IL-10. In contrast, deficiency of tissue plasminogen activator or plasminogen activator inhibitor had little effect on DAH. We conclude that Serp-1 blocks pristane-induced lung hemorrhage by enhancing LXR-regulated M2 macrophage polarization and KLH4-regulated IL-10 production. In view of the similarities between DAH in pristane-treated mice and SLE patients, Serp-1 may represent a potential new therapy for this severe complication of SLE.

The study of distance changes between lumbar bi-cortical pedicle screws and anterior large vessels in patients with lumbar spondylolisthesis.

OBJECTIVE: This paper was a anatomical radiographic study of distance between lumbar bi-cortical pedicle screws (BPSs) and anterior large vessels (ALVs) in patients with lumbar spondylolisthesis, and to provide clinical basis for evaluating the safety of bi-cortical pedicle screw implantation during lumbar spondylolisthesis. METHODS: Complete Computed tomography (CT) data of 104 patients with grade I lumbar spondylolisthesis (L4 52 and L5 52) and 107 non-spondylolisthesis patients (control group) were collected in this study. The distances between lumbar 4,5(L4,5) and sacrum 1(S1) BPSs and ALVs (abdominal aorta, inferior vena cava, left and right common iliac artery, internal and external iliac artery) were respectively measured at different transverse screw angles (TSAs) (L4:5°,10°; L5:10°,15°; S1:0°,5°,10°) and analyzed by SPSS (v25.0). There were three types of distances from the anterior vertebral cortex (AVC) to the ALVs (DAVC-ALV): DAVC-ALV N, DAVC-ALV ≥ 0.50 cm, and DAVC-ALV < 0.50 cm; these different distances represented non-contact, distant and close ALV respectively. RESULTS: We calculated the incidences of screw tip contacting large vessels at different TSAs and provided the appropriate angle of screw implantation. In non-spondylolisthesis group, in L4, the appropriate left TSA was 5°, and the incidence of the close ALV was 4.62%. In S1, the appropriate left TSA was 0° and the incidence of the close ALV was 22.4%, while the appropriate right TSA was 10° and the incidence of the close ALV was 17.8%. In L4 spondylolisthesis group, in L4, the appropriate left TSA was 5°, and the incidence of the close ALV was 3.8%. In L5 spondylolisthesis group, in S1, the appropriate left TSA was 0° and the incidence of the close ALV was 19.2%, while the appropriate right TSA was 10° and the incidence of the close ALV was 21.2%. The use of BPS was not appropriate on the right side of L4 or on the either side of L5 both in spondylolisthesis and control group. In patients with lumbar 4 spondylolisthesis, the incidences of screw tip contacting large vessels were less than the control group in both L4 and 5. In patients with lumbar 5 spondylolisthesis, the incidences of screw tip contacting large vessels were less than the control group in L5, while there were no significant difference in S1. CONCLUSION: It is very important that considering the anatomical relationship between the AVC and the ALVs while planning BPSs. The use of BPS does not apply to every lumbar vertebra. In patients with lumbar spondylolisthesis and non-spondylolisthesis patients, the incidences of screw tip contacting large vessels are different.

The diagnostic pitfalls of lumbar disc herniation---- malignant sciatic nerve tumour: two case reports and literature review.

BACKGROUND: Sciatica pain is a typical symptom of lumbar disc herniation (LDH), but some neurogenic and malignant tumours surrounding the sciatic nerve can also cause similar symptoms. These tumours are often misdiagnosed or even mistreated as LDH in clinical practice. CASE PRESENTATION: In our clinical practice, we found two patients with malignant tumours who were misdiagnosed with LDH. One patient complained of pain and numbness in the right lower limb. The primary diagnosis was LDH, and the patient underwent posterior lumbar interbody fusion surgery. After the operation, the symptoms were not alleviated. Then, diffuse large B-cell lymphoma involving the soft tissue and the sciatic nerve was identified. Another patient who manifested with radiating pain in the right lower limb was diagnosed with LDH at Chengde Central Hospital. He received regular conservative treatment for approximately 6 months, but his symptoms were not relieved, and then he was referred to our hospital. A malignant peripheral nerve sheath tumour (MPNST) of the sciatic nerve was diagnosed, and he received cisplatin (DDP) chemohyperthermia. CONCLUSIONS: Descriptions of tumour lesions involving the sciatic nerve and misdiagnosed as LDH in the literature are rare. In the reported literature, 7 patients were misdiagnosed with LDH, and all patients presented with sciatica. Among them, 4 patients only received surgical treatment, 1 patient only underwent neurolysis, and 2 patients received both surgical and chemotherapy treatment. Their low incidence and similar clinical manifestations to LDH make malignant tumours involving the sciatic nerve easy to misdiagnose. When the clinical symptoms and signs are inconsistent with the imaging findings, we need to be aware of non-discogenic sciatica, including tumours involving the sciatic nerve. Furthermore, tumours that grow near the exit of the sciatic notch may be misdiagnosed because of their deeper location and because they are covered with gluteal muscles. Sometimes sciatica caused by sciatic nerve tumours is only distal, without any radicular distribution. This pain is more severe than that caused by LDH, and this pain is not related to the position of the lumbar spine. Thus, it is beneficial to perform a detailed physical examination of the sciatic nerve to avoid this kind of misdiagnosis.

