Heparin Binding Protein (HBP) is released to blood circulation from activated neutrophils in bacterial infections. It is a potential inducer of vascular leakage and precludes the development of septic shock. Filgrastim induces the production of new neutrophils and modulates their bacterial-killing activity. We evaluated the effect of filgrastim on HBP –concentrations in critically ill patients with acute respiratory failure.59 critically ill patients with acute respiratory failure were included in this randomised, double-blind, placebo-controlled study of filgrastim 300 micrograms/day or corresponding placebo for 7 days. Plasma samples were drawn on baseline, day 4 and day 7. HBP –concentrations, absolute leukocyte and neutrophil counts were measured.The median [IQR] HBP concentrations were 23.6 ng/ml [13.9-43.0 ng/ml], 25.1 ng/ml [17.7-35.5 ng/ml] and 15.9 ng/ml [12.6-20.7 ng/ml] in patients receiving filgrastim on baseline, day 4 and day 7, respectively. The HBP concentrations in placebo group were 21.6 ng/ml [16.9-28.7 ng/ml], 13.9 ng/ml [12.0-19.5 ng/ml] and 17.8 ng/ml [13.6-20.9 ng/ml]. At day 4, the filgrastim group had significantly higher HBP –concentrations when compared to placebo group (p < 0.05). No correlation between HBP –concentrations and absolute neutrophil count or P/F –ratios was found.Filgrastim treatment is associated with increased circulating HBP levels compared to placebo, but the absolute neutrophil count or the degree of oxygenation failure did not correlate with the observed plasma HBP –concentrations.Clinicaltrials.gov NCT01713309 Plasma leakage from the vasculature is an important step in the development of septic shock in patients with infections . HeparinIn capillary leakage such as septic shock, burns or erysipelas caused by group A Streptococci, elevated levels of HBP have been documented -8. The lPulmonary infections and septic infections are responsible for more than a third of acute respiratory failure leading to ICU admission . In bactWe hypothesized that increased number of circulating neutrophils in response to G-CSF treatment would be associated with increased HBP concentrations in plasma compared to placebo. Accordingly, we analyzed the HBP concentrations in critically ill patients with acute respiratory failure who participated in a prospective, randomised, double-blind study of G-CSF vs. placebo.This is a study of plasma HBP –levels of a previously published trial of G-CSF in critically ill patients . The stuDuring a 16 –month period from February 1997 to June 1998 altogether 636 patients were admitted to the mixed 10 –bed ICU. 59 consecutive patients were included into the study. The inclusion criteria were 1. age >18 years, 2. admission <12 h before study inclusion, 3. intubation <48 h of study inclusion, 4. expected ICU stay >48 h, 5. Informed consent signed. The exclusion criteria were 1. age <18 years, 2. admission >12 h before study inclusion 3. Intubated >48 h before study inclusion, 4. expected stay in ICU <48 h, 5. no informed consent, 6. pregnant or nursing, 7. administration of filgrastim, sargramostin or other biological response modifiers within 7 days, 8. participation in another medicinal trial.9/L filgrastim/placebo was administered every other day and if neutrophil count exceeded 75x109/L filgrastim/placebo was discontinued. The study personnel were blinded to study patients’ leukocyte and neutrophil counts until the data had been analyzed. Other intensive care treatments were performed according to the written standard operating procedures of the ICU.The study was a prospective, randomised, double-blind, placebo-controlled trial of filgrastim in patients with acute respiratory failure requiring intubation. The patients were treated with subcutaneous injections of 300 micrograms of filgrastim or corresponding placebo (1 ml of 0.9% NaCl –solution) once daily for 7 days or until discharge from the ICU. If the neutrophil count exceeded 50x10Blood samples were drawn at study entry (baseline), day 4 and day 7 afterwards. The samples were taken before administration of the study drug. The sampling was performed only during the ICU stay and if a patient was discharged by day 7, blood collection was also discontinued. The blood samples were drawn and placed immediately in ice and centrifuged at +4°C. The plasma was stored in −70°C until further analysis. Total leukocyte count and absolute neutrophil count were determined in routine analysis of Helsinki University Central Hospital laboratory. The plasma HBP was determined by enzyme-linked immunosorbent assay as described earlier . The detThe primary endpoint of the main study were number of adverse events, the number of patients developing ARDS, disseminated intravascular coagulation or acute renal failure during days 1–28, and changes in MOD score . For thiThe data are presented as median and interquartile ranges (IQR) or absolute numbers and ranges, as appropriate. The normality of distribution of parameters was tested with Kolmorogov-Smirnov one-sample test. In case of non-normal distribution, the parameters were transformed to natural logarithm before analysis. The difference between groups is calculated by one-way analysis of variance with a p value <0.05 considered statistically significant. The correlations between parameters were calculated from values transformed to natural logarithm. Intraclass correlation coefficient (ICC) was used to examine the correlation between HBP -levels, P/F –ratio, SOFA –score, WBC and neutrophil count as well as inflammatory mediators.Altogether 59 patients were included into the study, 30 in the filgrastim group and 29 in the placebo group. Due to an error in the preparation of randomisation envelopes number 45 was omitted and only 59 patients were randomised instead of the intended 60 patients. The baseline characteristics of the patients are presented in the Table The plasma HBP –concentrations, absolute neutrophil counts and white blood cell counts at baseline, day 4 and day 7 are presented in Figure The absolute neutrophil count and white blood cell counts were similar in filgrastim and placebo groups at baseline but at days 4 and 7 there were significant increase in filgrastim group  regardless of filgrastim treatment. The HBP –concentrations were above 15 ng/ml in 44 of the 59 patients at baseline. No correlations between HBP –concentrations and absolute neutrophil count or P/F –ratio could be demonstrated Figure . The conOur study was the first to assess HBP –levels in critically ill patients receiving filgrastim or placebo in a controlled study. The HBP –levels were similar in both treatment groups at baseline, but differed at day 4. The difference in HBP –levels did not persist to day 7, and HBP –concentrations did not correlate with absolute neutrophil count or P/F –ratios.The concentrations of HBP were elevated in both groups already at the baseline samples. In healthy volunteers without infections and in patients with local infections only, the concentrations of HBP have been around 7 ng/ml compared to 24 ng/ml and 22 ng/ml in filgrastim and placebo groups at baseline, respectively . These bHBP is an important mediator of bacterial infections transforming to septic shock. In septic situations, HBP is released from activated neutrophils by exocytosis of granule compartments. Filgrastim stimulates the production of new neutrophils in bone marrow and enhances the effector functions of mature neutrophils . As HBP The correlation of HBP concentrations to WBC count has been studied in previous trials ,5. In a The M1 –protein mediated release of neutrophil granule contents is responsible for subsequent lung injury . As HBP Our study had some limitations. The number of patients was low and, accordingly, there is considerable risk to type II error, i.e. not detecting a true difference even though it is present. Due to the limited number of patients in this randomized pilot study, we were not able to analyze HBP –levels in subgroups of patients with bacterial infections, other inflammatory processes, or with non-infectious causes of acute respiratory failure. Second, the sampling was not primarily designed for measurements of plasma HBP –concentrations, but rather to show the change in organ failure score before and after the intervention. As the decline in HBP occurs within days in critically ill patients , more frFilgrastim treatment is associated with increased circulating HBP levels compared to placebo, but the absolute neutrophil count or the degree of oxygenation failure did not correlate with the observed plasma HBP –concentrations.Plasma levels of heparin-binding protein (HBP) were elevated in critically ill patients with acute respiratory failure requiring intubation at baseline.A significant decrease in HBP –levels in placebo –treated patients was observed at day 4.HBP –levels did not correlate with absolute white blood cell or neutrophil counts.HBP –levels did not correlate with worsening of oxygenation measured by P/F –ratio.HBP: Heparin-binding protein; IQR: Interquartile range; G-CSF: Granulocyte colony stimulating factor; ICU: Intensive care unit; APACHE II: Acute physiology and chronic health evaluation; SOFA: Sequential organ failure assessment; CV: Coefficient of variation; ARDS: Acute respiratory distress syndrome; DIC: Disseminated intravascular coagulopathy; WBC: White blood cell; ICC: Intraclass correlation coefficient; P/F –ratio: Arterial oxygen tension to fraction of inspired oxygen –ratio.Hansa Medical AB has filed a patent application on the use of HBP as a diagnostic tool in sepsis. HH is listed as inventor. KMK, LL and VP declare no competing interests.KMK has performed statistical analysis of the HBP data and is responsible for the draft writing and submission of the manuscript. HH and LL have contributed in the planning of the study, are responsible for the determination of HBP –concentrations, contributed on the analysis of the data and writing of the manuscript. VP is responsible for the planning, execution and reporting of the original study and contributed in HBP data analysis and manuscript preparation. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/51/prepub


