[Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism].

Objective: To establish a high glucose senescent model of human dermal fibroblasts (HDFs), and to investigate the effects of exosomes derived from human decidua mesenchymal stem cells (dMSCs) on the proliferation, migration, and apoptosis of senescent HDFs and possible mechanism. Methods: The experimental research method was used. From January to March 2021, discarded foreskin tissue was collected for isolation and culture of primary HDFs from 4 male phimosis patients (aged 18-22 years) admitted for circumcision in the Fourth Medical Center of the PLA General Hospital. The 6th passage of HDFs were taken and divided into low glucose group and high glucose group according to the random number table, and subsequently cultured in low-glucose complete medium and high-glucose complete medium, respectively, with medium changed every 72 h without subculturing. After 10 days of culture, the cells were taken and measured for cellular senescence using the ß-galactosidase kit at 24 h after seeding; the expression of senescence-related proteins p16 and p53 was assessed by Western blotting at 48 h after seeding; cell proliferation was detected at 24, 48, and 72 h after seeding using the cell counting kit 8 (CCK-8) method; the cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) staining method, cell cycle and apoptosis were measured by flow cytometry after 48 h of seeding; Transwell experiment was used for the calculation of cell migration rate at 24 h after seeding. The human dMSCs were taken and cultured for 48-72 h from which the exosomes were extracted by differential high speed centrifugal method. The morphology of dMSC exosomes was observed by transmission electron microscopy, the particle size distribution of dMSC exosomes was measured by nanoparticle tracking analysis, and the expression of dMSC-exosomes marker proteins CD9 and tumor susceptibility gene101 (TSG101) were detected by Western blotting. The dMSC exosomes and high-glucose complete medium-induced senescent HDFs were co-cultured for 24 hours, then PKH67 kit was used to detect the uptake of exosomes by HDFs. High-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group, high glucose+low concentration of exosomes group, and high glucose+high concentration of exosomes group according to the same method above. The high-glucose complete medium with equal volume of phosphate buffered saline, dMSC exosomes with final concentration of 50 µg/mL, and dMSC exosomes with final concentration of 100 µg/mL were added to the corresponding groups for conventional cell culture, respectively. After grouped, the cell proliferation, cell cycle and apoptosis as well as cell migration were detected by CCK-8 method and EdU staining method, flow cytometry, and Transwell experiment at the corresponding time points as before, respectively. Based on the previous results, high-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group and high glucose+high concentration of exosomes group for the same treatment. After being grouped and cultured for 48 h, real-time fluorescent quantitative polymerase chain reaction was used to evaluate the mRNA expression of senescent-related microRNA (miR)-145-5p, miR-498, miR-503-5p, calcium/calmodulin dependent protein kinase 1D (CAMK1D), phosphates and tensin homologue deleted on chromosome ten (PTEN) gene, and Cyclin D1 in high glucose alone group and high glucose+high concentration of exosomes group. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, and independent sample t test. Results: At 24 h after seeding, the rate of ß-galactosidase-positive staining of HDF in high glucose group was (38.4±4.2)%, which was significantly higher than (16.5±2.2)% of low glucose group (t=4.65, P<0.01). At 48 h after seeding, the expression levels of senescence-related proteins p16 and p53 both were significantly higher in HDFs of high glucose group than those in low glucose group (with t values of 11.85 and 3.02, respectively, P<0.05 or P<0.01). At 0, 24, 48, and 72 h after seeding, the cell proliferation viability of HDFs in high glucose group was all significantly lower than in low glucose group (with t values of 4.13, 9.90, and 15.12, respectively, P<0.01). At 48 h after seeding, the rate of EdU-positive staining of HDFs in high glucose group was obviously lower than that of low glucose group (t=3.83, P<0.05). At 48 h after seeding, the percentage of G2/M+S subpopulations in three subpopulations (G0/G1, S, and G2/M) of HDF cycle was significantly lower in high glucose group than that in low glucose group (t=8.74, P<0.01). At 24 h after seeding, the number of HDFs migrated through the filter membrane to the lower chamber was 37±6 in high glucose group, which was significantly less than 74±7 in low glucose group (t=8.42, P<0.01). At 48 h after seeding, the HDF apoptosis rate was significantly higher in high glucose group than in low glucose group (t=8.48, P<0.01). The dMSC exosomes were cup-shaped or round vesicles with well-defined edges and uniform size distribution. The size of dMSC exosomes was basically in the range of 80-200 nm. Exosomal markers including CD9 and TSG101 were positively presented on the dMSC exosomes. After being co-cultured for 24 hours, the dMSC exosomes were taken up intracellularly by HDFs and mainly distributed around the nucleus of HDFs. After being grouped and cultured for 24, 48, and 72 h, the HDF proliferation viabilities in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 6.36, 6.10, 7.76, 8.92, 12.17, and 10.74, respectively, P<0.01), the HDF proliferation viability in high glucose+high concentration of exosomes group was significantly higher than in high glucose+low concentration of exosomes group (with t values of 7.92, 4.82, and 4.72, respectively, P<0.01). After being grouped and cultured for 48 h, the percentages of EdU-positive HDFs in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 5.32 and 9.88, respectively, P<0.01), the percentage of EdU-positive HDFs in high glucose+high concentration of exosomes group was notably higher than in high glucose+low concentration of exosomes group (t=5.27, P<0.01). After being grouped and cultured for 48 h, the proportion of G0/G1 subpopulation in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was distinctly lower (with t values of 3.81 and 4.31, respectively, P<0.05), while the proportion of G2/M+S subpopulation was markedly higher (with t values of 3.81, 4.31, respectively, P<0.05) than in high glucose alone group. After being grouped and cultured for 24 h, the number of HDFs migrated through the filter membrane in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was significantly higher than in high glucose alone group (with t values of 10.14 and 13.39, respectively, P<0.01), the number of HDFs migrated through the filter membrane in high glucose+high concentration of exosomes group was significantly increased than in high glucose+low concentration of exosomes group (t=6.27, P<0.01). After being grouped and cultured for 48 h, the HDF apoptosis rates in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly lower than in high glucose alone group (with t values of 3.72 and 5.53, respectively, P<0.05 or P<0.01). After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of miR-145-5p and miR-498 were both obviously higher (with t values of 13.03 and 8.90, respectively, P<0.01), while the mRNA expression level of miR-503-5p was significantly lower (t=3.85, P<0.05) in high glucose+high concentration of exosomes group. After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of CAMK1D and PTEN gene were both significantly lower (with t values of 8.83 and 5.97, respectively, P<0.01), while the mRNA expression level of Cyclin D1 was significantly higher in high glucose+high concentration of exosomes group (t=4.03, P<0.05). Conclusions: The dMSC exosomes are capable of improving cell proliferation and migration, and inhibiting cell apoptosis of high-glucose senescent HDFs. This may be related to the mechanism by which the increased expressions of intracellular miR-145-5p and miR-498 inhibit the expression of CAMK1D and PTEN gene, and the decreased expression of miR-503-5p promote the expression of Cyclin D1.