A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental Lupus.

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.

Irisin Attenuates Neuroinflammation and Prevents the Memory and Cognitive Deterioration in Streptozotocin-Induced Diabetic Mice.

Diabetes mellitus (DM) patients experience memory and cognitive deficits. The mechanisms underlying this dysfunction in the brain of DM patients are not fully understood, and therefore, no optimized therapeutic strategy has been established so far. The aim of the present study was to assess whether irisin was able to improve memory and cognitive performance in a streptozotocin-induced diabetic mouse model. A diabetic mouse model was established and behavioral tests were performed. We also set up primary cultures for mechanism studies. Western blots and EMSA were used for molecular studies. Significant impairment of cognition and memory was observed in these DM mice, which could be effectively prevented by irisin cotreatment. We also found upregulated levels of GFAP protein, reduced synaptic protein expression, and increased levels of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in the brains; however, irisin significantly attenuated these cellular responses. Meanwhile, our results demonstrated that irisin inhibited the activation of P38, STAT3, and NFκB proteins of DM mice. Furthermore, our results suggested that irisin might regulate the function of P38, STAT3, and NFκB in hippocampal tissues of DM mice. Collectively, irisin inhibited neuroinflammation in STZ-induced DM mice by inhibiting cytokine release and improving their cognitive function. Our findings revealed the mechanism of irisin's anti-inflammatory effect in the CNS.

The Association between Depression and Type 1 Diabetes Mellitus: Inflammatory Cytokines as Ferrymen in between?

The depression incidence is much higher in patients with diabetes mellitus (DM), and the majority of these cases remain under-diagnosed. Type 1 diabetes mellitus (T1D) is now widely thought to be an organ-specific autoimmune disease. As a chronic autoimmune condition, T1D is characterized by T cell-mediated selective loss of insulin-producing ß-cells. The age of onset of T1D is earlier than T2D, and T1D patients have an increased vulnerability to depression due to its diagnosis and treatment burden occurring in a period when the individuals are young. The literature has suggested that inflammatory cytokines play a wide role in both diseases. In this review, the mechanisms behind the initiation and propagation of the autoimmune response in T1D and depression are analyzed, and the contribution of cytokines to both conditions is discussed. This review outlines the immunological mechanism of T1D and depression, with a particular emphasis on the role of tumor necrosis factor-α (TNF-α), IL-1ß, and interferon-γ (IFN-γ) cytokines and their signaling pathways. The purpose of this review is to highlight the possible pathways of the cytokines shared by these two diseases via deciphering their cytokine cascades. They may provide a basic groundwork for future study of the possible mechanism that links these two diseases and to develop new compounds that target the same pathway but can conquer two diseases.

Developmental neurotoxicity and immunotoxicity induced by graphene oxide in zebrafish embryos.

Graphene oxide (GO) has emerged as the worldwide promising candidate for biomedical application, such as for drug delivery, bio-sensing and anti-cancer therapy. This study was focused on the zebrafish and RAW264.7 cell line as in vivo and in vitro models to assess the potential developmental neurotoxicity and immunotoxicity of GO. No obvious acute developmental toxicity was observed upon treatments with 0.01, 0.1, and 1 µg/mL GO for five consecutive days. However, decreased hatching rate, increased malformation rate, heart beat rate and hypoactivity of locomotor behavior were detected when exposed to 10 µg/mL GO. Also, RT-PCR analysis revealed that expressions of genes related to the nervous system were up-regulated. The potential risk of GO for developmental neurotoxicity may be ascribed to the high level of oxidative stress induced by high concentration of GO. Most importantly, the mRNA levels of immune response associated genes, such as interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ) were significantly increased under environmental concentration exposure. The activation of pro-inflammatory immune response was also observed in macrophage cell line. Taken together, our results demonstrated that immunotoxicity is a sensitive indicator for assessment of bio-compatibility of GO.

