A chemiluminescence immunoassay for the detection of NT-proBNP.

BACKGROUND: The aim of the study was to assess the analytical performance of the HISCL NT-proBNP assay, a newly developed chemiluminescence immunoassay, for the detection of NT-proBNP. METHODS: The within-run and total imprecision of the NT-proBNP assay were determined with HISCL cardiac marker controls. The linear ranges of the NT-proBNP assays were evaluated based on the CLSI EP6-A document using selected serum samples. Two hundred serum samples were evaluated to compare the HISCL NT-proBNP and Elecsys NT-proBNP assays. Five additional high NT-proBNP concentrations serum samples were evaluated to assess if there was high-dose hook effect in the HISCL NT-proBNP assay. RESULTS: The total and within-run imprecision values of the HISCL NT-proBNP assay were 5.85%, 0.81%, 2.56% and 0.54% and 6.07%, 0.73%, 2.61% and 0.59% at 6.1, 129.83, 3732.84and39737.33 pg/ml, respectively. The assay was verified to be linear for NT-proBNP levels ranging between 6.1 and 39737.33 pg/ml. The assay comparison showed that HISCL NT-proBNP = 0.9803 × Elecsys NT-proBNP -4.383. The sensitivity of HISCL NT-proBNP was 87.23%, and the specificity was 85.61%. The AUC of HISCL NT-proBNP (0.90 (95% CI, 0.86-0.93)) did not differ from that of Elecsys NT-proBNP(0.89 (95% CI, 0.85-0.93)) (P = 0.638). The results of five high NT-proBNP concentrations samples (44448, 54206, 55634, 55728 and 109406 pg/ml, measured with the Elecsys NT-proBNP assay) tested with HISCL NT-proBNP assay were all displayed with ">40000 pg/ml". CONCLUSIONS: The HISCL NT-proBNP chemiluminescence immunoassay showed good analytical and diagnostic performance for the detection of NT-proBNP and could be used in routine clinical practice.

Treponema pallidum promotes macrophage polarization and activates the NLRP3 inflammasome pathway to induce interleukin-1ß production.

BACKGROUND: The involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1ß production and its influence on macrophage polarization triggered by T. pallidum. RESULTS: The results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1ß and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1ß secretion in T. pallidum-treated macrophages, and the observed production of IL-1ß occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1ß by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1ß release from T. pallidum-treated macrophages. CONCLUSIONS: These findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1ß secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1ß production in macrophages in response to T. pallidum infection involves K+ efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.

Correlation between polymorphisms in toll-like receptor genes and the activity of hepatitis B virus among treatment-naïve patients: a case-control study in a Han Chinese population.

BACKGROUND: Because of the high prevalence and absence of cure for infection, chronic hepatitis B virus (HBV) infection has been acknowledged as a pressing public health issue. Toll-like receptors (TLRs) activate the human innate immune system and the polymorphisms in TLRs may alter their function. The present study aimed to investigate the association between TLR polymorphisms and disease progression of chronic HBV infection. METHODS: During the study period, 211 treatment-naïve patients with chronic HBV infection were recruited, and blood samples were collected from each individual. Matrix-assisted laser desorption/ionization time of flight mass spectrometry was employed to genotype the selected TLR polymorphisms after human genome extraction. In addition, HbsAg, TNF-α, and IL-6 levels were quantified using enzyme linked immunosorbent assay (ELISA). Statistical analyses were conducted to investigate the association between TLR polymorphisms and hepatitis activity, liver function parameters, HbsAg level, and cytokine level. RESULTS: We did not observe any mutations in rs4986790, rs4986791, and rs5743708 among all study subjects. A logistic regression revealed that mutations in rs3804099 and rs4696480 were associated with milder hepatitis activity. Consistent with the logistic regression, improved liver function parameters and reduced level of both HbsAg and cytokines were also correlated with the mutant carriers of rs3804099 and rs4696480. CONCLUSIONS: TLR mutations were significantly associated with milder hepatitis activity among patients with chronic HBV infection. Therefore, we conclude that the activation of TLR pathways may further intensify the inflammation of hepatocytes, and leads to progression of disease.