[Vigilance against a highly lethal insecticide chlorfenapyr poisoning (report of 4 cases and literature review)].

In order to improve the clinical attention to the poisoning of chlorfenapyr, the diagnosis and treatment strategy of chlorfenapyr poisoning were discussed. This paper collected 4 cases of chlorfenapyr in the emergency department of the Second Hospital of Hebei Medical University and 4 cases of literature review, summarized the clinical characteristics of pesticide poisoning cases containing chlorfenapyr in China, and summarized and analyzed the clinical data of the cases. Seven of the 8 patients died from poisoning by chlorfenapyr. Exposure to chlorfenapyr through respiratory tract and digestive tract showed high mortality. Fever, hyperhidrosis, elevated muscle enzymes and progressive central nerve damage were its prominent clinical characteristics. Most of the initial symptoms of exposure were not serious. Some patients, especially those with low exposure dose, had a relatively stable stage with or without clinical diagnosis and treatment. In case of sweating, obvious fever and disturbance of consciousness, the condition would deteriorate rapidly, respiratory and circulatory failure and eventually die. With the increase of production capacity and market launch, people have more opportunities to be exposed to chlorfenapyr. It is urgent to strengthen the basic and clinical research of chlorfenapyr poisoning; Attention should be paid to the observation and treatment in the initial stable stage of poisoning, which can be used as a reference for the treatment of oxidative phosphoric acid dissolving coupling agent (sodium pentachlorophenol) poisoning.