"Lupoid hepatitis" in SLE patients and mice with experimental lupus.

The unusual subset of patients with severe hepatitis, hypergammaglobulinemia, arthritis, and LE cells in the blood reported by Henry Kunkel and others suggested to these investigators that "lupoid" hepatitis might share pathogenic mechanisms with SLE. More than half a century later, the etiology of autoimmune hepatitis remains unclear. The occurrence of autoimmune hepatitis in a small fraction (about 3%) of SLE patients in our lupus cohort and in two mouse models of SLE supports their conclusion that lupoid hepatitis may be share pathogenic mechanisms with SLE. The development of autoimmune hepatitis in mice with pristane-induced lupus provides an opportunity to further explore the potential link between these two autoimmune disorders.

STAT3 signaling contributes to the high effector activities of interleukin-15-derived dendritic cells.

Dendritic cells (DCs) are important innate and adaptive immune effectors, and have a key role in antigen presentation and T-cell activation. Different lineages of DCs can be developed from hematopoietic progenitors following cytokine signaling, and the various lineages of DCs display distinct morphology, phenotype and functions. There has been limited information on differential cytokine-mediated molecular signaling in DCs. Analyses of surface molecules by flow cytometry and quantitative RNA profiling revealed differences between DCs derived from interleukin-4 (IL-4) versus IL-15 signaling, yet both lineages of DCs exhibited similar levels of surface molecules key to immune activation. Functional assays confirmed that IL-15-derived DCs elicited greater antigen-specific, primary and secondary CD8 and CD4 T-cell responses than did IL-4-derived DCs. Importantly, IL-15 DCs secreted substantial amounts of proinflammatory cytokines, including IL-6, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNFα), which helped polarize a strong T-cell response. Assessment of signaling pathways revealed that IL-15 DCs exhibited a lower levels of activated signal transducer and activator of transcription 5 (STAT5), STAT6 and extracellular signal-regulated kinase 1/2 than IL-4 DCs, but after lipopolysaccharide (LPS)/TNFα treatment, the STAT3 and p38 mitogen-activated protein kinase (MAPK) activities were significantly enhanced in the IL-15 DCs. Surprisingly, contrary to the canonical IL-15-mediated STAT5 signaling pathway in lymphoid cells, IL-15 did not mediate a strong STAT5 or STAT3 activation in DCs. Further analysis using specific inhibitors to STAT3 and p38 MAPK pathways revealed that the STAT3 signaling, but not p38 MAPK signaling, contributed to IFN-γ production in DCs. Therefore, while IL-15 does not promote the STAT signaling in DCs, the increased STAT3 activity after LPS/TNFα treatment of the IL-15 DCs has a key role in their high IFN-γ effector activities.

[Clinical evaluations of neoadjuvant chemotherapy with DN and FP regimens for patients with middle or lower thoracic locally advanced esophageal squamous cell carcinoma].

OBJECTIVE: To explore the efficacies and side effects of neoadjuvant chemotherapy with DN (docetaxel plus cisplatin) and FP (nedaplatin plus cisplatin) regimens for patients with upper or middle thoracic locally advanced esophageal squamous cell carcinoma. METHODS: From January 2008 to January 2012, a total of 124 patients with upper or middle thoracic locally advanced esophageal squamous cell carcinoma were randomized into DN group (n = 64) and FP group (n = 60). Both groups received neoadjuvant chemotherapy. The treatment schedule was recycled every 3 weeks. After 2 cycles, those with potential surgical resection underwent surgery. RESULTS: The 2-year overall, locoregional relapse-free and distant metastasis-free survival rates in DN and FP groups were 71.1% and 66.7%, 65.0% and 63.0%, 78.3% and 74.3% respectively (P > 0.05). The incidence of leucopenia was higher in DN group than that in FP group (P < 0.05). And the occurrence rates of gastrointestinal toxicity and mucositis were significantly higher in FP group than those in DN group. No perioperative mortality occurred with a low incidence of postoperative complications. The rates of overall response, resection, postoperative complications and pathological complete rates response were similar in two groups. And the rates of downstage and R0 resection were significantly higher in DN group than those in FP group (P < 0.05). CONCLUSION: For patients with middle or lower thoracic locally advanced esophageal squamous cell carcinoma, neoadjuvant chemotherapies of docetaxel and nedaplatin may achieve excellent outcomes in clinical response and 2-year survival rate. And the side effects are clinically acceptable.

Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity.

Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.

Pleiotropic IFN-dependent and -independent effects of IRF5 on the pathogenesis of experimental lupus.

Genetic polymorphisms of IFN regulatory factor 5 (IRF5) are associated with an increased risk of lupus in humans. In this study, we examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF5(-/-) mice shared many features with type I IFN receptor (IFNAR)(-/-) and TLR7(-/-) mice: production of anti-Sm/RNP autoantibodies, glomerulonephritis, generation of Ly6C(hi) monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation following pristane injection was greatly diminished in IRF5(-/-) mice, and Th cell differentiation was deviated from Th1 in wild-type mice toward Th2 in IRF5(-/-) mice. Th cell development was skewed similarly in TLR7(-/-) or IFNAR(-/-) mice, suggesting that IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not recruited to the site of inflammation in IRF5(-/-) or MyD88(-/-) mice, but were recruited normally in IFNAR(-/-) and TLR7(-/-) mice. In striking contrast to wild-type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF5(-/-) mice, suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data indicate that altered production of IFN-I and other cytokines in IRF5(-/-) mice prevents pristane from inducing lupus pathology by broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I-independent role of IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets.

MEK1/2 and ERK1/2 mediated lung endothelial injury and altered hemostasis promote diffuse alveolar hemorrhage in murine lupus.

Objective: About 3% of lupus patients develop severe diffuse alveolar hemorrhage (DAH) with pulmonary vasculitis. B6 mice with pristane-induced lupus also develop DAH, but BALB/c mice are resistant. DAH is independent of TLR signaling and other inflammatory pathways. This study examined the role of the mitogen-activated protein kinase pathway (MEK1/2-ERK1/2, JNK, p38). Methods: B6 and BALB/c mice were treated with pristane ± inhibitors of MEK1/2 (trametinib/GSK1120212, "GSK"), ERK1/2 (SCH772984, "SCH"), JNK, or p38. Effects on lung hemorrhage and hemostasis were determined. Results: GSK and SCH abolished DAH, whereas JNK and p38 inhibitors were ineffective. Apoptotic cells were present in lung from pristane-treated mice, but not mice receiving pristane+GSK and endothelial dysfunction was normalized. Expression of the ERK1/2-regulated transcription factor Egr1 increased in pristane-treated B6, but not BALB/c, mice and was normalized by GSK. Pristane also increased expression of the anticoagulant genes Tfpi (tissue factor pathway inhibitor) and Thbd (thrombomodulin) in B6 mice. The ratio of tissue factor ( F3 ) to Tfpi increased in B6 (but not BALB/c) mice and was normalized by GSK. Circulating Thbd protein increased in B6 mice and returned to normal after GSK treatment. Consistent with augmented endothelial anticoagulant activity, pristane treatment increased tail bleeding in B6 mice. Conclusion: Pristane treatment promotes lung endothelial injury and DAH in B6 mice by activating the MEK1/2-ERK1/2 pathway and impairing hemostasis. The hereditary factors determining susceptibility to lung injury and bleeding in pristane-induced lupus are relevant to the pathophysiology of life-threatening DAH in SLE and may help to optimize therapy.

MEK1/2- and ERK1/2-Mediated Lung Endothelial Injury and Altered Hemostasis Promote Diffuse Alveolar Hemorrhage in Murine Lupus.