Development of tissue inflammation accompanied by NLRP3 inflammasome activation in rabbits infected with Treponema pallidum strain Nichols.

BACKGROUND: The inflammasome responses in Treponema pallidum infection have been poorly understood to date. This study aimed to investigate the expression of the nucleotide-binding leucine-rich receptor protein 3 (NLRP3) inflammasome in the development of tissue inflammation in rabbits infected with T. pallidum. METHODS: Forty-five rabbits were randomly assigned to a blank group or an infection group, and the latter was divided into no benzathine penicillin G (BPG) and BPG treatment subgroups. Rabbits in the infection group were injected intradermally with 0.1 mL of a 107/mL T. pallidum suspension at 10 marked sites along the back, and the blank group was treated with normal saline. The BPG treatment subgroup received 200,000 U of BPG administered intramuscularly twice, at 14 d and 21 d post-infection. The development of lesions was observed, and biopsies of the injection site and various organs, including the kidney, liver, spleen, lung, and testis, were obtained for NLRP3, caspase-1, and interleukin-1ß (IL-1ß) mRNA analysis during infection. Blood was also collected for the determination of IL-1ß concentration. RESULTS: Rabbits infected with T. pallidum (both the BPG treatment and no BPG treatment subgroups), exhibited NLRP3 inflammasome activation and IL-1ß secretion in cutaneous lesions, showing a trend in elevation to decline; NLRP3 mRNA expression reached a peak at 18 d in the BPG treatment subgroup and 21 d in the no BPG treatment subgroup and returned to "normal" levels [vs. the blank group (P > 0.05)] at 42 d post-infection. The trend was similar to the change in cutaneous lesions in the infected rabbits, which reached a peak at 16 d in the BPG treatment subgroup and 18 d in the no BPG treatment subgroup. NLRP3, caspase-1, and IL-1ß mRNA expression levels were slightly different in different organs. NLRP3 inflammasome activation was also observed in the kidney, liver, lung, spleen and testis. IL-1ß expression was observed in the kidney, liver, lung and spleen; however, there was no detectable level of IL-1ß in the testes of the infected rabbits. CONCLUSIONS: This study established a clear link between NLRP3 inflammasome activation and the development of tissue inflammation in rabbits infected with T. pallidum. BPG therapy imperceptibly adjusted syphilitic inflammation.

Novel predictors of neurosyphilis among HIV-negative syphilis patients with neurological symptoms: an observational study.

BACKGROUND: Known predictors of neurosyphilis were mainly drawn from human immunodeficiency virus (HIV)-infected syphilis patients, which may not be applicable to HIV-negative populations as they have different characteristics, particularly those with neurological symptoms. This study aimed to identify novel predictors of HIV-negative symptomatic neurosyphilis (S-NS). METHODS: From June 2005 to June 2015, 370 HIV-negative syphilis patients with neurological symptoms were recruited, consisting of 191 S-NS patients (including 123 confirmed neurosyphilis and 68 probable neurosyphilis patients) and 179 syphilis/non-neurosyphilis (N-NS) patients. Clinical and laboratory characteristics of S-NS were compared with N-NS to identify factors predictive of S-NS. Serum rapid plasma reagin (RPR), Treponema pallidum particle agglutination (TPPA), and their parallel testing format for screening S-NS were evaluated. RESULTS: The likelihood of S-NS was positively associated with the serum RPR and TPPA titers. The serum TPPA titers performed better than the serum RPR titers in screening S-NS. The optimal cut-off points to recognize S-NS were serum RPR titer ≥1:4 and serum TPPA titer ≥1:2560 respectively. A parallel testing format of a serum RPR titer ≥1:2 and serum TPPA titer ≥1:1280 screened out 95.8% of S-NS and all confirmed cases of neurosyphilis. S-NS was independently associated with male sex, serum RPR titer ≥1:4, serum TPPA titer ≥1:2560, and elevated serum creatine kinase. Concurrence of these factors increased the likelihood of S-NS. CONCLUSIONS: Quantitation of serum TPPA is worthwhile and performs better than serum RPR in screening S-NS. Serum RPR, serum TPPA, male sex, and serum creatine kinase can predict S-NS. Moreover, patients with both a serum RPR titer <1:2 and a serum TPPA titer <1:1280 have a low probability of S-NS, suggesting that it is reasonable to reduce lumbar punctures in such individuals.

