Inhibitory Effects of Myrtucommuacetalone 1 (MCA-1) from Myrtus communis on Inflammatory Response in Mouse Macrophages.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.

Activation of interleukin-6 and -8 expressions by methylmercury in human U937 macrophages involves RelA and p50.

The accumulation of macrophages has been observed around lesions of the brain in patients with Minamata disease. In this condition, mercury has been detected histochemically in macrophages throughout the brain. However, the role of macrophages in the neurotoxicity of methylmercury (MeHg) and the molecular mechanisms of their response to MeHg exposure remain to be elucidated. Here, we investigated how MeHg affects the expression of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 in cultured human U937 macrophages. Compared with controls, IL-6 and IL-8 mRNA expression was maximally induced in U937 macrophages after treatment with 10 µM MeHg for 6 h. The protein secretion of IL-6 and IL-8 was significantly stimulated by MeHg in U937 macrophages. Results from luciferase reporter assay indicated functional activation of nuclear factor kappa B and the involvement of subunit RelA and p50 in MeHg-induced IL-6 and IL-8 activation, which was confirmed by siRNA knockdown experiments. MeHg exposure at 4 µM also significantly induced IL-8 expression in U-87 MG cells at mRNA and protein level, indicating that IL-8 induction might be a general mode of action of MeHg treatment among different cell types. These results indicate a possible involvement of an early inflammatory response, including IL-6 and IL-8 expression in the pathogenesis of MeHg. N-acetyl-l-cysteine suppressed MeHg-induced activation of IL-6 and IL-8 mRNA expression in U937 macrophages, indicating the effectiveness of N-acetyl-l-cysteine as a therapeutic drug in MeHg-induced inflammation. Copyright © 2016 John Wiley & Sons, Ltd.

Suppression of methylmercury-induced IL-6 and MCP-1 expressions by N-acetylcysteine in U-87MG human astrocytoma cells.

AIMS: The aim of this study was to clarify the involvement of oxidative stress in methylmercury (MeHg)-induced pro-inflammatory cytokine expressions and the suppressive effects of N-acetylcysteine (NAC) in MeHg-induced cytokine expression. MATERIALS AND METHODS: Using U-87-MG human astrocytoma cell line, interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 expressions induced by 4 µM MeHg were measured at mRNA and protein levels. Hydrogen peroxide (H2O2) and superoxide anion (O2(-)) were quantified by flow-cytometry analysis. To examine the suppressive effects of NAC on the cytokine expressions among different timing of NAC treatment, cells were treated with 0.5 or 5mM NAC before, simultaneously, or after MeHg administration. KEY FINDINGS: MeHg exposure at 4 µM, a non-cytotoxic concentration, significantly induced MCP-1 and IL-6 expressions at both mRNA and protein levels. A significant increase of H2O2 production but not O2(-) was observed. MeHg-induced expression of MCP-1 and IL-6 mRNA was reduced by 10-20% in the presence of 5mM NAC (co-treatment experiment) compared to cells treated with MeHg only. Pre-treatment of cells with 0.5 or 5mM NAC at 0.5 or 1h and its subsequent washout before MeHg addition suppressed MCP-1 and IL-6 cytokine expressions. Post-treatment of cells with NAC after MeHg addition also suppressed the cytokine induction, but the magnitude of suppression was evidently lower than in co-treated cells even though the H2O2 generation was almost completely suppressed by NAC. SIGNIFICANCE: NAC may effectively suppress the MeHg-induced cytokine production through both, inhibition of reactive oxygen species as well as extracellular chelation of MeHg in astrocytes.

N­acetyl­L­cysteine reduces arsenite­induced cytotoxicity through chelation in U937 monocytes and macrophages.