Correction: Deacetylation of HSPA5 by HDAC6 leads to GP78-mediated HSPA5 ubiquitination at K447 and suppresses metastasis of breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Prucalopride inhibits lung cancer cell proliferation, invasion, and migration through blocking of the PI3K/AKT/mTor signaling pathway.

Lung cancer is the main cause of cancer incidence and mortality around the world. Prucalopride is an agonist for the 5-hydroxytryptamine 4 receptor, but it was unknown whether prucalopride could be used to treat lung cancer. To investigate the biological effects of prucalopride on proliferation, apoptosis, invasion, and migration of lung cancer cells, and its underlying molecular mechanism in the progression of lung cancer, we performed this study. The Cell Counting Kit 8 assay was used to measure the proliferation of A549/A427 lung cancer cells treated with prucalopride. Transwell assay was applied to evaluate cell invasion and migration. Cell apoptosis was detected by flow cytometry and Western blot analyses. The expression levels of related proteins in the PI3K/AKT/mTor signaling pathway were analyzed by Western blotting. Prucalopride inhibited the proliferation, invasion, and migration of A549/A427 human lung cancer cells. It also induced autophagy and apoptosis and decreased the expression of the phosphorylated protein kinase B (AKT) and mammalian target of rapamycin (mTor) in these cells. This study implied an inhibitory role for prucalopride in the progression of human lung cancer.

miR-520h is crucial for DAPK2 regulation and breast cancer progression.

This corrects the article DOI: 10.1038/onc.2015.168.

Relieving effect of Artemisia argyi aqueous extract on immune stress in broilers.

This experiment aimed to investigate the relieving action of Artemisia argyi aqueous extract (AAE) on immune stress in broiler chickens. A 2 × 2 factorial design was used to test the effect of 2 dietary treatments (adding 0 or 1000 mg/kg AAE) and 2 immune stress treatments (injecting saline or lipopolysaccharide (LPS)). A total of 96 one-day-old Arbor Acres (AA) broilers were randomly divided into four treatment groups with six replicates, four birds in each replicate. Broilers in Treatment groups 1 and 2 were fed with the basal diet, and those in Treatment groups 3 and 4 were fed with the experimental diet supplemented with 1000 mg/kg AAE. On days 14, 16, 18 and 20, broilers in both Treatments 1 and 3 were injected intra-abdominally with LPS solution at the dose of 500 µg LPS per kg BW with the LPS dissolved in sterile saline at a concentration of 100 µg/ml, and those in Treatments 2 and 4 were injected intra-abdominally with equal amount of sterile 0.9% saline. Blood samples were collected on days 21 and 28. The results showed that dietary supplementation of AAE prevented reductions in average daily gain (ADG) and average daily feed intake (ADFI) of broilers caused by LPS on d 15-21. On day 21, the injection of LPS increased serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT); meanwhile, feeding AAE reduced the rise of CORT caused by LPS. Immune parameters such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), immunoglobulin G (IgG) and immunoglobulin A (IgA) were also improved by LPS, but the content of IL-2 and IgG in broilers fed with AAE diet was significantly lower than that of broilers fed with control diet. All the data suggested that diets supplemented with AAE could relieve the immune stress response of broilers.

miR-520h is crucial for DAPK2 regulation and breast cancer progression.