Evaluation of the diagnostic performance of an immunochromatographic test for Chlamydia trachomatis.

Objectives: To evaluate the diagnostic performance of different brands of immunochromatographic test (ICT) reagents for Chlamydia trachomatis using homogenized samples to provide a reference for reagent quality control. Methods: Eight commercially available ICT reagents were evaluated, of which three used the latex method and five used the colloidal gold method. Analytical performance evaluation using a pure culture broth of C. trachomatis, as well as clinical application validation using cervical epithelial cell samples acquired from the research subjects, were conducted. The concentration of C. trachomatis was quantified using a nucleic acid amplification test. Results: The limit of detection (LOD) of different ICT reagents in the analytical performance evaluation varied from 9.5 × 103 to 1 × 105 IFU/mL, and only one reagent met the LOD specified in the manufacturer's instructions. Likewise, only one reagent in the clinical application validation achieved the analytical LOD, four reagents were 2.1-4.2-fold of the analytical LODs, and three reagents failed to detect positive results in clinical samples. Conclusions: The diagnostic performance of different methods and different brands of ICT reagents in clinical practice was different from the manufacturer's instructions and the results of laboratory evaluation. The diagnostic performance of reagents should be evaluated before they are actually used in clinical practice.

Treponema pallidum protein Tp0136 promoting MMPs/TIMPs imbalance via PI3K, MAPK and NF-κB signalling pathways in HDVSMCs.

The invasive capability of Treponema. pallidum is central to its infection process. Matrix metalloproteinases (MMPs), which are specifically inhibited by the tissue inhibitors of metalloproteinases (TIMPs), play a pivotal role in promoting pathogenic invasion by destroying tissue barriers within the body. This study aimed to explore the effect of T. pallidum protein Tp0136 on the balance of MMPs/TIMPs in human dermal vascular smooth muscle cells (HDVSMCs) and the related underlying mechanisms. A number of in vitro studies were conducted to access the impact of recombinant Tp0136 protein on the balance of MMPs/TIMPs in HDVSMCs. The involvement of the PI3K, MAPK, and NF-κB signaling pathways in this process was also investigated. Tp0136 induced the mRNA and protein expressions of MMP1 in HDVSMCs in a concentration-dependent way. In addition, MMP1/TIMP1 and MMP1/TIMP2 ratios were also increased. Furthermore, the study demonstrated that treatment of HDVSMCs with Tp0136 activated the PI3K, MAPK, and NF-κB signaling pathways. Inhibition of PI3K, JNK, P38, and NF-κB, suppressed MMP1 expression and reduced the induction of MMP1/TIMP1 and MMP1/TIMP2 ratios by Tp0136. These findings demonstrate that Tp0136 enhanced the expression of MMP1 involving the PI3K, MAPK, and NF-κB signaling pathways in HDVSMCs, and thus generated the unbalance of MMPs/TIMP, which could contribute to the early spread of T. pallidum and pathogenesis of syphilis.

Mice with Fabp4-Cre ablation of Arid5b are resistant to diet-induced obesity and hepatic steatosis.