Psidium guineense Swartz (Araçá-do-campo) and five antimicrobials  against twelve strains of Staphylococcus aureus with a resistant phenotype previously determined by the disk diffusion method. Four S. aureus strains showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between aqueous extract of P. guineense and antimicrobial agents, by the checkerboard method. The criteria used to evaluate the synergistic activity were defined by the fractional inhibitory concentration index (FICI). All S. aureus strains were susceptible to P. guineense as determined by the microdilution method. The combination of the P. guineense extract with the antimicrobial agents resulted in an eight-fold reduction in the MIC of these agents, which showed a FICI ranging from 0.125 to 0.5, suggesting a synergistic interaction against methicillin-resistant Staphylococcus aureus (MRSA) strains. The combination of the aqueous extract of P. guineense with cefoxitin showed the lowest FICI values. This study demonstrated that the aqueous extract of P. guineense combined with beta lactamics antimicrobials, fluoroquinolones, and carbapenems, acts synergistically by inhibiting MRSA strains.The aim of this study was to evaluate the antimicrobial activity of aqueous extract of  Staphylococcus aureus is an important pathogen both in community acquired and nosocomial infections and a common etiological agent of infections of many different tissues and organs  [Staphylococcus aureus strains now pose serious problems to hospitalized patients and their care providers. The organism has successfully evolved numerous strategies for resisting, the action to practically all antimicrobial agents [The widespread emergence of bacteria resistance to a large number of antimicrobial agents poses major health problems because of difficulties in treatment . Staphylyelitis) . The mull agents . Thus, il agents .The screening of plant extracts and phytochemicals for antimicrobial activity has shown that higher plants, especially their secondary metabolites, are a source to provide structurally diverse bioactive compounds with different pharmacological activities, including antimicrobials .According to Hemaiswarya et al. , plants-Psidium guineense Swartz (Myrtaceae), popularly known as wild guava, is a shrub native and widely dispersed throughout Tropical America. In Brazil, it mainly occurs across the coastline [oastline , 10.P. guineense to be effective against Streptococcus mutans strains.Few studies have been focused on the phytochemical profile and pharmacological activity of this plant; however, it is widely used in folk medicine in regions of South America to treat infections of the gastrointestinal and genitourinary tract . These aP. guineense and to determine the synergistic potential of the combination of this extract with five antimicrobial agents against MRSA strains.The aim of the present study was to evaluate the antimicrobial activity of the aqueous extract of Psidium guineense Swartz (leaves) that were collected in November/2009 from Moreno city, Pernambuco, Brazil, (08°07′07′′S-35°05′32′′W). This plant was identified by the biologist Olivia Cano, and the voucher specimen was deposited in the Herbarium Dárdano de Andrade Lima, Instituto Agronômico de Pernambuco and indexed under registration 83.564.The plant materials used in this study consisted of The leaves were air-dried for two weeks and then grinded into fine powder using an electric dry mill. A total of 50 g of the powder was soaked in 1000 mL of boiled distilled water and extracted exhaustively at room temperature for 48 hours. The extracts were filtered through Whatman No. 2 filter paper. The aqueous extract was lyophilized to obtain a dry powder extract.P. guineense using standard procedures [2O ) to investigate the presence of saponins, flavonoids, tannins, phenylpropanoids, alkaloids, condensed proanthocyanidins, leucoanthocyanidins, and iridoids and (n-BuOH–Me2CO-buffer phosphate pH = 5.0 (40 : 50 : 10 v/v)) to evaluate the presence of sugars.The preliminary phytochemical screening was carried out by various secondary metabolites present on aqueous extract of ocedures –14. The P. guineense (1000 μg/mL) were prepared in distilled water. Concentrations (10 to 30 μg/mL) were measured. The standard pyrogallol (1000 μg/mL) was prepared in distilled water. 80 microliters of each solution was transferred to a 25 mL flask containing distilled water (10 mL) and Folin-Ciocalteu's reagent (1 mL). The volume was completed with anhydrous sodium carbonate solution (10.75% w/v), resulting in final concentrations of 3.2 μg/mL of pyrogallol. A standard curve was determined with concentrations between 1.6 and 4.8 μg/mL (r2 = 0.999). Final results were given as pyrogallol equivalents. The samples were scanned in a UV/VIS spectrophotometer , and measurements were obtained 15 min after addition of the sodium-carbonate solution and scanning ranging from 500 to 900 nm wavelength. Distilled water was used as a blank. All measurements were performed in triplicate. The total polyphenol content (TPC) was determined colorimetrically by using Folin-Ciocalteu's reagent and expressed as pyrogallol, according to the Brazilian Pharmacopoeia with modifications . Thus, sS. aureus strains (n = 11) were isolated from clinical specimens and food. The standard strain used was S. aureus ATCC 25923. Strains were isolated in sheep blood agar and after identification they were stored in brain heart infusion (BHI) plus glycerol 20% v/v [S. aureus strains used in this study showed a resistant phenotype, by the diffusion method, to several antimicrobial agents such as beta-lactams, aminoglycosides, macrolides, fluoroquinolones, tetracycline, chloramphenicol, and lincosamides. These strains were cultured onto Mueller Hinton Agar (MHA)  and incubated at 37°C for 18 hours. Single colonies were selected and inoculated into Mueller-Hinton broth  to turbidity comparable to that of 0.5 McFarland standard, which is equivalent to a bacterial count of approximately 108 CFU/mL. 20% v/v . The S. 7 CFU/mL.After that, the bacterial suspension was diluted in saline (1 : 10) to obtain a final inoculum of 10μg/mL); amoxicillin/clavulanic acid ; cefoxitin ; ciprofloxacin ; gentamicin .The standard reference powders of ampicillin; amoxicillin/clavulanic acid; cefoxitin; ciprofloxacin; gentamicin; meropenem were provided by Eurofarma Laboratório LTDA, Brazil. Resistance was defined for each case: ampicillin . Five microliters of bacterial suspension were inoculated in each well to give a final concentration of 104 CFU/mL. P. guineense extract and antimicrobial agents concentrations ranged from 7.25 to 1000 μg/mL and 3.12 to 400 μg/mL, respectively. The growth inhibition was demonstrated by optical density at 630 nm using a microplate reader (Thermo plate—TP Reader). Considering the total growth (100%) in the control well (MHB + bacteria), the percentage of growth reduction was attributed to the remaining wells. The MIC was reported as the lowest concentration of P. guineense extract or antimicrobial agents that inhibited the bacterial growth after 24 h of incubation at 37°C. In order to determine the Minimal Bactericidal Concentration (MBC), the contents of the well that showed higher or equal than 70% of growth inhibition were seeded into MHA. After 24 h of incubation at 37°C, the number of surviving S. aureus was determined. The MBC was defined as the lowest extract concentration at which 99.9% of the bacteria have been killed. All experiments were carried out in duplicate on two different days.The Minimal Inhibitory Concentration (MIC) test was performed by the microdilution broth method, following the recommendations established by Clinical Laboratory Standards Institute, , with soP. guineense and antimicrobial agents were tested by the checkerboard method. The appropriate dilution of P. guineense extract and antimicrobial agents were performed into MHB. From these dilutions, one hundred microliters were added in 96-well microplates to obtain a final concentration equal to MIC or six dilutions lower than MIC to P. guineense and nine dilutions lower than MIC to antimicrobial agents. Each well received 5 μL of the bacterial suspensions (107 CFU/mL). Plates were incubated for 24 hours. Interpretation of the data was achieved by calculating the fractional inhibitory concentration index (FICI) as follows:  + . The combination was considered to be synergistic when the FICI was ≤0.5, additive when it was 0.5 to ≤1, and antagonistic when ≥2 [Combinations of  when ≥2 .Psidium guineense extract showed the presence of tannins, flavonoids, condensed proanthocyanidins, leucoanthocyanidins, and sugar. On the other hand, the presence of alkaloids, phenylpropanoid, and saponins was not verified. The aqueous extract of P. guineense yielded 3.7% (w/w) over 50 g of plant material.The phytochemical profile from the P. guineensis show an important amount of such secondary metabolites. Thus, the total polyphenol content was 21.62 ± 0.40 g% (1.51%) as pyrogallol equivalent.Concerning the total polyphenol content, the results of this study suggest that the aqueous extract of P. guineense extract and of the ampicillin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, gentamicin, and meropenem against twelve S. aureus strains are shown in MIC and MBC values of P. guineense showed a strong activity against all S. aureus strains with MIC values between 250 and 500 μg/mL. The S. aureus strains revealed a resistance profile against most antimicrobial agents tested, in particular to the beta-lactam antibiotics. The MIC values for ampicillin and cefoxitin ranged from 3.12 to 400 μg/mL which showed to be less effective against the S. aureus strains tested. For amoxicillin/clavulanic acid, the values ranged from 3.12 to 50 μg/mL. Among all S. aureus strains tested, seven showed to be resistant to ciprofloxacin. For meropenem, MIC values ranged from 3.12 to 25 μg/mL. All strains showed to be sensitive to gentamicin, except LFBM 26, LFBM 28, LFBM 33. Four strains of S. aureus  showed resistance to all antimicrobial agents tested. This resistance profile selected the microorganisms for the study of the interaction between P. guineense extract and antimicrobial agents. The values of MBC were higher than MIC in one dilution.The aqueous extract of P. guineense with ampicillin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, and meropenem, against S. aureus strains  are listed in P. guineense extract (250 μg/mL) enhanced the antistaphylococcal activity of all antimicrobial agents. The synergistic activity was detected by an eight-fold decrease in the MIC of the antimicrobial agents in the combination and determined by the FICI ≤0.5.The MICs obtained by the combination of the aqueous extract of P. guineense and cefoxitin showed the lowest FICI whose values ranged from 0.125 to 0.5. The MIC of cefoxitin  was lowered to 1/512 (combined MIC ranged 0.19–0.78 μg/mL), when it was used in combination with the aqueous extract of P. guineense (MIC 250 μg/mL). For S. aureus LFBM 26 and LFBM 33 strains, this combination was also efficient on reducing the MIC of P. guineense extract to 1/8 × MIC (31.25 μg/mL) or 1/4 × MIC (62.25 μg/mL), respectively.The combination of the aqueous extract of S. aureus strains by meropenem was enhanced by P. guineense extract (FICI 0.5). This combination was more effective against LFBM 28 (FICI 0.25) which individual MIC of P. guineense (250 μg/mL) lowered to 1/4 × MIC.The inhibition of the growth of all the S. aureus LFBM 33 strain, the MIC value of ciprofloxacin  was lowered to 0.78 μg/mL when combined with P. guineense extract (1/4 × MIC) enhancing the antistaphylococcal activity of this fluoroquinolone (FICI 0.25). All other combinations of the aqueous extract of P. guineense with antimicrobial agents demonstrated synergistic activity, enhancing its activity, with FICI 0.5.For Phytochemicals have great potential as antimicrobial compounds and have been proven to have great therapeutic potential . Some seSeveral studies have investigated the interactions between antibiotics and phytochemicals or crude extracts against MRSA strains , 8, 20.In the Myrtaceae family, there is a large variety of phytochemicals with antimicrobial activity which include terpenes present in essential oils, flavonoids , and tanThe genus Psidium is native to America but is distributed worldwide. Their specimens are traditionally used in the prevention or treatment of a large number of diseases. Preparations of twigs or leaves of Psidium species are extensively used for the control of gastrointestinal and respiratory disorders as well as in the treatment of skin damage .Psidium ssp. that explain its use for the treatment of infectious diseases of the digestive, respiratory, urinary tract as well as the skin and soft tissues. Within this genus, P. guajava is the most studied and used species [P. guineense, an indigenous plant of Brazil, because it is characterized by its ample spectrum of uses including gastrointestinal disorders, infection of the genitourinary tract, treatment of colds, bronchitis, and ulcers [Ethnopharmacological evaluation demonstrated the antimicrobial activities of  species . Thus, wd ulcers .