MicroRNAs (miRNAs) are small RNAs that suppress gene expression by their interaction with 3'untranslated region of specific target mRNAs. Although the dysregulation of miRNAs has been identified in human cancer, only a few of these miRNAs have been functionally documented in breast cancer. Thus, defining the important miRNA and functional target involved in chemoresistance is an urgent need for human breast cancer treatment. In this study, we, for the first time, identified a key role of miRNA 520h (miR-520h) in drug resistance. Through protecting cells from paclitaxel-induced apoptosis, expression of miR-520h promoted the drug resistance of human breast cancer cells. Bioinformatics prediction, compensatory mutation and functional validation further confirmed the essential role of miR-520h-suppressed Death-associated protein kinase 2 (DAPK2) expression, as restoring DAPK2 abolished miR-520h-promoted drug resistance, and knockdown of DAPK2 mitigated cell death caused by the depletion of miR-520h. Furthermore, we observed that higher level of miR-520h is associated with poor prognosis and lymph node metastasis in human breast cancer patients. These results show that miR-520h is not only an independent prognostic factor, but is also a potential functional target for future applications in cancer therapeutics.

Deacetylation of HSPA5 by HDAC6 leads to GP78-mediated HSPA5 ubiquitination at K447 and suppresses metastasis of breast cancer.

Heat-shock protein 5 (HSPA5) is a marker for poor prognosis in breast cancer patients and has an important role in cancer progression, including promoting drug resistance and metastasis. In this study, we identify that the specific lysine residue 447 (K447) of HSPA5 could be modified with polyubiquitin for subsequent degradation through the ubiquitin proteasomal system, leading to the suppression of cell migration and invasion of breast cancer. We further found that GP78, an E3 ubiquitin ligase, interacted with the C-terminal region of HSPA5 and mediated HSPA5 ubiquitination and degradation. Knock down of GP78 significantly increased the expression of HSPA5 and enhanced migration/invasive ability of breast cancer cells. Knock down of histone deacetylase-6 (HDAC6) increased the acetylation of HSPA5 at lysine residues 353 (K353) and reduced GP78-mediated ubiquitination of HSPA5 at K447 and then increased cell migration/invasion. In addition, we demonstrate that E3 ubiquitin ligase GP78 preferentially binds to deacetylated HSPA5. Notably, the expression levels of GP78 inversely correlated with HSPA5 levels in breast cancer patients. Patients with low GP78 expression significantly correlated with invasiveness of breast cancer, advanced tumor stages and poor clinical outcome. Taken together, our results provide new mechanistic insights into the understanding that deacetylation of HSPA5 by HDAC6 facilitates GP78-mediated HSPA5 ubiquitination and suggest that post-translational regulation of HSPA5 protein is critical for HSPA5-mediated metastatic properties of breast cancer.

Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification.

OBJECTIVE/METHOD: Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). RESULTS: Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. CONCLUSION: This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic.

MiR-520h-mediated FOXC2 regulation is critical for inhibition of lung cancer progression by resveratrol.

Resveratrol, a phytochemical found in various plants and Chinese herbs, is associated with multiple tumor-suppressing activities, has been tested in clinical trials. However, the molecular mechanisms involved in resveratrol-mediated tumor suppressing activities are not yet completely defined. Here, we showed that treatment with resveratrol inhibited cell mobility through induction of the mesenchymal-epithelial transition (MET) in lung cancer cells. We also found that downregulation of FOXC2 (forkhead box C2) is critical for resveratrol-mediated suppression of tumor metastasis in an in vitro and in vivo models. We also identified a signal cascade, namely, resveratrol-∣miRNA-520h-∣PP2A/C-∣Akt → NF-κB → FOXC2, in which resveratrol inhibited the expression of FOXC2 through regulation of miRNA-520h-mediated signal cascade. This study identified a new miRNA-520h-related signal cascade involved in resveratrol-mediated tumor suppression activity and provide the clinical significances of miR-520h, PP2A/C and FOXC2 in lung cancer patients. Our results indicated a functional link between resveratrol-mediated miRNA-520h regulation and tumor suppressing ability, and provide a new insight into the role of resveratrol-induced molecular and epigenetic regulations in tumor suppression.

Effects of dietary polysavone (Alfalfa extract) and chlortetracycline supplementation on antioxidation and meat quality in broiler chickens.