Mice with global deletion of Arid5b expression are lean and resistant to diet-induced obesity, and Arid5b is required for adipogenesis in a variety of in vitro models. To determine whether the lean phenotype of Arid5b-/- mice can be explained by its absence in adipose tissues, we generated mice with Fabp4-mediated ablation of Arid5b. Arid5b expression was ablated in adipocytes, from Fabp4-CREpos; Arid5bFLOX/FLOX (FSKO) mice. FSKO mice were not lean when maintained on standard chow, but males were resistant to weight gains when placed on high-fat diets (HFD). This was mainly due to decreased lipid accumulation in subcutaneous (inguinal) white adipose tissue (IWAT), and the liver. Lipid accumulation proceeded normally in gonadal WAT (GWAT) and glucose intolerance developed to the same degree in FSKO and WT controls when subjected to HFD. CD68-positive macrophages were also significantly reduced in both inguinal and gonadal fat depots. RNA-Seq analysis of IWAT adipocytes from FSKO mice on HFD revealed significant decreases in the expression of genes associated with inflammation. Although Arid5b expression was normal in livers of FSKO mice, tissue weight gains and triglyceride accumulation, and expression of genes involved in lipid metabolism were markedly reduced in livers of FSKO mice on HFD. These results suggest that Arid5b plays a critical role in lipid accumulation in specific WAT depots, and in the inflammatory signaling from WAT depots to liver that lead to lipid accumulation and hepatic steatosis.

Modulator recognition factor-2 is required for adipogenesis in mouse embryo fibroblasts and 3T3-L1 cells.

Previous study showed that mice lacking modulator recognition factor-2 (Mrf-2) were lean, with significant decreases in white adipose tissue. One postulated mechanism for the lean phenotype in Mrf-2 knockout mice is a defect in adipogenesis. In order to investigate this further, we examined the effects of Mrf-2 deficiency on adipogenesis in vitro. In mouse fibroblasts (MEFs) derived from Mrf-2(-/-) embryos, and in 3T3-L1 cells after knockdown of Mrf-2 by small interference RNA (siRNA) there was a potent inhibition of hormone-induced lipid accumulation, and significant decreases in the expression of the adipogenic transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor-gamma and the mature adipocyte genes they control. Transduction of Mrf-2(-/-) MEFs with a retroviral vector expressing the longer Mrf-2 splice variant (Mrf-2B) stimulated both gene expression and lipid accumulation. Because 3T3-L1 cells are committed to the adipocyte lineage, we used this simpler model system to examine the effects of Mrf-2 deficiency on adipocyte maturation. Analyses of both mRNA and protein revealed that knockdown of Mrf-2 in 3T3-L1 cells prolonged the expression of C/EBP homologous protein-10, a dominant-negative form of C/EBP. Consistent with these findings, suppression of Mrf-2 also inhibited the DNA-binding activity of C/EBPbeta. These data suggest that Mrf-2 facilitates the induction of the two key adipogenic transcription factors C/EBPalpha and peroxisome proliferator-activated receptor-gamma indirectly by permitting hormone-mediated repression of the adipogenic repressor C/EBP homologous protein-10.

Products of 12/15-lipoxygenase upregulate the angiotensin II receptor.

Angiotensin II and its type 1 receptor (AT1R) play important roles in the pathogenesis of renal disease and diabetic nephropathy. The 12/15-lipoxygenase pathway of arachidonate metabolism and its lipid products have also been implicated in diabetic nephropathy. However, it is unclear whether 12/15-lipoxygenase regulates expression of AT1R. In cultured rat mesangial cells, we found that the 12/15-lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) increased AT1R mRNA and protein expression, primarily by stabilizing AT1R mRNA. Pretreatment with 12(S)-HETE also amplified the signaling effects of angiotensin II, likely due to the increased AT1R expression. Levels of AT1R protein expression decreased when 12/15-lipoxygenase was knocked down with specific short hairpin RNA (shRNA) compared with control cells. Similarly, levels of the AT1 receptor, but not the AT2 receptor, were significantly lower in mesangial cells and glomeruli derived from 12/15-lipoxygenase knockout mice compared with control mice. Reciprocally, stable overexpression of 12/15-lipoxygenase increased AT1R expression in cultured mesangial cells. In vivo, modified siRNA targeting 12/15-lipoxygenase reduced glomerular AT1R expression in a diabetic mouse model. Interestingly, angiotensin II induced greater levels of 12/15-lipoxygenase, TGF-beta1, and fibronectin (FN) in AT1R-overexpressing mesangial cells compared with control cells. Therefore, oxidized lipids generated by the 12/15-lipoxygenase-mediated metabolism of arachidonic acid can enhance AT1R expression in mesangial cells and augment the profibrotic effects of angiotensin II.