In the present study, in order to clarify the preventive mechanism of N­acetyl­L­cysteine (NAC) on arsenite­induced apoptosis in U937 cells, which lack functional p53, the cytotoxicity among U937 cells [monocytes and 12­O­tetradecanoylphorbol­13­acetate (TPA)­treated macrophages] receiving NAC treatment at different times post arsenite treatment was examined. TPA­treated macrophages were more resistant to arsenite­induced apoptosis than monocytes, which may be associated with the induction of Bcl­2 expression. Pretreatment with 20 mM NAC prior to arsenite exposure suppressed apoptosis up to 75% in the monocytes and 100% in the macrophages. However, 6­h NAC pretreatment and subsequent washing out of NAC from the culture medium prior to arsenite treatment did not inhibit the arsenite­induced apoptosis. Post­treatment by NAC up to 1 h following arsenite exposure almost completely inhibited the cytotoxic effects of arsenite in U937 monocytes and macrophages. The results of the current study indicate that the preventive mechanism of NAC on arsenite­induced apoptosis in U937 monocytes and macrophages mainly involves chelation of arsenite in culture medium.

New inhibitors of ROS generation and T-cell proliferation from Myrtus communis.

Phytochemical investigation on Myrtus communis Linn. afforded myrtucommuacetalone (1) with an unprecedented carbon skeleton and a new phloroglucinol-type compound, myrtucommulone M (2), along with four known constituents 3-6. Their structures were established by extensive analyses of NMR and mass spectral data as well as by single-crystal X-ray diffraction studies. These constituents were evaluated for their ability to modulate the immune response, based on their effects on various components of immune system. Compounds 1 and 5 exhibited significant inhibitory effect against nitric oxide (NO(•)) production. Compound 1 also exhibited significant antiproliferative activity (IC50 < 0.5 µg/mL) against T-cell proliferation. Myricetin (3) exerted a significant inhibition (IC50 = 1.6 µg/mL) on zymosan-stimulated whole blood phagocytes ROS production. Compounds 1 and 3 were active against PMA-stimulated ROS generation.

EGFR mutations and human papillomavirus in lung cancer.

Our previous study reported a frequent detection of human papillomavirus (HPV) genome in primary lung adenocarcinomas of the recurrent patients who were responsive to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, suggesting that HPV presence in lung cancer may be related to a genetic background related to EGFR mutations. The present study examined the association between the HPV presence and mutations in exons 19 and 21 of EGFR gene in Japanese lung cancer patients. Thirteen (31%) out of 42 cases had EGFR mutations. Although these mutations were tended to be observed in females, non-smokers, or adenocarcinomas, there was no statistically significant associations. HPV DNA was found in 7/42 (17%) lung tumors. The frequency of HPV presence did not differ in histological types. The presence of HPV DNA was significantly related to EGFR mutations (P=0.021), especially in adenocarcinomas of the lung (P=0.014). HPV-positive lung tumors accounted for 38% and 7% of those with and without EGFR mutations, respectively. Our results suggest that EGFR mutations are associated with HPV presence in Japanese patients with lung cancer.

Suppression of c-Fos protein and mRNA expression in pentylenetetrazole-induced kindled mouse brain by isoxylitones.

An early immediate gene c-fos has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes occurring during kindling. We have evaluated the effects of novel anticonvulsant isomeric compounds isoxylitones [(E/Z)-2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine] on the c-Fos protein and mRNA expression in the brain samples of kindled mice and compared it with the normal and untreated kindled groups. Kindling was induced in male NMRI mice by repeated administration of sub-convulsive dose (50 mg/kg) of pentylenetetrazole (PTZ) until a seizure score of 4-5 was achieved. The c-Fos expression was quantified by combination of immunohistochemistry and RT-PCR protocols. Both the immunohistochemical and RT-PCR analysis revealed a marked increase in the expression of c-fos mRNA and protein in the brain regions tested in case of PTZ-kindled control group compared to normal control. In contrast, the isoxylitone (30 mg/kg)-treated group demonstrated significant reduction of c-Fos expression compared to PTZ-kindled control animals. However, low expression of c-fos mRNA was only detected in the thalamus of the isoxylitone-treated brain samples. Based on these observations, we suggest that isoxylitones may have the capacity to control the seizure pattern by mechanism such as the suppression of c-Fos protein and mRNA levels in different regions of the brain. Further investigations to explore the mechanism of action of these compounds are under process.

Same-day versus delayed vitrectomy with lensectomy for the management of retained lens fragments.