OBJECTIVE: About 3% of patients with lupus develop severe diffuse alveolar hemorrhage (DAH) with pulmonary vasculitis. C57BL/6 (B6) mice with pristane-induced lupus also develop DAH, but BALB/c mice are resistant. DAH is independent of Toll-like receptor signaling and other inflammatory pathways. This study examined the role of the MEK1/2 pathway (MEK1/2-ERK1/2, JNK, p38). METHODS: B6 and BALB/c mice were treated with pristane with or without inhibitors of MEK1/2 (trametinib/GSK1120212 [GSK]), ERK1/2 (SCH772984 [SCH]), JNK, or p38. Effects on lung hemorrhage and hemostasis were determined. RESULTS: GSK and SCH abolished DAH, whereas JNK and p38 inhibitors were ineffective. Apoptotic cells were present in lung samples from pristane-treated mice but not in mice receiving pristane and GSK, and endothelial dysfunction was normalized. Expression of the ERK1/2-regulated transcription factor early growth response 1 increased in pristane-treated B6, but not BALB/c, mice and was normalized by GSK. Pristane also increased expression of the anticoagulant genes Tfpi and Thbd in B6 mice. The ratio of Tfpi to tissue factor (F3) to Tfpi increased in B6 (but not BALB/c) mice and was normalized by GSK. Circulating thrombomodulin protein levels increased in B6 mice and returned to normal after GSK treatment. Consistent with augmented endothelial anticoagulant activity, pristane treatment increased tail bleeding in B6 mice. CONCLUSION: Pristane treatment promotes lung endothelial injury and DAH in B6 mice by activating the MEK1/2-ERK1/2 pathway and impairing hemostasis. The hereditary factors determining susceptibility to lung injury and bleeding in pristane-induced lupus are relevant to the pathophysiology of life-threatening DAH in systemic lupus erythematosus and may help to optimize therapy.

Divergent molecular strategies drive evolutionary adaptation to competitive fitness in biofilm formation.

Biofilm is a group of heterogeneously structured and densely packed bacteria with limited access to nutrients and oxygen. These intrinsic features can allow a mono-species biofilm to diversify into polymorphic subpopulations, determining the overall community's adaptive capability to changing ecological niches. However, the specific biological functions underlying biofilm diversification and fitness adaptation are poorly demonstrated. Here, we launched and monitored the experimental evolution of Pseudomonas aeruginosa biofilms, finding that two divergent molecular trajectories were adopted for adaptation to higher competitive fitness in biofilm formation: one involved hijacking bacteriophage superinfection to aggressively inhibit kin competitors, whereas the other induced a subtle change in c-di-GMP signaling to gain a positional advantage via enhanced early biofilm adhesion. Bioinformatics analyses implicated that similar evolutionary strategies were prevalent among clinical P. aeruginosa strains, indicative of parallelism between natural and experimental evolution. Divergence in the molecular bases illustrated the adaptive values of genomic plasticity for gaining competitive fitness in biofilm formation. Finally, we demonstrated that these fitness-adaptive mutations reduced bacterial virulence. Our findings revealed how the mutations intrinsically generated from the biofilm environment influence the evolution of P. aeruginosa.

[Evaluation of induction chemotherapy with vinorelbine plus cisplatin (NP) or docetaxel plus cisplatin (TP) combined with concurrent chemoradiotherapy for patients with locally advanced nasopharyngeal carcinoma].

OBJECTIVE: To compare the efficacy and side effects of induction chemotherapy with vinorelbine plus cisplatin (NP) or docetaxel plus cisplatin (TP) combined with concurrent chemoradiotherapy in treating locally advanced nasopharyngeal carcinoma (NPC). METHODS: From January 2005 to December 2009, 146 patients with locally advanced nasopharyngeal carcinoma treated in our department were randomized into NP group (76 patients) or TP group (70 patients). Both groups received two cycles of induction chemotherapy and concurrent chemoradiotherapy. After three weeks of induction chemotherapy, the patients received concurrent chemoradiotherapy. The chemotherapy was recycled every three weeks. Two groups were treated with intensity-modulated radiation therapy. RESULTS: The short-term efficacy of NP group was similar to that of TP group. The 3-year overall survival rates, disease-free-survival rates, locoregional relapse-free survival rates and distant metastasis-free survival rates in the NP and TP groups were 84.2% and 82.9%, 71.1% and 74.3%, 89.5% and 91.4%, 81.6% and 77.1%, respectively (P > 0.05). The occurrence rates of leucopenia, anemia and acute mucositis were significantly higher in the TP group than those in the NP group (P < 0.05). The gastrointestinal toxicity, dermatitis and liver toxicity were similar in the two groups. CONCLUSIONS: The efficacy of NP regimen induction chemotherapy plus concurrent chemordiotherapy for advanced NPC is similar to that of TP regimen. The toxicity of the NP regimen is lower than that of NP regimen, tolerable, and with a good compliance.

A novel monocyte differentiation pattern in pristane-induced lupus with diffuse alveolar hemorrhage.