Suppressed Expression of CXCL14 in Hepatocellular Carcinoma Tissues and Its Reduction in the Advanced Stage of Chronic HBV Infection.

INTRODUCTION: CXCL14 was a significantly under-expressed mRNA in hepatocellular carcinoma tissues according to our microarray analysis, as well as head and neck squamous cell carcinoma and cervical squamous cell carcinoma. CXCL14 was considered a tumor suppressor in some studies; however, its role in HBV infection has not been identified. METHODS: CXCL14 mRNA expression was quantified from 20 male HCC patients, and the fold change in cancer tissues was calculated by comparisons with normal adjacent tissues. Overall, 212 patients with chronic HBV infection and 180 HBV-free controls were recruited to investigate the association between CXCL14 polymorphisms and HBV progression as well as liver function parameters. Serum CXCL14 levels were determined by enzyme-linked immunosorbent assay (ELISA), and comparisons were made between different HBV status and different CXCL14 genotypes. RESULTS: The mRNA expression of CXCL14 was 0.33-fold in HCC tissues when compared with adjacent tissues. The frequencies of rs2237062 and rs2547, but not rs2237061, were significantly different between patients with mild hepatitis and moderate-to-severe hepatitis. Moreover, rs2237062 and rs2547 polymorphisms correlated with impaired liver function parameters. ELISA results suggested that HBV-free controls had the highest level of CXCL14, while mild hepatitis patients had low levels, and patients with moderate-to-severe hepatitis had the lowest level. GA+AA genotypes of rs2547 were associated with reduced levels of serum CXCL14 because it introduced a stop codon at residue 109. CONCLUSION: CXCL14 was significantly suppressed in HBV-related HCC tissues, and its polymorphisms were linked with advanced stage chronic HBV infection and impaired liver function.

Enhanced proatherogenic responses in macrophages and vascular smooth muscle cells derived from diabetic db/db mice.

Diabetes is associated with enhanced inflammatory responses and cardiovascular complications such as atherosclerosis. However, it is unclear whether similar responses are present in cells derived from experimental animal models of diabetes. We examined our hypothesis that macrophages and short-term cultured vascular smooth muscle cells (VSMCs) derived from obese, insulin-resistant, and diabetic db/db mice would exhibit increased proatherogenic responses relative to those from control db/+ mice. We observed that macrophages from db/db mice exhibit significantly increased expression of key inflammatory cytokines and chemokines as well as arachidonic acid-metabolizing enzymes cyclooxygenase-2 and 12/15-lipoxygenase that generate inflammatory lipids. Furthermore, VSMCs derived from db/db mice also showed similar enhanced expression of inflammatory genes. Expression of inflammatory genes was also significantly increased in aortas derived from db/db mice. Both macrophages and VSMCs from db/db mice demonstrated significantly increased oxidant stress, activation of key signaling kinases, and transcription factors cAMP response element-binding protein and nuclear factor-kappaB, involved in the regulation of atherogenic and inflammatory genes. Interestingly, VSMCs from db/db mice displayed enhanced migration as well as adhesion to WEHI mouse monocytes relative to db/+. Thus, the diabetic milieu and a potential hyperglycemic memory can induce aberrant behavior of vascular cells. These new results demonstrate that monocyte/macrophages and VSMCs derived from db/db mice display a "preactivated" and proinflammatory phenotype associated with the pathogenesis of diabetic vascular dysfunction and atherosclerosis.

Risk factors and molecular epidemiology of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in Xiamen, China.