μg/mL, moderate activity MIC values between 600–1500 μg/mL, and weak activity above 1500 μg/mL. Comparing with literature results, the aqueous extract of P. guineense (250–500 μg/mL) has a strong activity against MRSA strains tested. According to Sartoratto et al. , strong P. guineense indicated the presence of flavonoids, hydrolysable, and condensed tannins distributed throughout the leaf tissue [Psidium guineense plays an important role on biological properties of this extract.The phytochemical analysis from the leaves of f tissue . These rP. guineense, with the activity against clinical isolated Streptococcus mutans strains.Neira González et al.  associat Flavonoids have the ability to complex with proteins and bacterial cells forming irreversible complexes mainly with nucleophilic amino acids. This complex often leads to inactivation of the protein and loss of its function .Tannins are not crystallizable substances and when they are in the aqueous system, form colloidal solutions. The antimicrobial activity of tannins can be summarized as follows: (i) binding with proteins and adhesins, inhibiting enzymes, (ii) complexation with the cell wall and metal ions, and (iii) disruption of the plasmatic membrane .Synergism of natural products and antimicrobial agents is a thrust area of phytomedicinal research, developing novel perspective of phytopharmaceuticals. The synergism of plant-derived compounds and antimicrobial agents has been evaluated previously against pathogenic microorganisms. The approach is not exclusive for extract combinations, since effective combinations between single natural products, essential oils or extracts with chemosynthetics or antibiotics have been described , 29.In addition to achieving these synergistic effects, the combinations of two or more compounds are essential for the following reasons: (1) to prevent or suppress the emergence of resistant strains, (2) to decrease dose-related toxicity, as a result dosage, and (3) to attain a broad spectrum of activity .P. guineense extract on MRSA strains was observed for its combinations with beta-lactams, carbapenems, and fluoroquinolones.In this study, a growth inhibitory effect of P. guineense extract and beta-lactam may help to reduce the amount of antimicrobial agents used and deliver a medicine with similar or greater potency as antimicrobial. More importantly, since phytochemicals are structurally different from antimicrobial agents and often have different modes of action, they may provide new means of studying the mechanisms of bacterial control at a molecular level. With the increase prevalence of multidrug resistant S. aureus, synergism testing using various combinations of phytochemicals with antimicrobial agents could be a powerful tool in helping to select appropriate antimicrobial therapy [The combination of  therapy , 29.The indiscriminate use of antimicrobial agents in the treatment of bacterial infections has led to the emergence and spread of resistant strains, and it resulted in a great loss of clinical efficacy of previously effective first-line antimicrobials which results in shifting of antimicrobial treatment regimen to second-line or third-line antimicrobial agents that are often more expensive with many side effects . In factArctostaphylos uva-ursi is found to markedly reduce the MIC of beta-lactam agents against MRSA. According to Hemaiswarya et al. [There is a wide list of phytochemicals which act as inhibitors, and a few of them are glycosylated flavones suppressing topoisomerase IV activity, myricetin inhibiting DnaB helicase, allicin inhibiting RNA synthesis. Corilagin, a polyphenol from a et al. , there aS. aureus thereby restoring the activity of penicillin. The combination of EGCg with ampicillin/sulbactam reduced the MIC90 to 4 μg/mL from its initial value of 16 μg/mL [The polyphenol epigallocatechin gallate (EGCg) from green tea is believed to be synergic with beta-lactam agents since both attacked the same target site, namely, peptidoglycan. EGCg inhibits the penicillinase produced by μg/moL, both reduced the MIC for oxacillin to 1/2–1/16 of those in the absence of the polyphenols.Hatano et al.  investigIt is interesting to note that most plant secondary metabolites have weak antimicrobial activity, several orders of magnitudes less than that of common antimicrobial agents produced by bacteria and fungi. In spite of the fact that plant-derived antimicrobials are less potent, plants fight infections successfully. Hence, is becomes apparent that plants adopts a synergistic mechanism between their compounds .According to Rosales-Reyes et al. , the useTherefore, it is speculated that the efficacy of a crude extract may be due to the interplay between the different active constituents that may be present in the extract leading to better activity and/or decrease in potential toxicity of some individual constituents .P. guineense extract and a synergistic effect in combination with antimicrobial agents were reproducibly observed and might be an interesting alternative therapy for infectious diseases caused by MRSA strains. In addition, more studies, including toxicity tested in vivo, need to be conducted on this plant before therapeutic treatments are implemented.An antibacterial effect of


Although diffuse large B-cell lymphoma (DLBCL) usually occurs in the lymph nodes, approximately 30–40% of the time it can have an extranodal site of involvement and it can arise in nearly every body site such as intestine, bone, breast, liver, skin, lung, and central nervous system. Muscle involvement of DLBCL is especially uncommon, comprising 0.5% of extranodal NHL. We report a case of a 72-year-old man with extranodal DLBCL of a unique manifestation in the calf muscle, involving predominantly the gastrocnemius muscle. The patient achieved complete response and remained free of local recurrence or metastasis following diagnosis. Non-Hodgkins lymphoma (NHL) is the most common form of lymphoma, with an estimated 65,980 new cases of NHL diagnosed in America during 2009, resulting in approximately 19,500 NHL deaths . AlthougA 72-year-old male initially presented to his general practitioner with complaints of right calf pain and swelling that started while doing maintenance on his roof. Physical examination revealed a firm mass in the right calf, measuring approximately 10 cm, nontender, with no warmth or erythema. Distally, the calf was grossly neurovascularly intact, with no inguinal adenopathy. Magnetic resonance imaging (MRI) of the right calf showed diffuse increased signal intensity of the medial gastrocnemius muscle that was associated with a central 3 cm region of abnormal signal intensity, possibly due to contusion and muscular injury. A repeat MRI in one or two months was advised to ensure stability of the findings. The repeat MRI showed marked enlargement, to 11.5 × 6.0 × 8.5 cm, of the mass involving the medial gastrocnemius muscle, suspicious for sarcoma . The adjAn incisional biopsy was obtained from the mass and stained with hematoxylin and eosin and immunostains specific to certain cancers. The biopsy tissue contained diffuse infiltrate of large malignant cells with a high nucleus-to-cytoplasm ratio and scanty cytoplasm. In addition, the nuclei were round, with prominent nucleoli and high mitotic activity. The differential diagnosis based on morphology included sarcoma, Merkel cell carcinoma, and lymphoma. Immunoperoxidase staining results were consistent with malignant B-cell lymphoma . LaboratBone marrow aspiration revealed normocellular marrow (50%) with adequate trilineage hematopoesis, and no evidence of lymphoma, immunoglobulin heavy chain gene rearrangements, or immunoglobulin kappa light chain gene rearrangements. Bilateral marrow trephine biopsy sections were adequate and normocellular (50%) for age. Granulopoietic and erythroid maturation were adequately present with an M : E ratio of 3 : 1. Megakaryocytes were normal in number and appearance. No focal lesions were identified. Trabecular bone is normal. Cytogenetic analysis showed a male karyotype with loss of the Y chromosome in 4 of 20 mitotic cells, most likely an age-related phenomenon. No additional clonal cytogenetic changes were detected. Additionally, the 14q32/IGH translocation was not detected above established background limits by FISH analysis. 2), doxorubicin (50 mg/m2), vincristine (1.4 mg/m2), and cyclophosphamide (750 mg/m2). The chemotherapy was administered intravenously every three weeks for a total of six cycles. Subsequently, the patient received three cycles of rituximab (375 mg/m2) administered intravenously every three months. In addition, the patient received radiation therapy via 6-field IMRT technique. A dose of 32.4 Gy was given in eighteen 1.8 Gy fractions. The radiotherapy field was then reduced to a smaller field which covered the MRI-defined postchemotherapy residual tumor, and an additional 12.6 Gy was given to this volume. This brought the total radiation dose of the residual disease to 45 Gy. Six MV photons were used for all treatments. The patient was started on aggressive chemotherapy with rituximab (375 mg/mThe follow-up PET scan obtained one month after the end of treatment showed tremendously decreased activity in the right lower extremity, with only a residual amount of activity noted along the medial gastrocnemius muscle that could have been caused by some mild physiological activity. The posttreatment serum LDH value was 407 U/L. The subsequent CT scan reported some residual hypoattenuation involving the proximal medial gastrocnemius muscle and loss of muscle mass consistent with muscle atrophy. Otherwise, there was no evidence of soft tissue masses or fluid collection. The one-year follow-up PET scan revealed no evidence of disease recurrence.DLBCL is an aggressive form of non-Hodgkins lymphoma, and comprises approximately 30% of all lymphomas. This type of lymphoma occurs predominantly in men over 64 years of age and typically presents as a fast-growing lymph node or mass with or without “B” symptoms, including fever, weight loss, and night sweats. Extranodal presentation most commonly involves the gastrointestinal tract or bone marrow, and less commonly occurs in the skeletal muscle. When skeletal muscle involvement is seen, it is predominantly in upper arm muscles and glutei . The sta E  DLBCL, in which the lymphoma was limited to one site and had not spread to other organs or lymph nodes. The biologic grade was high grade, with a proliferation index of 70% as measured by Ki67 expression. The patient received 6 cycles of R-CHOP, without any treatment-related complications, followed by radiation and three subsequent rituximab infusions. He achieved complete response and remained free of local recurrence or metastasis in the 16 months following diagnosis. This supports the concept that correct diagnosis and early chemotherapy with R-CHOP and radiation, followed by rituximab infusions, are appropriate treatments in early-stage primary skeletal muscle lymphoma.This patient presented with a fast-growing right calf mass, without “B” symptoms. Upon biopsy, the patient was found to have stage IA