1. A total of 360 1-d-old male commercial Arbor Acre broiler chickens were randomly assigned to 5 groups (6 replicates of 12 birds each) to evaluate the dietary effects of polysavone (0·5, 1·0 and 1·5 g/kg), a natural extract from alfalfa, and 0·15 g/kg chlortetracycline (CTC) on growth performance, antioxidation and meat quality of broiler chickens. 2. Over the 6-week study, feed intake increased significantly with CTC supplementation and final body weight (BW) was significantly higher for 1·0 g/kg polysavone and 0·15 g/kg CTC treatments. Feed:gain ratio was not significantly affected by the dietary treatments. 3. At 3 weeks of age, serum total superoxide dismutase (T-SOD) activity for all polysavone treatments was significantly higher than controls, liver T-SOD activity in 1·5 g/kg polysavone group was significantly higher than the control and CTC groups, and serum glutathione peroxidase (GSHPx) activity for 1·5 g/kg polysavone and liver GSHPx activity in all polysavone groups were significantly higher than CTC. 4. At 6 weeks of age, serum and liver T-SOD activity in 1·5 g/kg polysavone group and liver GSHPx activity for all polysavone treatments were higher significantly than the control and CTC groups, and serum malondialdehyde (MDA) content for all polysavone treatments was significantly lower than CTC. 5. Breast muscle T-SOD activity and pH value at 6 weeks of age were significantly higher and MDA content was significantly lower in 1·0 and 1·5 g/kg polysavone groups than in the control and CTC groups. Breast muscle shear force was significantly lower in l·5 g/kg polysavone group compared with the control, and drip loss for all polysavone treatments was significantly lower than CTC. 6. It was indicated that polysavone modulates antioxidation and modifies meat quality, but with no adverse effect on performance of broiler chickens, and that CTC can be beneficial to performance but has no beneficial effect on antioxidant function or meat quality.

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk.

AIMS: To develop a PCR-based assay to detect Prototheca zopfii (P. zopfii) and its mastitis-related subtype (genotype 2) directly from milk samples. METHODS AND RESULTS: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA-binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10(2) colony-forming units (CFU) ml(-1) for P. zopfii and 5 × 10(3) CFU ml(-1) for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. CONCLUSIONS: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.

Development of an imaging-planning program for screen/film and computed radiography mammography for breasts with short chest wall to nipple distance.

OBJECTIVE: Imaging breasts with a short chest wall to nipple distance (CWND) using a traditional mammographic X-ray unit is a technical challenge for mammographers. The purpose of this study is the development of an imaging-planning program to assist in determination of imaging parameters of screen/film (SF) and computed radiography (CR) mammography for short CWND breasts. METHODS: A traditional mammographic X-ray unit (Mammomat 3000, Siemens, Munich, Germany) was employed. The imaging-planning program was developed by combining the compressed breast thickness correction, the equivalent polymethylmethacrylate thickness assessment for breasts and the tube loading (mAs) measurement. Both phantom exposures and a total of 597 exposures were used for examining the imaging-planning program. RESULTS: Results of the phantom study show that the tube loading rapidly decreased with the CWND when the automatic exposure control (AEC) detector was not fully covered by the phantom. For patient exposures with the AEC fully covered by breast tissue, the average fractional tube loadings, defined as the ratio of the predicted mAs using the imaging-planning program and mAs of the mammogram, were 1.10 and 1.07 for SF and CR mammograms, respectively. The predicted mAs values were comparable to the mAs values, as determined by the AEC. CONCLUSION: By applying the imaging-planning program in clinical practice, the experiential dependence of the mammographer for determination of the imaging parameters for short CWND breasts is minimised.

The role of the VEGF-C/VEGFR-3 axis in cancer progression.

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) (also called VEGFR-3) is activated by its specific ligand, VEGF-C, which promotes cancer progression. The VEGF-C/VEGFR-3 axis is expressed not only by lymphatic endothelial cells but also by a variety of human tumour cells. Activation of the VEGF-C/VEGFR-3 axis in lymphatic endothelial cells can facilitate metastasis by increasing the formation of lymphatic vessels (lymphangiogenesis) within and around tumours. The VEGF-C/VEGFR-3 axis plays a critical role in leukaemic cell proliferation, survival, and resistance to chemotherapy. Moreover, activation of the VEGF-C/VEGFR-3 axis in several types of solid tumours enhances cancer cell mobility and invasion capabilities, promoting cancer cell metastasis. In this review, we discuss the novel function and molecular mechanism of the VEGF-C/VEGFR-3 axis in cancer progression.

Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments.

OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.

The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K/Akt pathway.

Interleukin-6 (IL-6), a multifunctional cytokine, has recently been implicated in human cervical cancer, though the mechanism remains elusive. This study demonstrates that the anti-apoptotic protein Mcl-1 and IL-6 was concomitantly expressed in human cervical cancer tissues and cell lines, but not in normal cervix tissues. Upon IL-6 treatment, Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated peaking at 4-8 h in human cervical cancer C33A cells. Supporting this observation, using anti-IL-6 or anti-IL-6 receptor antibody to interrupt the IL-6 autocrine loop in SiHa cells significantly reduced cellular level of Mcl-1. This study hypothesizes that the expression of Mcl-1 in cervical cancer cells is regulated by IL-6. The matter of which signaling pathways transduced by IL-6 is responsible for the Mcl-1 up-regulation is further investigated herein. Blocking the STAT3 or MAPK pathway with dominant-negative mutant STAT3F or the MEK inhibitor PD98059 failed to inhibit IL-6-mediated Mcl-1 expression. Meanwhile, the IL-6-induced Mcl-1 up-regulation was effectively abolished by treatment with PI 3-K inhibitors, LY294002. Additionally, overexpression of dominant-negative (dn) Akt in C33A cells could inhibit the IL-6-induced increase of Mcl-1. Finally, overexpression of IL-6 in C33A cells caused a markable resistance to apoptosis induced by doxorubicin or cisplatin. Transient transfection of IL-6-overexpressed cells with a mcl-1 antisense vector, leading to the attenuation of their apoptosis-resistant activity. In conclusion, the data herein suggest that IL-6 regulated the mcl-1 expression via a PI 3-K/Akt-dependent pathway that may facilitate the oncogenesis of human cervical cancer by modulating the apoptosis threshold.

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Progression of symptoms in the early and middle stages of Huntington disease.

OBJECTIVE: To delineate the progression of symptoms in the early and middle stages of Huntington disease (HD). DESIGN: A survey of individuals with symptomatic HD completed by a first-degree relative. SETTING: The National Huntington Disease Research Roster for Patients and Families, Indianapolis, Ind. PARTICIPANTS: The survey included 1238 individuals with a minimum of a 6-year history of symptomatic HD. MEASURES: Participating families completed a series of surveys, including the Affected Individual Questionnaire, which consists of 19 physical, emotional, and cognitive signs commonly thought to occur during disease progression. The respondent indicates if each of the symptoms occurred and, if so, at what time during the course of the disease: (1) within 1 year, (2) within 2 to 5 years, (3) within 6 to 10 years, (4) after more than 10 years, (5) has not occurred, or (6) "don't know." RESULTS: The symptoms are categorized into 6 onset periods. Involuntary movements are grouped alone as the earliest reported symptom. The second group is composed entirely of mental and emotional symptoms, including sadness, depression, and difficult to get along with. The third group includes clumsiness, sexual problems, lack of motivation, and suspiciousness/paranoia. As the disease progresses, a variety of motor, emotional/behavioral, and cognitive symptoms are experienced, including unsteadiness, trouble holding onto things, trouble walking, changes in sleeping patterns, delusions and hallucinations, intellectual decline, and memory loss. With the approach of late-stage HD, affected individuals begin to experience speech difficulty and weight loss. In the late stage, patients lose bowel and bladder control. CONCLUSIONS: Even though the symptoms of HD are fairly well characterized, their progression, especially in the early and middle stages, remains uncertain. Clarification of the disease progression is vital to improved understanding of the pathogenesis of HD and to the evaluation of therapeutic agents that are designed to slow the progression of disease. The results of this study assist in clarifying HD progression from early involuntary movements and emotional changes to more overt motor symptoms and difficulty with activities of daily living.

Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.