OBJECTIVES: The aim of this study was to evaluate the risk factors for pneumonia due to extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) and to analyse the molecular epidemiology of ESBL-KP in Xiamen, China. METHODS: A case-control study was conducted at Zhongshan Hospital from January 2014 to August 2015. Medical records of patients with nosocomial pneumonia caused by K. pneumoniae were collected. A total of 40 cases with ESBL-KP infection and 90 controls with non-ESBL-KP infection were included. The sequence types (STs) of the 40 ESBL-KP strains were determined by multilocus sequence typing (MLST). RESULTS: Univariate analysis primarily revealed an association between the following seven risk factors and ESBL-KP infection (P<0.10): length of hospitalisation; use of cephalosporins; use of quinolones; presence of a nasogastric tube; presence of an intravenous catheter; mechanical ventilation; and cerebrospinal fluid drainage. Furthermore, multivariate analysis revealed that use of cephalosporins and presence of a nasogastric tube were independent risk factors for ESBL-KP infection (P<0.05), with adjusted odds ratios of 3.473 [95% confidence interval (CI) 1.105-10.911; P=0.033] and 2.488 (95% CI 1.083-5.715; P=0.032), respectively. MLST identified 28 STs. The main STs were ST23 (10.0%) and ST37 (10.0%); three novel STs were identified. CONCLUSIONS: Use of cephalosporins and presence of a nasogastric tube are independent risk factors for ESBL-KP infection. In addition, the discovery of three novel STs serves as a reminder to continuously monitor outbreaks of ESBL-KP infection.

Serological Response Predicts Normalization of Cerebrospinal Fluid Abnormalities at Six Months after Treatment in HIV-Negative Neurosyphilis Patients.

This study aimed to determine whether a serological response could predict the normalization of cerebrospinal fluid (CSF) abnormalities at 6 months after treatment in human immunodeficiency virus (HIV)-negative neurosyphilis patients. A total of 123 neurosyphilis patients were recruited at baseline, 58 of these patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study. Before treatment, the CSF rapid plasma reagin (RPR) titer, CSF Treponema pallidum particle agglutination (TPPA) titer, CSF leukocyte count, and CSF protein concentration were correlated with both serum RPR and TPPA titers. At 6 months after treatment, 28 and nine patients achieved serological responses of RPR and TPPA tests, respectively. The sensitivities of the serological response of RPR and TPPA tests for identifying the normalization of CSF abnormalities were 60.0∼83.3% and 17.1~22.2%, respectively; and 75.0∼91.3% of patients showing serological response of RPR test also achieved CSF normalization, suggesting that the serological response could predict CSF normalization to some degree. Particularly, in patients with ≥8-fold decreases in the serum RPR titer, the CSF RPR, CSF leukocyte count, and CSF protein concentration had normalized, and follow-up lumbar puncture could be reduced considering the resolution of neurological symptoms.

Re-evaluating the sensitivity of the rabbit infectivity test for Treponema pallidum in modern era.

BACKGROUND: The rabbit infectivity test (RIT) was previously described as a highly-sensitive method for clinically detecting Treponema pallidum. But our primary study indicated this result may have changed in current antibiotics era. METHODS: By inoculating rabbits testis with cerebrospinal fluid (CSF) (n=63) and exudate from hard chancre lesions (n=13), we re-evaluated the sensitivity of RIT in modern era. All isolated T. pallidum strains from the RIT were performed for the strain type based on "CDC subtype/tp0548" method. Chi-square and Fisher's exact tests were used to determine the statistical significance of differences across data sets. RESULTS: Result indicated that 2 of 63 CSF (2/63, 3.17%) and 5 of 13 lesion exudate samples (5/13, 38.47%) were positive in the RIT, with a much longer time to detection for CSF samples. Only 1 of 28 samples from patients who admitted treatment with antibiotics prior to clinical exam was positive in the RIT; while 6 of 48 patients, who admitted no recent exposure to antibiotics or was unclear about the medical history, were positive in RIT. DNA sequence analysis revealed 6 strains of 14d/f subtype and one strain of 14a/f subtype. CONCLUSIONS: In conclusions, RIT is no longer a highly sensitive method for detecting T. pallidum in clinical samples as before, and is not inadequately considered to be a reference method for measuring the sensitivity of other new methods, such as the PCR. These data represent the first reexamination of the sensitivity of RIT in the post-antibiotic era with a large clinical sample.