Staphylococcus aureus (MRSA) taking additional factors like stay in a health care facility into account.The present study aimed to investigate the dose response relationship between the prescriptions of antimicrobial agents and infection/colonization with methicillin resistant Multi-centre retrospective study on a cohort of patients that underwent microbiological diagnostics in Belgium during 2005. The bacteriological results retrieved from 17 voluntary participating clinical laboratories were coupled with the individual antimicrobial consumption patterns (July 2004-December 2005) and other variables as provided by pooled data of health insurance funds. Multivariate analysis was used to identify risk factors for MRSA colonization/infection.A total of 6844 patients of which 17.5% died in the year 2005, were included in a logistic regression model. More than 97% of MRSA was associated with infection , and only a minority with screening/colonization (1.59%). Factors (95% CI) significantly (p≤<0.01) associated with MRSA in the final multivariate model were: admission to a long term care settings (2.79–4.46); prescription of antibiotics via a hospital pharmacy (1.30–2.01); age 55+ years (3.32–5.63); age 15–54 years (1.23–2.16); and consumption of antimicrobial agent per DDD (defined daily dose) (1.25–1.40).The data demonstrated a direct dose-response relationship between MRSA and consumption of antimicrobial agents at the individual patient level of 25–40% increased risk per every single day. In addition the study indicated an involvement of specific healthcare settings and age in MRSA status. Staphylococcus aureus (MRSA) S. aureus strains In hospitals, antimicrobial resistance leads to increased healthcare costs primordially due to a higher morbidity and mortality from infectious diseases, and increased length of stay. This has been demonstrated, among other pathogens, for methicillin resistant The objective of this multicentre retrospective cohort study was to investigate a dose-effect relationship between consumption of antimicrobial agents and MRSA infection/colonisation at the individual patient level, taken into account variables such as class of antimicrobials administered, age and type of healthcare setting.S. aureus isolate (infection/colonization) was retained to minimize confounding through underlying disease and/or severity of infection.The microbiological results were retrieved from 17 voluntary participating clinical laboratories during 2005 in Belgium (convenience sample based upon willingness of participants). These bacteriological results were coupled with the individual antimicrobial consumption patterns during this observation period, and extended with a half year prior to the first laboratory observations (July 2004–December 2005). Briefly, national registry numbers were provided by the laboratories for each patient that underwent reimbursed bacteriological examinations. Via an encrypted key code, antimicrobial prescription records from the Belgian reimbursement agency (RIZIV-INAMI) were coupled. The Intermutualistic Agency (IMA-AIM) served as third trusted party (TTP). For every patient, only the first S. aureus was assumed to be done according to the laboratory internal routine methodology, and the definition of MRSA versus methicillin susceptible S. aureus (MSSA) was based on the susceptibility testing result for oxacillin (cefoxitin). All participating labs were at the time of survey certified by a mandatory external quality control organisation . For the purposes of this study, infection was attributed if microbiological results were obtained from clinical samples, i.e. not categorized by the laboratory as: ‘surveillance’; ‘screening’; or ‘unknown’.Data collected included a unique patient identification number, sample date, sample site (matrix), identification up to (sub)species level, and antibiogram. The latter susceptibility testing results were mainly done by the Kirby Bauer disk diffusion technique according to CLSI guidelines , which was often performed with semi-automated systems (e.g. SIRScan). Modifications were present according to the manufacturer for deviations in disk charge or diameter. The majority of Belgian hospitals worked with Neosensitabs  for producing these antibiograms. The detection of S. aureus was isolated, were obtained from seven Belgian health insurance funds (via IMA-AIM). Consumption data were categorized using the Anatomical Therapeutic Chemical (ATC) classification  up to four digits (e.g. J01C) and accordingly transformed into defined daily doses (DDD). Following additional patient characteristics were included in the analysis; age, sex, and admission to an acute, long term care (e.g. nursing homes), or other healthcare facility. Only observations prior to the moment of sampling (minimum one day) were considered for the inference analysis.Antimicrobial prescription records for patients in which S. aureus presence (MRSA). Single factors with a p-value <0.20 were retained for a stepwise forward multiple-factor analysis. Factors were recategorized due to analytical restrictions and conform the age groups applied by the Belgian IMA.Univariate logistic regression was used to presumptively identify risk factors for oxacillin resistance For all analyses, the significance level was set at α = 0.05. All tests were performed using the statistical software package STATA version 10.0 .Sectorial committee of the Belgian Federal Social Security as well as by the jointed ethical committee of the Scientific Institute of Public Health (WIV-ISP) and the Veterinary and Agrochemical Research centre (CODA-CERVA).None of the participants  provided an informed consent because data from laboratories and reimbursement organizations were encrypted by a third trusted party to ensure patient confidentiality. This consent procedure and the entire study protocol was approved by the Initially, a total of 92 117 samples  was reported in the J01C class of beta-lactams, penicillins  followed by J01M quinolones , J01X other antimicrobials  and J01D other beta-lactams . Amoxicilline & enzyme inhibitor , nifurtoinol , ciprofloxacine  and cefuroxime  were the drugs most frequently prescribed. , and retention of the index samples for which an oxacillin test result was available, a total of 6 844 http://www.nsih.be/download/AB/multicentrstudienov2008.pdf).More detailed information on prescribed antimicrobial classes and molecules, as well as on the microbiological results can be found elsewhere in Dutch and French  by age as follows: 0–9y: 629 (55); 10–19y: 276 (25); 20–29y: 417 (43); 30–39y: 452 (76); 40–49y: 518 (97); 50–59y: 679 (187); 60–69∶806 (259); 70–79y: 1410 (568); 80–89y: 1328 (657); 90–99y: 324 (210); 100–104y: 5 (4). Of these index patients 17.5% (n = 1200) died in 2005. Within the latter group, 51.2% (n = 614) were MRSA positive whereas in the patients who survived 2005 only 28.1% (1568/5644) were found to have MRSA .A total of 6844 index-patients with When comparing MRSA and MSSA individuals, factors univariately significantly associated with MRSA were as follows : health care facility type : 2.55, 1.71–3.80) compared to no admission ( = reference), female gender ., antimicrobial consumption (minimum 1 day prior to sample date) in ambulant care  and intramural , total DDD (minimum 1 day prior to sample date) ; 0–9 DDDs: 1.48–2.12; 9–24 DDDs: 2.27–3.22; 24–59 DDDs: 2.34–3.33; 59–1191.5 DDDs: 3.88–5.47), and age .In the final retained multivariate model following factors were independently associated with MRSA colonization/infection: admission to a long term care settings; supply by a hospital pharmacy, age, and consumption of antimicrobial agent per DDD .The present study aimed to investigate the dose-relationship between antimicrobial consumption and resistance. The relationship is well studied at aggregated level We demonstrated that hospitalisation is not a risk factor for MRSA as such in acute care, although prescription of antimicrobials in hospitals clearly is. On the other hand, residency in a long term care facility was identified as a risk factor for MRSA in our study. A decline of MRSA in nursing homes from 19% in 2005 to 13% in 2010, along with a parallel decline in Belgian hospitals has been documented General practitioners (GP) can consult enclosed susceptibility results for empiric treatments of MRSA infections in the community. However, a substantial selection bias might overestimate the resistance situation in the field since, and as stated previously, only patients that underwent bacteriological examinations were included here. Studies S. aureus antibiograms in the community. This study and the conclusions only concern patients that underwent reimbursed microbiology. Although this bias might have favoured resistance in our isolates, the narrow OR range (1.25–1.40) and thus strong relationship between consumption of antimicrobial agents and MRSA provides a clear quantitative assessment which will help clinicians to improve their understanding of resistance. The clinical relevance can be further exemplified by the crude mortality found here , in line with earlier investigations Our results on antimicrobial drug resistance patterns cannot be extrapolated to the Belgian community as such. Since GPs will treat the majority of patients in ambulatory care empirically, the microbiology reported in this study will not be representative for Another weaknesses of the study is the exclusion of a substantial number of patients due to incompatibility of the original data, and the absence of molecular typing methods (e.g. multiplex PCR) to confirm the identification of MRSA. The variety in the applied phenotyping methodologies for identification (e.g. selective agar) and susceptibility testing over different laboratories is a limitation inherent to the chosen study design.The results of the present analysis confirm the importance of health care settings and age on the presence of MRSA. Clinicians should consider that antimicrobial consumption inherently bears an individual risk for their own patient, besides the resistance induction at the population level that has been known for a long time.Table S1Staphylococcus aureus strains (n patients = 7309) retrieved from 16 Belgian clinical laboratories (2005).Disk diffusion susceptibility profiles of 15 442 (DOCX)Click here for additional data file.