PURPOSE: To evaluate whether performing same-day pars plana vitrectomy versus delayed pars plana vitrectomy affects visual outcomes and ocular morbidity of patients with retained lens fragments after a complicated cataract surgery. METHODS: Retrospective, comparative case series of 172 eyes of 171 patients with retained lens fragments undergoing 3-port pars plana vitrectomy using 20-, 23-, or 25-gauge instrumentation between 2005 and 2008. Outcome measures included best-corrected visual acuity at 6 months, final best-corrected visual acuity, and postoperative complications such as cystoid macular edema, intraocular pressure elevation, retinal detachment, vitreous hemorrhage, choroidal hemorrhage, and endophthalmitis. RESULTS: The median age was 75 ± 0.8 years. The mean time to vitrectomy for the delayed group was 15 ± 2 days. The preoperative logarithm of the minimum angle of resolution best-corrected visual acuity for immediate vitrectomy was 0.73 ± 0.09 versus 0.72 ± 0.06 for delayed vitrectomy. Six-month logarithm of the minimum angle of resolution acuity was 0.44 ± 0.09 for same-day vitrectomy compared with 0.44 ± 0.05 for delayed vitrectomy (P = 0.97, 2-tailed t-test). Of 59 eyes undergoing immediate vitrectomy, 17 (29%) experienced postoperative complications, while 38 of 113 eyes (34%), experienced complications if undergoing delayed vitrectomy (Fisher exact test, P = 0.61). Overall, the most common complication was cystoid macular edema occurring in 25 of 172 eyes (15%). CONCLUSION: The outcomes of same-day pars plana vitrectomy appear to be similar to non-same-day pars plana vitrectomy. The risks and benefits related to the timing of vitrectomy after a complicated cataract surgery should be carefully discussed with each patient. Further investigation is warranted to establish an optimal time for surgical planning.

Human papillomavirus and Epstein-Barr virus infections in breast cancer from chile.

BACKGROUND: Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas (BCs) around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV-16 viral load/physical status and E6/E7 expressions were determined. RESULTS: The amplification of a housekeeping gene showed that 46/55 samples (84%) had amplifiable DNA. HPV-16 was detected in 4/46 BCs (8.7%) and EBV was detected in 3/46 (6.5%) BCs. The analysis of HPV-16 physical status showed that this virus was integrated in all of the tumors with a relatively low viral load (range: 0.14 to 33.8 copies/cell). E6 and E7 transcripts, however, were not detected in any HPV-16 positive specimens. Using a Cox-regression model, we found a statistically significant association between EBV presence and poor survival (p = 0.013). CONCLUSIONS: The findings in this study suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of BC.

High-risk human papillomavirus in mammary gland carcinomas and non-neoplastic tissues of Mexican women: no evidence supporting a cause and effect relationship.

Human papillomavirus (HPV) has been implicated in breast carcinogenesis. Consecutive and non-selected mastectomy specimens from Mexican patients harboring breast carcinomas were sampled in order to look for the presence of HPV DNA. HPV-16 was detected in 6 (10%) of 60 breast carcinomas. Two of these also had HPV genome in adjacent non-neoplastic mammary-tissues. Seven cases had HPV DNA only in non-neoplastic tissue specimens. HPV DNA was also detected in 4 (25%) of 10 tumor-bed specimens without residual neoplastic lesions that were obtained from patients who underwent neoadjuvant chemotherapy or neoadjuvant chemotherapy/radiotherapy. HPV-positive tumors tended to be smaller in size, than HPV-negative tumors (p=0.047). Histological distributions of HPV-positive and -negative cases showed no significant difference. Although all the HPV-16 DNA were found integrated, its low viral load rendered it difficult to incriminate this virus in breast carcinogenesis. However, the possibility that HPV infection occurred during carcinoma development cannot be ruled out.

Anatomic and visual outcomes in early versus late macula-on primary retinal detachment repair.