Elevated glucose and diabetes promote interleukin-12 cytokine gene expression in mouse macrophages.

Inflammation is emerging as an important mechanism for micro- and macrovascular complication of diabetes. The macrophage plays a key role in the chronic inflammatory response in part by generating particular cytokines. IL-1beta, IL-6, IL12, IL-18, TNFalpha, and interferon-gamma are produced primarily in macrophages and have been associated with accelerated atherosclerosis and altered vascular wall function. In this study, we evaluated the effect and mechanism of high glucose (HG) on gene expression of these cytokines in mouse peritoneal macrophages (MPM). HG led to a 2-fold increase in the mRNA expression of these cytokines, with IL-12 showing the highest activation (5.4-fold) in a time-dependent (3-12 h) and dose-dependent (10, 17.5, and 25 mmol/liter) manner. The effects were specific to HG because mannitol and 3-O-methyl-glucose had no effect on cytokine mRNA expression. HG also increased IL-12 protein accumulation from MPM. We also explored the role of induced and spontaneous diabetes on inflammatory cytokine expression in MPM. Increases in expression in MPM of multiple inflammatory cytokines, including a 20-fold increase in IL-12 mRNA, were observed in streptozotocin-induced type 1 diabetic mice as well as type 2 diabetic db/db mice, suggesting that cytokine gene expression is increased by hyperglycemia in vivo. We next explored potential mechanisms of HG-induced increases in IL-12 mRNA. HG increased the activity of protein kinase C, p38 MAPK (p38), c-Jun terminal kinase, and inhibitory-kappaB kinase in MPM. Furthermore, inhibitors of these signaling pathways significantly reduced HG-induced IL-12 mRNA expression in MPM. These results provide evidence for a potentially important mechanism linking elevated glucose and diabetes to inflammation.

A chimeric humanized single-chain antibody against the type I insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-I.

Insulin-like growth factors (IGFs) stimulate breast cancer proliferation, motility, and survival. The type I IGF receptor (IGF1R) mediates the effects of IGF-I. Thus, inhibition of IGF1R activation could inhibit IGF action in breast cancer cells. A single-chain antibody directed against IGF1R (IGF1R scFv-Fc) has been shown to partially inhibit xenograft growth of MCF-7 cells in athymic mice. In this study, we have examined the effects of scFv-Fc on IGF1R signaling in the estrogen receptor-positive (ER+) MCF-7 breast cancer cells in vitro and in vivo. The antibody stimulated IGF1R activation in vitro in MCF-7 cells and was unable to block IGF-I effects. The antibody also stimulated proliferation of MCF-7 cells in monolayer growth assays. To determine how scFv-Fc could stimulate in vitro growth yet inhibit in vivo tumor growth, we examined the effect of scFv-Fc on IGF1R expression. In MCF-7 cells, scFv-Fc down-regulated IGF1R levels after 2 h, and the levels were greatly reduced after 24 h. In contrast, IGF-I treatment over the same time period did not affect IGF1R levels. Twenty-four-h pretreatment of cells with scFv-Fc blocked IGF-I mediated phosphorylation of insulin receptor substrate-1 and subsequent extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phosphatidylinositol 3'-kinase activation. In contrast, cells treated with 5 nM IGF-I for 24 h still retained the ability to further activate downstream signaling pathways in response to IGF-I. Moreover, pretreatment of MCF-7 cells with scFv-Fc rendered them refractory to further proliferation induced by additional IGF-I. Twenty-four-h pretreatment of cells with scFv-Fc also inhibited IGF-I stimulated anchorage-independent growth. scFv-Fc did not enhance antibody-dependent cell-mediated cytotoxicity. In vivo, treatment of mice bearing MCF-7 xenograft tumors with scFv-Fc resulted in near complete down-regulation of IGF1R. Our data show that scFv-Fc stimulates biochemical activation of IGF1R, then causes receptor down-regulation, making MCF-7 cells refractory to additional IGF-I exposure. These results indicate that such chimeric single-chain antibodies against IGF1R have future potential in breast cancer therapy by causing down-regulation of receptor.