There are no time trends in prevalence, unawareness, treatment, and control of hypertension in Switzerland. The objective of this study was to analyze these trends and to determine the associated factors.Population-based study conducted in the Canton of Geneva, Switzerland, between 1999 and 2009. Blood pressure was measured thrice using a standard protocol. Hypertension was defined as mean systolic or diastolic blood pressure ≥140/90 mmHg or self-reported hypertension or anti-hypertensive medication. Unawareness, untreated and uncontrolled hypertension was determined by questionnaires/blood pressure measurements. Yearly age-standardized prevalences and adjusted associations for the 1999–2003 and 2004–2009 survey periods were reported. The 10-year survey included 9,215 participants aged 35 to 74 years. Hypertension remained stable (34.4%). Hypertension unawareness decreased from 35.9% to 17.7% (P<0.001). The decrease in hypertension unawareness was not paralleled by a concomitant absolute increase in hypertension treatment, which remained low (38.2%). A larger proportion of all hypertensive participants were aware but not treated in 2004–2009 (43.7%) compared to 1999–2003 (33.1%). Uncontrolled hypertension improved from 62.2% to 40.6% between 1999 and 2009 (P = 0.02). In 1999–2003 period, factors associated with hypertension unawareness were current smoking , male gender , hypercholesterolemia , and older age . In 1999–2003 and 2004–2009, obesity and diabetes were negatively associated with hypertension unawareness, high education was associated with untreated hypertension , and male gender with uncontrolled hypertension . Sedentarity was associated with higher risk of hypertension and uncontrolled hypertension in 1999–2003.Hypertension prevalence remained stable since 1999 in the canton of Geneva. Although hypertension unawareness substantially decreased, more than half of hypertensive subjects still remained untreated or uncontrolled in 2004–2009. This study identified determinants that should guide interventions aimed at improving hypertension treatment and control. Hypertension is one of the major causes of disease burden worldwide Trends in hypertension prevalence differ according to geographic and population characteristics and definition of arterial hypertension. In the United States, the last NHANES 1988–2008 analysis reported an increase in prevalence from 23.9% in 1988–1994 to 28.5% in 1999–2000. The prevalence remained stable since then To control the public health burden of hypertension, several guidelines recommend the screening, treatment and control of high BP In Switzerland, there are no recent time trends in hypertension treatment, control and unawareness. We analyzed the 1999–2009 trends from a large ongoing population-based study conducted in Switzerland.Specifically, the aims of this study were to assess 10-year trends changes in hypertension prevalence, unawareness, treatment and control for the adult population living in the canton of Geneva, Switzerland. We also aimed to identify factors associated with these four outcomes.‘Bus Santé’ is an ongoing cross-sectional population-based study, which collects information on cardiovascular risk factors, diet and physical activity. Yearly, a representative stratified sample of 500 men and 500 women from the population of the Geneva Canton is recruited and studied The Subjects are selected independently throughout each year to represent the canton’s approximately 100’000 male and 100’000 female non-institutionalized residents aged 35 to 74 years. Eligible subjects are identified using a standardized procedure using an annual residential list established by the local government. This listing includes all potential eligible participants except persons living illegally in the country. Stratified random sampling based on the list by gender within 10-year age strata is proportional to the corresponding population distributions. Selected subjects are mailed an invitation to participate, and, if they do not respond, up to 7 telephone attempts are made at different times on various days of the week. If telephone contact is unsuccessful, 2 more letters are mailed. Subjects not reached are replaced using the same selection protocol. Subjects who refuse to participate are not replaced. Each participant receives several self-administered, standardized questionnaires covering the risk factors for the major lifestyle chronic diseases, socio-demographic characteristics, educational and occupational histories, and reproductive history for women. The 1999–2009 mean participation rate was 60% (range: 55%–65%).http://epidemiologiepopulation.hug-ge.ch/video_busSante.html). In a temperature-controlled room, body weight is measured with the subject lightly dressed without shoes using a medical scale (precision 0.5 kg), and standing height is measured using a medical gauge (precision 1 cm). BP is measured thrice in the sitting position on the right arm after at least 10 minutes rest using a standard protocol. Between 1999 and 2003, BP was measured using a manual mercury sphygmomanometer. Since 2004, BP was measured using a validated automated oscillometric sphygmomanometer .Each participant brings along their filled-in questionnaires, which are checked for correct completion by trained interviewers  on Detection, Evaluation, and Treatment of High Blood Pressure Physical activity levels were quantified using a physical activity frequency questionnaire (PAFQ), developed in the Geneva general adult population and validated using heart rate monitoring Alcohol consumption  was estimated using a validated food frequency questionnaire Self-reported information on education was categorized as high  and low (Elementary school or apprenticeship). Citizenships were categorized as Swiss and non-Swiss according to self-reported nationality. Self-reported information on lifestyle, medical history and comorbidities included smoking status , history of myocardial infarction, diabetes, and hypercholesterolemia. Diabetes and hypercholesterolemia were defined as follows: positive responses to the questions: “Have you ever been told that you had diabetes/high cholesterol?” and “If so, are you taking a drug for it?”.; non-manual, employed; manual, independent; manual, employed; women/man-at-home; retired or jobless.Since 2005, socioeconomic information is collected in the ‘Bus Santé’ study. Self-reported monthly household income is collected using the following ranges: <3′000 CHF; 3′000–4′999 CHF; 5′000–6′999 CHF; 7′000–9′499 CHF; 9′500–13′000 CHF; >13′000 CHF (Swiss Francs CHF≈1.1 US$≈0.80€ as on January 2012). Job position information was categorized as non-manual, manager or independentStatistical analyses were performed using Stata 11.0 . Continuous variables were expressed as mean ± standard deviation (SD). Categorical variables were expressed as number of subjects and percentage. To determine whether the prevalence, unawareness, treatment and control of hypertension changed between 1999 and 2009, yearly data were used and trend test performed. To test the associations between potential determinants (e.g. smoking status) and study outcomes (e.g. untreated hypertension), period 1999–2003 and 2004–2009 were used. The periods were so defined because of the publication of the last Joint National Committee (JNCVII) on Detection, Evaluation, and Treatment of High Blood Pressure report (2003) In the years 2005 through 2008 the annual average number of participants was lower than the other years because another cohort study was conducted relying on the same infrastructure. This is why sample size is smaller for the 2004–2009 than for the 1999–2003 period. Trends in prevalence of hypertension, hypertension unawareness, untreated and uncontrolled hypertension were further determined by income level in a sub-population taking into account information on job position status.The ‘Bus Santé’ study complied with the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the University of Geneva . All participants gave written informed consent.2. Twelve percent had a BMI of 30 kg/m2 or more (obesity). Twenty-six percent had hypercholesterolemia, 6.1% diabetes, 1.9% a history of myocardial infarction. About half of the participants reported high education level.A total of 9,215 subjects were included in the analyses (50% women). Overall mean age was 51.5 yrs . There were 4,402 subjects between 35–49 yrs (47.8%), 3,462 between 50–64 yrs (37.6%) and 1,351 between 65–74 yrs (14.7%) Table 1.Compared to participants, non-participants were more likely to be women , older (52.7 years (SD 11.1) vs 51.5 (SD 10.8), p value <0.001) and to have the Swiss citizenship . There was no difference with respect to smoking status .The mean systolic and diastolic BP were 125.9 mmHg (19.4) and 78.0 mmHg (11.2), respectively. The percentages of participants by BP categories were as follow: systolic and diastolic BP<120/<80, 33.4%; systolic or diastolic BP 120–139/80–89, 38.1%; systolic or diastolic BP 140–159/90–99, 20.7%; and systolic or diastolic BP≥160/≥100, 7.8%.The number of participants were 6,020 (65.3%) and 3,195 (34.7%) in the 1999–2003 and 2004–2009 survey period, respectively. The prevalence of smoking status, diabetes, hypercholesterolemia, education level, Swiss citizenship, and alcohol consumption differed significantly between the two periods. The mean diastolic BP was lower in the second period than the first period , and the overall distribution of the grades or stages of hypertension differed between the two periods., S2) in both men and women. The prevalence of hypertension unawareness decreased between 1999 and 2009, in men and in women specifically (p<0.001). In 1999, 35.90% of hypertensive participants were unaware of having hypertension, whereas 17.7% of hypertensive participants were unaware of having hypertension in 2009., The increasing tendancy of the prevalence of untreated hypertension observed in men and in women specifically was only statistically significant  when men and women participants were combined and thus power increased. The prevalence of uncontrolled hypertension decreased between 1999 and 2009 (p<0.05); from 62.2% to 40.6% in men and women combined, from 60.6% to 49.6% in men, and from 72.2% to 37.8% in women.The overall age-standardized prevalence of hypertension was 34.4%. This prevalence remained stable between 1999–2009 . To adjust for the effect of job position, trends with household monthly income were further adjusted for job position. After full adjustment, no clear trends were found.Information on monthly household income and job position collected since 2005 was available for 2,024 (22.0%) participants whose main characteristics are presented in Hypertension is the most prevalent cardiovascular disorder in high-income countries, where it affects 20% to 50% of the adult population Our analyses showed no significant changes in hypertension prevalence in adults from 1999 to 2009. A 1993–2000 analysis conducted in the same source population reported, a decline in hypertension prevalence in men and women Hypertension awareness improved between 1999 and2009 in the Geneva population. Similar favorable trends have been reported in other, yet scarce, longitudinal reports on hypertension awareness Figure S2). The observed gain in hypertension awareness is therefore unlikely to translate into public health benefit if no action is undertaken to control BP. We found that BP was not controlled in half of hypertensive treated subjects, which corresponds to the control rate reported in 2009 in a Swiss city population-based study (48%) Elevated BP is a risk factor for coronary heart disease, heart failure, stroke, peripheral arterial disease, and renal failure Hypertension treatment initiation and intensification once hypertension is detected are challenged by both patient- and physician-related factors, which may vary across regions After adjustment, participants with high education level were at increased risk of untreated hypertension than participants with lower level of education. The associations between education and hypertension awareness, treatment and control are inconsistent. Some reports showed an inverse association or no association between education level and hypertension awareness, treatment or control Our results have to be interpreted within the context of the Swiss health care system. Health insurance is compulsory for all citizens of Switzerland (7 millions) and insurance premiums are paid independently of earnings When interpreting the findings of this study, one has to keep in mind its limitations. Non participants slightly differed from participants with respect to sex, age and Swiss citizenship. Given that our results suggested that age, smoking status and gender are significant predictors of study outcomes, these differences may somewhat limit the generalisability of our findings. Several informations were determined by the use of questionnaires. By nature of its reliance on self-reported data, this is a source of possible bias. Similarly to other large population-based studies, white coat effect, white coat hypertension, or masked hypertension could not be determined in this analysis. BP was measured with two different methods; mercury sphygmanometer (1999–2003) and semi-automatic oscillometer (2004–2009). Although semi-automatic oscillometer are calibrated with mercury sphygmanometer, we cannot exclude that some of the differences observed between the two periods are attributable to the method of BP measurement. Yet, it is also possible that the 2003 antihypertension guidelines  In conclusion, in a representative sample of the canton of Geneva, Switzerland, the prevalence of hypertension remained stable between 1999–2003 and 2004–2009. Population-based primary prevention measures are needed to decrease the burden of hypertension in this region. While favorable trends in hypertension unawareness and uncontrolled hypertension occurred during this period, about half of hypertensive subjects were not treated or had uncontrolled high BP in the latest 2004–2009 survey period. Factors associated with untreated and uncontrolled hypertension in our analyses could guide the implementation of targeted interventions aimed at reducing these rates.Figure S1Flow chart of age-standardized prevalences of hypertension, unawareness, untreated, and uncontrolled hypertension, by survey period.(EPS)Click here for additional data file.Figure S2Age-standardized prevalences of the four mutually exclusive categories of participants with hypertension, by survey period.(EPS)Click here for additional data file.Table S1Age-standardized prevalences (95%CI) of hypertension, hypertension unawarenness, untreated and uncontrolled hypertension, by survey year and gender .(DOCX)Click here for additional data file.Table S2Crude numbers of participants by hypertension, hypertension unawarenness, untreated and uncontrolled hypertension groups, by survey year and gender .(DOCX)Click here for additional data file.Table S3Patients’ characteristics among subjects with socioeconomic information .(DOCX)Click here for additional data file.Table S4Age-standardized prevalence (95%CI) of hypertension, hypertension unaeareness, untreated hypertension, and uncontrolled hypertension by monthly household income and job position, and adjusted trends with income .(DOCX)Click here for additional data file.