PURPOSE: The purpose of this study was to describe the single surgery anatomic success rates and visual outcomes of primary macula-on retinal detachment repaired within 24 hours compared with later than 24 hours. METHODS: This is a retrospective, comparative, interventional, consecutive case series. All eyes underwent primary surgical repair of the macula-on retinal detachment with a scleral buckle, pars plana vitrectomy, or combination of both procedures. The duration from the initial examination to the time of surgical repair was categorized as early (<24 hours) versus late (>24 hours). RESULTS: Sixty-six eyes, 42 phakic and 24 preoperative pseudophakic, had retinal detachment repair with a median time to surgery of 1.0 ± 2.1 days (0.8 ± 0.4 days in early group versus 3.7 ± 2.2 days in late group, P < 0.005). The overall single surgery anatomic success rate was 59 of 66 eyes (89%). The single surgery anatomic success rate between the early (32 of 37 [87%]) versus late (27 of 39 [93%]) repair groups showed no statistical difference (P = 0.45). The mean time of follow-up was 13.1 months (range 0.9-39.2 months) with the mean final logarithm of the minimum angle of resolution best-corrected visual acuity showing no statistical difference between the 2 groups (early [0.10 ± 0.02] versus late [0.12 ± 0.03], t-test; P = 0.52). The rates of postoperative glaucoma (P = 0.5) and hemorrhage (P = 0.19) did not differ significantly between the 2 groups. CONCLUSION: Delaying the repair of primary macula-on retinal detachment by more than 24 hours does not appear to cause worse visual or anatomic outcomes compared with early (<24 hours) surgical intervention. There was no significant difference in the complication rates between the two groups.

Human papillomavirus is frequently detected in gefitinib-responsive lung adenocarcinomas.

A number of studies have reported the presence of human papillomavirus (HPV) in lung carcinoma. Interestingly, its detection rate appears to differ histologically and geographically. The present study examined 30 adenocarcinomas and 27 squamous cell carcinomas of the lung in a southern area of Japan, and detected high-risk HPV genome in 9 (30%) adenocarcinomas and 2 (7%) squamous cell carcinomas, using PCR with SPF10 primers and INNO-LiPA HPV genotyping assay. The difference of HPV detection rates in adenocarcinomas and squamous cell carcinomas was statistically significant (P=0.044, Fisher's exact test). HPV-16 was the most prevalent HPV genotype, and was detected in 27% (8/30) of adenocarcinomas and in 7% (2/27) of squamous cell carcinomas. High-risk-HPV positive carcinomas had decreased proportions of pRb (P=0.107) and significantly increased proportions of p16INK4a expressing cells (P=0.031) when compared to HPV-negative lung carcinomas. All HPV-16-positive cases were considered to have an integrated form of HPV-16 but its viral load was low (geometric mean = 0.02 copy per cell). In 20 additional adenocarcinomas treated with gefitinib, a tyrosine kinase inhibitor specific for epidermal growth factor receptor, the presence of HPV was examined. Note that East Asian ethnicity is a predictive factor of gefitinib response. High-risk HPV genome was found in 75% (6/8) of adenocarcinomas with complete or partial response to gefitinib but was not found in the remaining 12, which did not respond to gefitinib. In conclusion, the present study suggests that high-risk HPV may be more strongly related to adenocarcinomas, particularly gefitinib-responsive adenocarcinomas, when compared to squamous cell carcinomas. However, its low viral load makes it difficult to determine the etiological significance of these findings.

Analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis.

Several independent studies have presented evidence for the involvement of human papillomaviruses (HPV) in the aetiology of human breast cancer, while others have reported the opposite findings. Here, we have analysed by a high sensitive multiplex PCR-based method the prevalence of alpha mucosal and beta cutaneous HPV DNA in 90 ductal lavages, colostrum and milk. Ten of the 70 DLs analyzed (14%) contained a single or multiple beta HPV types, while DNA from mucosal high-risk HPV types was detected in only one sample (1/70). A strong reduction of HPV positivity in DL fluids was observed in 45 specimens collected after removal of the superficial layers of the nipple epidermis. All DLs were negative for the mucosal low-risk HPV types 6 and 11. Finally, HPV positivity was low in colostrum and milk. Our data show that DNA of alpha mucosa and beta cutaneous HPV types are rarely present in the breast fluids and suggest that a direct role of HPV in breast carcinogenesis is unlikely.