A negative nontreponemal and/or specific antitreponemal IgM test does not exclude active infectious syphilis: evidence from a rabbit infectivity test: A case report.

BACKGROUND: The diagnostic criteria for active infectious syphilis in the clinic are important matter of controversy and debate. So far, clinicians habitually do use the negative results of the nontreponemal and/or the specific antitreponemal IgM as the evidences of disease-free or active infection-free status. METHOD: We present a case study involving a patient who was admitted to Zhongshan Hospital because of cerebral infarct. Clinical examination indicated he had a history of latent syphilis with negative nontreponemal and specific antitreponemal IgM tests. The cerebrospinal fluid sample from the patient was inoculated into seronegative New Zealand rabbit. RESULTS: Motile Treponema pallidum was detected by a rabbit infectivity test in the patient's cerebrospinal fluid. This syphilis strain was confirmed by DNA subtyping form of "centers for disease control subtype/tp0548 sequence type", and the strain type was 14d/f. Treatment with benzathine penicillin provided no apparent benefit, but treatment with aqueous crystalline penicillin G, especially recommended for neurosyphilis, led to disease regression. No evidence of cerebral infarct was observed during a 2-year follow-up period. CONCLUSION: The definitive differential diagnosis of active infectious syphilis should be reconsidered. Moreover, selecting the appropriate penicillin preparation is important because T pallidum can reside in sequestered sites. It is necessary to treat a patient with known invasion of the central nervous system with aqueous crystalline penicillin G, if previous treatment for syphilis failed and patients had some clinical neurological presentation that is otherwise unexplained, but that could represent neurosyphilis. Additional studies are needed to confirm the results in other syphilis patients.

Macrophage migration inhibitory factor as a novel cerebrospinal fluid marker for neurosyphilis among HIV-negative patients.

BACKGROUND: Neurosyphilis (NS) is difficult to diagnose, especially in syphilis patients with negative cerebrospinal fluid (CSF) rapid plasma reagin (RPR) or Venereal Disease Research Laboratory (VDRL) tests. METHODS: We conducted a cross-sectional study and an analysis of macrophage migration inhibitory factor (MIF) in syphilitic patients to identify a novel marker for the diagnosis of NS, with a focus on probable NS (NS with negative VDRL/RPR tests). For this purpose, CSF and serum MIF concentrations were determined in 43 NS and 43 syphilis/non-NS (N-NS) patients at the Zhongshan Hospital of the Medical College of Xiamen University from July 2014 to June 2015. Sixty-three blood donors were used as healthy controls. RESULTS: NS patients had higher CSF (median [IQR]: 8.77ng/ml [4.76-19.13]) and serum (52.58ng/ml [28.31-95.94]) MIF concentrations than N-NS patients did (4.08 [2.21-9.68] and 34.30 [19.77-59.75], respectively). Using a cut-off point of 6.63ng/ml, CSF MIF had a sensitivity of 74.42% and a specificity of 67.74% for the diagnosis of NS. The sensitivity was higher than that of CSF RPR (39.53%) and increased protein (48.84%) tests and similar to that of CSF pleocytosis (67.44%). Additionally, the sensitivity of CSF MIF, which was 92.31% for the diagnosis of probable NS, was higher than that of CSF pleocytosis (65.38%) and increased protein (53.85%) tests. By integrating all CSF parameters (pleocytosis, increased protein and MIF), the sensitivity would be improved to 100% by parallel testing, which would avoid missed diagnoses. Moreover, the specificity would be improved to 100% by the serial testing algorithm, which would again avoid misdiagnosis. CONCLUSIONS: CSF MIF concentrations can be used as a novel CSF marker to establish or exclude a diagnosis of NS.