Objectives. The aim of our study was to examine serum anti-Müllerian hormone (AMH) concentration in ovarian cancer patients in relation to clinicopathological features, such as a pathological subtype of the tumor, (FIGO) stage, grading, and overall 5-year survival. Material and Methods. We enrolled 72 epithelial ovarian cancer patients in our study, aged 45–79 years, who underwent optimal cytoreductive surgery. In all patients, serum AMH concentration was measured using a two-step sandwich type enzyme immunoassay before surgery. As a reference value for women over 45 years we accepted anti-Müllerian hormone concentration below 1 ng/mL. Results. In the whole group of patients with ovarian cancer, median serum concentration of AMH was 0.07 (0.0–0.37) ng/mL, whereas in the group of those with positive AMH values (≥0.14 ng/mL) it was 0.31 (0.15–0.73) ng/mL. No significant correlation was found between serum AMH levels and FIGO stage, histological subtype, or grading (P > 0.05). The analysis of five-year survival rate related to AMH levels showed no statistically significant differences. There were no differences in survival rates between patients with positive or negative serum AMH levels. Conclusion. Measurement of serum anti-Müllerian hormone levels was not useful in predicting clinicopathological features and survival in patients with ovarian cancer. Among cancers of female reproductive system, ovarian cancer became the second most common and is the fifth ranked cause of cancer-related mortalities in women in Europe and the United States . Althougβ (TGF-β). AMH signals through two transmembrane receptors, type II which is specific (present in Müllerian duct and gonads) and type I receptors, shared with the bone morphogenetic proteins family [Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), belongs to a larger family of transforming growth factor-s family , 5.AMH is expressed in the Sertoli cells of fetal testis from the seventh week of pregnancy , and itsIn women AMH is produced by granulosa cells, from preantral and antral follicles. Serum AMH levels are undetectable in newborns, increase during childhood and adolescence reaching its peak in the early 20s, and remain stable throughout reproductive period only to decrease during menopausal transition , 9. DuriStudies on mice had shown that AMH inhibits the transition from the primordial to the primary follicular stage. AMH paracrine signaling inhibits FSH-related follicle growth leading to selection of primary follicle. FSH and estradiol decrease AMH gene expression in granulosa cells . Recent The number of primordial follicles decreases with age of women. Lower serum AMH levels precede the increase of FSH and inhibin B levels and thus are the most accurate parameter of ovarian reserve in clinical practice –16. AMH In women undergoing oncologic treatment AMH is considered a useful marker of damage to the ovarian reserve , 21.The fact that AMH expression is restricted to ovarian granulosa cells in women led to establishing AMH levels as a serum marker of granulosa cell tumours (GTCs). Recent studies showed an increased serum AMH concentration in 76–93% of women with both primary and recurrent GTCs , 23.β family and its representatives play an important role in ovarian cancer carcinogenesis, the aim of this study was to examine AMH concentration in ovarian cancer patients in relation to clinicopathological features, such as a pathological subtype of the tumor, FIGO stage, grading, and overall 5-year survival.As anti-Müllerian hormone belongs to transforming growth factor-We enrolled 72 epithelial ovarian cancer patients aged 45–79 years (mean 58.5 ± 10 years) treated in Department of Gynecology and Obstetrics, Ludwik Rydygier Collegium Medicum in Bydgoszcz in the period of 2005-2006. Only women who underwent optimal cytoreductive surgery were considered for further analysis, all of them with residual cancerous focuses smaller than 1 cm in diameter. All patients were operated by experienced gynecological oncologist. The standard surgical protocol included tumorectomy, hysterectomy with bilateral salpingoovariectomy, pelvic lymphadenectomy, omentectomy, and appendectomy. Women who had ever used hormonal replacement therapy or had undergone ovulation induction were excluded. All patients were Caucasian, after menopause with BMI range from 19 to 30 kg/m².International Federation of Gynecology and Obstetrics (FIGO) ovarian cancer staging system was used to assess clinical stage of the disease. Early stages were confirmed in 16 patients  and advanced disease in 56 patients . All ovarian cancer cases were with histological confirmation, of which 59 (82%) serous, 5 (7%) mucinous, and 8 (11%) endometrioid. Histological examination was performed in the Department of Pathology, Antoni Jurasz University Hospital in Bydgoszcz, and histological grading was determined . After optimal cytoreductive surgery, all women underwent 6 courses of chemotherapy based on carboplatin and paclitaxel. Baseline characteristics of the study participants are shown in The Bioethical Committee at the Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University of Torun, have reviewed and approved this study. All participants have provided informed consent.Blood samples were collected (10 mL) after admission to the hospital, on the day before surgery. After centrifugation in standard conditions serum was obtained, aliquoted, and stored at −70°C until assayed.Serum AMH concentration was measured using a two-step sandwich type enzyme immunoassay . The imprecision of the assay was 12.3% at 0.2 ng/mL and 5.1% at 15.8 ng/mL. The lowest AMH concentration in a sample which could be detected with a 95% probability was 0.08 ng/mL (lower detection limit). Concentration of AMH below 0.14 ng/mL has been accepted as negative. Reference values for anti-Müllerian hormone for women over 45 years were less than 1 ng/mL .P value <0.05 was considered statistically significant. The Kolmogorov-Smirnov test was used to assess normality of distribution of investigated parameters. Data were expressed as mean ± standard deviation and median with 25th–75th percentiles. Comparison between the groups was performed by using the Mann-Whitney U test and the Kruskal-Wallis test for non-Gaussian distributed variables. Overall survival rate was examined for significance using log-rank test and Kaplan-Meier curves. P value of less than 0.05 for entry was applied. The effects of the variables were expressed as hazard ratios per 1 SD change to allow for a better comparability between the effect sizes of the different tested variables.Univariate and multivariate Cox regressions were performed. For the analysis, a forward selection with a All statistical analyses were performed using Statistica for Windows Statsoft 10.0. In the group of patients with ovarian cancer median serum concentration of AMH was 0.07 (0.0–0.37) ng/mL, whereas median concentration in the patients with positive AMH values (≥0.14 ng/mL) was 0.31 (0.15–0.73). Values equal to/above 0.14 ng/mL were found in 44 women (61%).Median concentrations of serum AMH in relation to FIGO stage did not differ significantly .No significant correlations were found between serum AMH concentration and histopathological subtype or grading Tables  and 4.The results of Cox regression of the predictive power of variables are shown in Tables N = 28) of women with serum concentration below the detection limit and another (N = 44) who displayed values over 0.14 ng/mL (P = 0.98). The analysis of five-year survival rate related to AMH levels showed no statistically significant differences; there were no differences in survival rates between patients with positive or negative AMH values . The results observed in our study were similar to those of age-specific AMH values found by others but in healthy women . Despite the lack of statistically significant differences, women with FIGO classifications I and II had lower AMH levels than women with FIGO stages III and IV. On the other hand, according to histological grading, highest AMH values were observed in women with G1 staging. Detailed analysis of concentrations of anti-Müllerian hormone has revealed no differences in the levels of AMH, depending on the type of cancer, clinical stage, and histological grade. There was no correlation between serum AMH and the five-year survival rate. There were no differences in years of survival of patients with AMH in serum compared to patients who were negative for anti-Müllerian hormone. In our study only advanced clinical stage according to FIGO was an independent poor prognostic factor. To the best of our knowledge there are no available studies demonstrating the usefulness of determination of AMH concentrations in the serum of patients with epithelial ovarian cancer. The search for alternative therapies in the treatment of ovarian cancer has led to research on biologically active substances which might inhibit the proliferation of the tumor. Masiakos et al. hypothesized that the anti-Müllerian hormone may serve as such factor because it causes apoptosis and the regression of the Müllerian ducts in embryos, by variation of ovarian tumor cell biology . Further In conclusion, AMH belongs to the family of transforming growth factor, which plays an important role in ovarian carcinogenesis. However, none of the limited studies could demonstrate the role of the anti-Müllerian hormone in the serum of patients with ovarian cancer. Similarly, in our study, we failed to show any benefits from the determination of serum AMH in women with ovarian cancer.Despite our findings concerning epithelial ovarian cancer, serum AMH levels remain well-established marker in granulosa-theca cell tumors. It is necessary to perform further studies to determine the tissue expression of AMH and type II receptor for AMH in ovarian cancer tissue and the potential usefulness of monoclonal antibodies against AMH and its receptor in the diagnosis and treatment of women with ovarian cancer.