Pristane causes chronic peritoneal inflammation resulting in lupus, which in C57BL/6 mice is complicated by lung microvascular injury and diffuse alveolar hemorrhage (DAH). Mineral oil (MO) also causes inflammation, but not lupus or DAH. Since monocyte depletion prevents DAH, we examined the role of monocytes in the disease. Impaired bone marrow (BM) monocyte egress in Ccr2-/- mice abolished DAH, confirming the importance of monocyte recruitment to the lung. Circulating Ly6Chi monocytes from pristane-treated mice exhibited increased annexin-V staining in comparison with MO-treated controls without evidence of apoptosis, suggesting that pristane alters the distribution of phosphatidylserine in the plasma membrane before or shortly after monocyte egress from the BM. Plasma membrane asymmetry also was impaired in Nr4a1-regulated Ly6Clo/- 'patrolling' monocytes, which are derived from Ly6Chi precursors. Patrolling Ly6Clo/- monocytes normally promote endothelial repair, but their phenotype was altered in pristane-treated mice. In contrast to MO-treated controls, Nr4a1-regulated Ly6Clo/- monocytes from pristane-treated mice were CD138+, expressed more TremL4, a protein that amplifies TLR7 signaling, and exuberantly produced TNFα in response to TLR7 stimulation. TremL4 expression on these novel CD138+ monocytes was regulated by Nr4a1. Thus, monocyte CD138, high TremL4 expression, and annexin-V staining may define an activated/inflammatory subtype of patrolling monocytes associated with DAH susceptibility. By altering monocyte development, pristane exposure may generate activated Ly6Chi and Ly6Clo/- monocytes, contributing to lung microvascular endothelial injury and DAH susceptibility.

DNA Methyltransferase Regulates Nitric Oxide Homeostasis and Virulence in a Chronically Adapted Pseudomonas aeruginosa Strain.

Opportunistic pathogens such as Pseudomonas aeruginosa adapt their genomes rapidly during chronic infections. Understanding their epigenetic regulation may provide biomarkers for diagnosis and reveal novel regulatory mechanisms. We performed single-molecule real-time sequencing (SMRT-seq) to characterize the methylome of a chronically adapted P. aeruginosa clinical strain, TBCF10839. Two N6-methyladenine (6mA) methylation recognition motifs (RCCANNNNNNNTGAR and TRGANNNNNNTGC [modification sites are in bold]) were identified and predicted as new type I methylation sites using REBASE analysis. We confirmed that the motif TRGANNNNNNTGC was methylated by the methyltransferase (MTase) M.PaeTBCFII, according to methylation sensitivity assays in vivo and vitro. Transcriptomic analysis showed that a ΔpaeTBCFIIM knockout mutant significantly downregulated nitric oxide reductase (NOR) regulation and expression of coding genes such as nosR and norB, which contain methylated motifs in their promoters or coding regions. The ΔpaeTBCFIIM strain exhibited reduced intercellular survival capacity in NO-producing RAW264.7 macrophages and attenuated virulence in a Galleria mellonella infection model; the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII occur frequently in P. aeruginosa as well as other bacterial species. Our work therefore provided new insights into the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed broad genome diversity among P. aeruginosa clinical strains and revealed their different regulatory traits compared to the laboratory strains. While current investigation of the epigenetics of P. aeruginosa is still lacking, understanding epigenetic regulation may provide biomarkers for diagnosis and facilitate development of novel therapies. Denitrification capability is critical for microbial versatility in response to different environmental stress conditions, including the bacterial infection process, where nitric oxide (NO) can be generated by phagocytic cells. The denitrification regulation mechanisms have been studied intensively at genetic and biochemical levels. However, there is very little evidence about the epigenetic regulation of bacterial denitrification mechanism. P. aeruginosa TBCF10839 is a chronically host-adapted strain isolated from a cystic fibrosis (CF) patient with special antiphagocytosis characteristics. Here, we investigated the regulatory effect of an orphan DNA MTase, M.PaeTBCFII, in P. aeruginosa TBCF10839. We demonstrated that the DNA MTase regulates the transcription of denitrification genes represented by NOR and affects antiphagocytic ability in bacteria. In silico analysis suggested that DNA methylation modification may enhance gene expression by affecting the binding of transacting factors such as DNR and RpoN. Our findings not only deepen the understanding of the role of DNA MTase in transcriptional regulation in P. aeruginosa but also provide a theoretical foundation for the in-depth study of the molecular mechanism of the epigenetic regulation on denitrification, virulence, and host-pathogen interaction.