If anterior cruciate ligament (ACL) reconstruction is to be performed, decision regarding graft choice and its fixation remains one of the most controversial. Multiple techniques for ACL reconstruction are available. To avoid disadvantages related to fixation devices, a hardware-free, press-fit ACL reconstruction technique was developed.The aim of this study was to evaluate clinical outcome and osteoarthritis progression in long term after ACL reconstruction with central third patellar-tendon autograft fixed to femur by press-fit technique.Fifty two patients met inclusion/excusion criteria for this study. The patients were assessed preoperatively and at 15 years after surgery with International Knee Documentation Committee Knee Ligament Evaluation Form, Lysholm knee score, Tegner activity scale and radiographs.p = 0.004) and 6.9 ± 1.4 (p = 0.005) respectively at follow-up. The IKDC subjective score improved from 60.1 ± 9.2 to 80.2 ± 8.1 (p = 0.003).Good overall clinical outcomes and self-reported assessments were documented, and remained good at 15 years. The mean Lysholm and Tegner scores improved from 59.7 ± 18.5 and 4.2 ± 1.0 preoperatively to 86.4 ± 5.6 (p > 0.05).According to IKDC objective score, 75% of patients had normal or nearly normal knee joints at follow-up. Grade 0 or 1 results were seen in 85% of patients on laxity testing. Degenerative changes were found in 67% of patients. There was no correlation between arthritic changes and stability of knee and subjective evaluation  press-fit fixation include unlimited bone-to-bone healing, cost effectiveness, avoidance of disadvantages associated with hardware, and ease for revision surgery. BPTB femoral press-fit fixation technique can be safely applied in clinical practice and enables patients to return to preinjury activities including high-risk sports. The anterior cruciate ligament (ACL) is regarded as critical to the normal functioning of the knee, and it is one of the most frequently injured ligaments in the human body. Its rupture affects knee stability, which may cause giving way symptoms, increased risk of meniscal injuries, and early onset of joint degeneration-5.When treating a torn ACL, many decisions must be made, especially if surgery is to be performed. The decision regarding graft choice and its fixation remains one of the most controversial. The graft could be autograft, allograft, or synthetic. These include patellar tendon, hamstring tendons, quadriceps tendon and others,6-11.Central third bone–patellar tendon– bone autograft fixed with interference screws has long been the graft of choice -14, despTo avoid disadvantages related to internal fixation devices, especially on femoral side, a hardware-free ACL reconstruction technique was developed. This technique uses the bone plugs on either end of the patellar tendon graft for press-fit fixation. The presented technique was originally developed in 1987 for femoral press-fit fixation and in 1989 for tibial press-fit fixation. AfterwaThe aim of this retrospective study was to evaluate the clinical outcome and osteoarthritis progression in the long term after ACL reconstruction with a central third patellar-tendon autograft fixed to the femur by press-fit technique.The entry inclusion criterion for this study was isolated ACL insufficiency combined with subjective knee instability. Patients who had had a previous injury or surgery on either knee, patients with bilateral ACL insufficiency; a posterior cruciate ligament (PCL) insufficiency; an injury of postero-lateral corner (PLC), lateral collateral ligament (LCL), or medial collateral ligament (MCL) tear of grade III were exc2.Seventy one patients met the entry inclusion criteria. The analyzed group consisted of 41 men and 30 women. The average patients’ age was 28 (range: 16–43 years). The mean period between the initial injury and surgery was 3.2 months . The BMI in the study group was 27.9 ± 3.8 kg/mThe miniarthrotomy transtibial technique using femoral press-fit fixation and tibial fixation with interference screw was performed. A diagnostic arthroscopy was carried out first. The middle third of the patella tendon (9–10 mm in diameter) was harvested with 25 mm to 30 mm of bone from the patella and tibial tubercle. The bone blocks were formed to a trapezoid shape by using an oscillating saw  to the ten-thirty position (for right knees) or the one-thirty position (for left knees) at the back of the intercondylar notch. The graft was then passed into the knee from outside-in using a pull-through suture and the bone blocks positioned in their tunnels by pulling and assisted with hammering using impactor inserted through a separate incision on thigh with the knee joint flexed up to 120°. The tibial bone plug was tapped into the femoral tunnel and fixed with press-fit. The patellar bone plug was secured in the tibial tunnel with an interference screw  score , difficulty with performing the duckwalk, and difficulty with squatting. Stiffness of the knee, catching and locking of the knee, and anterior knee pain as well as extent of donor- site morbidity were also recorded.Besides the parameters of the IKDC subjective score, we used other subjective outcome questions. “Would you have surgery again”, “Does your knee give way”Radiographic evaluation was performed according to IKDC evaluation form and included a standing weight-bearing anteroposterior view (45° of knee flexion) and lateThe clinical evaluations were performed by a physician not involved in the primary ACL reconstruction.χ2), the Mann–Whitney U test, Fisher’s exact test, the Pearson’s correlation coefficient and the Spearman's rank correlation coefficient. Significance was set at p < 0.05.The data was stored on a Microsoft Access database. Statistical analysis was performed using the Statistica 6 (StatSoft Inc) software program by medical statistician. Following tools were used: Pearson's chi-square test .5 patients were excluded because of an ACL injury of the contralateral knee, 4 patients 5.6% of 71) had a graft rupture due to an adequate trauma during the follow-up period, 4 patients were lost to follow-up. In 8 patients, during follow-up period, further surgeries to address the secondary restraints were conducted  to 131° of flexion (125 to 145). When compared with the contralateral limb, 17 of 52 (33%) patients had less than 5° of flexion loss, and 6 patients (11.5%) displayed between 5° and 15° of flexion loss. A flexion deficit of >15° was observed in 2 patients (3.8%). 7 patients (13%) had lost extension of 3°–5°, and in 3 patients (6%) an extension deficit of >5° was observed.p = 0.07).The mean anteroposterior translation measured with Rolimeter was 1.6 mm ± 1.3(a difference to the opposite knee). Instrumented test results were not associated with self-reported knee function . Thirty nine of 52 patients (75%) returned to their previous sport at the preinjury level, including high-risk sports such as soccer or skiing Table.The patients were asked to perform a single-legged hop for distance on the index and normal side. According to IKDC objective score a ratio of the index to normal knee was calculated: ≥90% (62% of cases), 89-76% (25% of cases), 75-50% (13% of cases). At the 15-year follow-up, 43 (83%) patients could squat normally, 7 (13%) others could do it with slight difficulty and 2 patients (4%) could squat with significant difficulty.When asked, 49 (95%) patients would have the surgery again. Subjectively, 2 patients that were seen for follow-up complained about instability. Anterior knee sensitivity (42% of patients reported the presence of kneeling pain) and donor site morbidity were the most often mentioned complaint. Numbness of the skin was reported by 37 (42%) patients.Radiographs were performed on 52 patients at 15 years. Degenerative changes were found in 37 (67%) patients. The results of the IKDC radiographic assessment are shown in TableThere was no correlation between radiographic changes and postoperative results in both subjective and objective scores Table. EctopicPostoperative complications were observed in 5 patients. One patient developed superficial wound infection, which resolved on treatment with antibiotics. Arthrofibrosis occurred in 4 patients, requiring intensive treatment, in 2 cases arthroscopical arthrolysis. There were 3 late arthroscopies, for arthrolysis to allow full extension.The success of the ACL reconstruction is influenced by different factors. One of them is the choice of the graft. It is clear that an ideal graft for ACL reconstruction does not exist. For many surgeons, bone–patellar tendon– bone autograft (BPTB) remains still the graft of choice. Although, the graft is criticized for resulting in significant harvest-site morbidity, over the years it has proved to be a stable graft that has longlasting biomechanical properties,15,35,36The primary stability of the graft is one of the most important issues in ACL reconstruction. It depends not only on the strength of the graft, but also on its fixation. Regarding BPTB, the graft can be fixed using different methods of fixation e.g.: interference screw, button, staple post and others. It can also be fixed with the use of press-fit technique without use of any hardware,22,25,37The femoral press-fit fixation in this study utilizes the native femoral ACL insertion as the point of fixation. The graft is protected additionally by the angulation of the bone block against the ligament graft thereby also minimizing the risk of tunnel misplacement or bone tunnel enlargement. The mosSimilar to other techniques it has also disadvantages and limitations. Comparing for example to hamstring techniques it is not a definitely cosmetic method, what may be an issue for some patients. The poor bone quality, especially in older women might be one of the possible limitations. The two incision operation method, may be technically demanding for beginners. However in our study we did not observe any complications related neither to bone quality nor to technical site.There is not a big number of studies presenting long-term results of ACL reconstruction,14,20,39In our study we present the 15-year results of femoral press-fit ACL reconstruction with patellar tendon autograft in a series of 52 patients. Good clinical results were documented in subjective and objective evaluation. The mean Lysholm and Tegner score were 86.3 and 6.8, respectively. These results are comparable to observations reported by others,14,35; iAccording to the IKDC standard evaluation form, the overall improvement was documented. 66.5% of 52 patients had normal or nearly normal knee (grade A or B) at the 15-year follow-up. At the time of follow-up 42 patients (81%) had 0 or 1+ on the Lachman test. There was also a marked reduction in the degree of pivot shift after the reconstruction; 43 patients (83%) had a value of 0 or 1+. Similar results have been noted in the literature after ACL reconstruction,20,37. TThe donor site morbidity seems to be the major concern of all techniques utilizing BPTB grafts. It includes complications such as anterior knee pain, pain when kneeling, patellar fracture, patellofemoral crepitation, numbness caused by damage of the infrapatellar branch of the saphenous nerve and possible loss of quadriceps strength,16,36,41The development of early osteoarthritis might be the main consequence of ACL rupture. We already know that ACL reconstruction does not prevent osteoarthritis, and the development of osteoarthritis post-ACL reconstruction has been reported for all types of reconstructions,3,36,44.In conclusion, most of our patients had a satisfactory outcome after ACL reconstruction using femoral press-fit technique, and the results are comparing well to the literature. This ACL reconstruction technique, after switching to arthroscopically assisted, is still used in our centre.We are aware of limitations associated with the present study. Limitations are related with restricted subject numbers and retrospective analysis. The small number of patients is the result of very strict criteria of inclusion. We aimed to analyze a very definite group of patients. In addition certain number of patients was lost to follow-up. Despite the limitations, in our opinion the present study allows to draw rational conclusions.Our study proved that the BPTB femoral press-fit fixation can be recommended as the fixation technique for ACL reconstruction.The advantages of this method include unlimited bone-to-bone healing, reduction of disadvantages associated with hardware, cost effectiveness, and ease for revision surgery. In our opinion the BPTB femoral press-fit fixation technique can be safely applied in clinical practice and allows patients to return to preinjury activities also including high-risk sports.The authors declare that they have no competing interests.WW – study design, data collection, manuscript preparation, data interpretation. MW – data collection, literature search. BK – data collection. SD – data interpretation. AC – statistical analysis, literature search. WT – statistical analysis, literature search. JW – data interpretation. All authors have read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/115/prepub


Tagetes erecta L.) has long been used as a medicinal herb for a number of therapeutic activities. In the present study, the cytotoxicities of ethanol and ethyl acetate extracts of marigold flowers and their inhibitory effects on elastase and tyrosinase enzymes were investigated. An MTT assay was performed to measure the cytotoxicity of these two extracts on the H460 lung cancer and the Caco-2 colon cancer cell lines. An elastase assay kit, based on the digestion of a non-fluorescent elastin substrate to highly fluorescent fragments by elastase, was used for the elastase inhibition assay. Tyrosinase inhibition activity was investigated using the dopachrome method with L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate. The data obtained in this study demonstrated that the extracts were nontoxic to H460 and Caco-2 cell lines. The elastase inhibition activities of ethanol (250 μg/ml) and ethyl acetate (125 μg/ml) extracts were found to be significantly higher than that of the negative control. The tyrosinase inhibition activities of ethanol and ethyl acetate extracts, in terms of the mean inhibition concentration (IC50), were 1,078 and 1,467 μg/ml, respectively. To the best of our knowledge, the present study has demonstrated for the first time that marigold flower extracts possess tyrosinase inhibition activity. The activities of ethanol and ethyl acetate extracts of marigold flowers were investigated in vitro and indicated that these extracts possess useful properties that may be of interest for cosmetic development.Marigold ( Tagetes erecta L.), an ornamental plant, belongs to the Asteraceae family and is commonly known as ‘Dow Ruang’ in Thailand. Numerous traditional uses of this plant have been reported. The whole plant has been used to treat bronchitis, rheumatic pain, cold and respiratory diseases, and as a stimulant and muscle relaxer  flowers were investigated for their cytotoxicity, total phenolic content (TPC) and inhibitory effects on elastase and tyrosinase enzymes.In the present study, ethanol and ethyl acetate extracts of marigold (® Elastase Assay kit (E-12056) was purchased from Molecular Probes , and mushroom tyrosinase, tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) were obtained from Sigma-Aldrich Chemie GmbH . Ethanol (EtOH), ethyl acetate (EtOAc), dimethylsulfoxide (DMSO) and sodium dodecyl sulfate (SDS) were purchased from Merck KGaA .The human lung cancer (NCI-H460) and colon cancer (Caco-2) cell lines were obtained from the American Type Culture Collection . MTT  was purchased from Sigma Chemical  and RPMI-1640 medium was obtained from Invitrogen Life Technologies . The EnzChekTagetes erecta L.) flowers were collected from Chiang Mai, Thailand in the winter of 2008 and authentication was performed by Dr Kongkanda Chayamarit in the Forest Herbarium . Voucher specimens have been deposited at the Herbarium Section .Marigold (Dried powder of the marigold flower (250 g) was separately extracted by Soxhlet’s apparatus  with two different solvents, ethyl acetate and ethanol. Each extract was subsequently filtered through Whatman No.4 paper and evaporated under a vacuum. The residues obtained from the ethyl acetate and ethanol extracts were named MF_EtOAc  and MF_EtOH , respectively. MF_EtOAc and MF_EtOH extracts were employed for further study.et al, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were grown in a humidified atmosphere of 5% COCaco-2 cells were cultured in Dulbecco’s modified Eagle medium to which 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine were added. The cells were incubated under the same conditions as the H460 cells and subcultured every week using 0.5% trypsin and 0.5 mM EDTA solution.The cytotoxicity of the MF_EtOH and MF_EtOAc extracts was assessed using the MTT assay. Mitochondrial respiration, an indicator of cell viability, reduced MTT from a yellow water-soluble dye to a purple insoluble formazan product by succinate dehydrogenase . Since M4 and 2×104 cells/well in 100 μl medium, respectively. Prior to testing, the cell culture medium was removed and cells were washed with phosphate-buffered saline (PBS). Experiments were initiated by adding 100 μl MF_EtOH or MF_EtOAc solution to each well. After 24 h of incubation, cells were washed with PBS and 100 μl MTT solution (0.5 mg/ml) was added to each well. Following a further 4 h of incubation, 100 μl SDS solution (10% w/v) was added to each well and the cells were incubated overnight. Absorbance was then measured at a wavelength of 590 nm using a Thermomax microplate reader . Untreated cells (0.5% DMSO) were used as the control group.Caco-2 and H460 cells were cultured separately in 96-well plates for 48 and 24 h at seeding densities of 5×10® Elastase Assay kit. This kit consisted of DQ™ elastin from bovine neck ligaments that was labeled with fluorescent BODIPY® FL conjugate, elastase from pig pancreas, N-methoxysuccinyl-ala-ala-pro-val-chloromethyl ketone (elastase inhibitor) and reaction buffer.The elastase inhibition activities of MF_EtOH and MF_EtOAc extracts were assayed using the EnzChek® FL conjugated with DQ™ elastin) to highly fluorescent fragments by elastase. The digestion products were subsequently measured using a fluorescence microplate reader .The principle was based on the digestion of non-fluorescent elastin substrate (BODIPYDMSO solution (5% v/v) was employed to enhance the solubility of MF_EtOH and MF_EtOAc extracts, respectively. The elastase inhibition activity of MF_EtOH was studied at three concentrations , while two concentrations of MF_EtOAc extract (50 and 125 μg/ml) were studied due to the limited solubility of MF_EtOAc. The test was performed in 96-well plates that were hidden from the light. DMSO (5%) and elastase inhibitor were used as a negative and a positive control, respectively.® Elastase Assay kit and described by Leu et al:50) was determined by interpolation of the dose-response curves.Where ‘A’ was enzyme activity in absence of the inhibitor and ‘B’ was enzyme activity in the presence of inhibitor. The concentration giving 50% tyrosinase inhibition activity (ICResults are presented as the mean ± standard deviation. An unpaired Student’s t-test (two-tailed) was used to test the significance of the difference between two mean values. One-way analysis of variance was used when more than two means were compared. P<0.05 was considered to indicate a statistically significant difference.The TPCs of MF_EtOH and MF_EtOAc extracts, expressed as gallic acid equivalents, are shown in To investigate whether the MF_EtOH and MF_EtOAc extracts affected the viability of H460 and Caco-2 cells, the MTT assay was performed. The cytotoxicity of the MF_EtOH and MF_EtOAc extracts is presented as a percentage of cell viability relative to the control group (100%). The mean and standard deviation of each experiment were calculated from 10 replicates.The viability of the H460 cells following treatment with different concentrations of MF_EtOH or MF_EtOAc extracts ranged between 87.78 and 126.2% . No signThe cytotoxicity studies of MF_EtOH and MF_EtOAc extracts on Caco-2 cells obtained similar results to the H460 cells . The CacAs shown in in vitro inhibition effects of various concentrations of MF_EtOH and MF_EtOAc extracts on tyrosinase activities are shown in 50 values of MF_EtOH and MF_EtOAc extracts against tyrosinase were 1,078 and 1,467 μg/ml, respectively. These observations suggested that the MF_EtOH extract exerted a more marked tyrosinase inhibition compared with that of the MF_EtOAc extract. However, at a concentration of 5,000 μg/ml, MF_EtOAc extract resulted in a higher tyrosinase inhibition of 90.6%, while the MF_EtOH extract exhibited 68.5% inhibition.The 50 values of MF_EtOH and MF_EtOAc extracts were 23- and 31-fold higher than that of kojic acid (IC50=47 μg/ml).Due to the aforementioned tyrosinase inhibition activity, kojic acid was selected as the positive control in this study. The ICThe phenolic compounds present in marigold flower extracts have previously been reported to markedly contribute to the antioxidant capacities of the extracts . As an aAccording to the ISO 10993-5 International Standard , a reducElastase, a member of the proteinase family, is mainly responsible for degrading elastin, a connective tissue component. Elastase activity increases with age, resulting in a reduction in elastin and further decreasing the elasticity of skin, which causes the appearance of wrinkles and stretchmarks . TherefoAlthough the tyrosinase inhibitory activities of MF_EtOH and MF_EtOAc extracts were significantly lower than that of kojic acid (P<0.05), the present study was, to the best of our knowledge, the first to demonstrate that ethanol and ethyl acetate extracts from marigold flowers possess tyrosinase inhibition activities.At present, tyrosinase inhibitors have important applications in cosmetics. For clinical usage, tyrosinase inhibitors are employed to treat dermatological disorders associated with melanin hyperaccumulation, and are essential in cosmetics for depigmentation . TherefoSince no cells were utilized in the elastase and tyrosinase inhibition assays, the solubility of MF_EtOH and MF_EtOAc extracts was able to be significantly enhanced using higher concentrations of DMSO. Therefore, while the cytotoxicity of these two extracts was studied at the maximum concentration of 100 μg/ml, the concentrations of the two extracts were as high as 250 and 5,000 μg/ml in the elastase and tyrosinase inhibition studies, respectively.The present study demonstrated that MF_EtOH and MF_EtOAc extracts possess elastase and tyrosinase inhibition effects and antioxidant activities. At present, numerous cosmetic companies utilize elastase, melanogenesis inhibitors and antioxidant ingredients in antiwrinkle, skin-lightening and antiaging products. Therefore, MF_EtOH and MF_EtOAc extracts have potential for use in cosmetic products in the future. Moreover, MF_EtOH and MF_EtOAc are plant extracts and, as a result, their use in cosmetic products may be particularly advantageous due to the relatively low incidence of side effects.Tagetes erecta L.) flowers were nontoxic to two human cell lines, lung cancer (H460) and colon cancer (Caco-2). Furthermore, the potential of these two extracts as sources of elastase and tyrosinase inhibitors was suggested. The in vitro activities of ethanol and ethyl acetate extracts from marigold flowers indicated that these two extracts possess useful properties and may be suitable for cosmetic applications as antiwrinkle, skin-lightening and antioxidant additives. Further studies may focus on evaluating the biological activities of marigold extracts in vivo for cosmetic development.In conclusion, the results obtained in the present study demonstrated that ethanol and ethyl acetate extracts from marigold (