Liposomes co-encapsulating doxorubicin and glucoevatromonoside derivative induce synergic cytotoxic response against breast cancer cell lines.

Cancer is one of the main causes of death in the world and thus a global public health problem. Among the treatments available for cancer are surgery, radiotherapy, and chemotherapy. Currently, there is increased interest in the combination of two or more antitumor agents to achieve a synergistic effect in cancer therapy. Doxorubicin (DOX), a chemotherapeutic which has a potent antineoplastic action, has been used in the treatment of various tumors. However, the use of DOX is limited, mainly due to the cardiotoxicity. Therefore, nanostructured systems, such as liposomes, have been developed to carry this drug and target the tumor region, since tumor tissues present enhanced permeability and retention for nanosystems. Cardiac glycosides, such as digitoxin, have recently shown great antitumor potential despite the low therapeutic index which may limit their use. Furthermore, some compounds of this class have low water solubility, which makes their in vivo administration difficult. In this context, liposomes represent a valid strategy to carry simultaneously antitumor drugs allowing their intravenous administration. In this study, liposomes loaded with glucoevatromonoside containing peracetylated glucose hydroxyl groups (GEVPG) and DOX at molar ratio of 1:1 (SpHL-GEVPG:DOX 1:1) were developed, and their chemical and physicochemical properties were evaluated. This formulation presented a combination index (CI) lower than 1 at inhibitory concentration of 90 % growth (IC90) for three human breast tumor lines evaluated (0.52 ± 0.39 for MDA-MB-231, 0.19 ± 0.13 for MCF-7, and 0.99 ± 0.09 for SKBR-3). These results indicate a synergistic cytotoxic effect of the GEVPG and DOX combination encapsulated in liposomes. In addition, SpHL-GEVPG:DOX 1:1 presented selectivity towards these cancer cells. Long-term in vitro cytotoxicity studies demonstrated that MDA-MB-231 surviving cells after treatment with SpHL-GEVPG:DOX 1:1 did not recover proliferation capacity after 21 d. From the studies of cell cycle and death pathway evaluation, it was observed that SpHL-GEVPG:DOX 1:1 arrested the cell cycle in the G2/M phase and similarly induced apoptosis and necrosis. However, SpHL-GEVPG:DOX at molar ratio of 1:1 showed lower induction of both apoptotic and necrotic pathways compared to free DOX and SpHL-DOX, suggesting that the mechanism of death involved may not be related to necrosis or apoptosis. Lastly, SpHL-GEVPG:DOX 1:1 showed a good storage stability for 90 d at 4 °C. Therefore, the results of the present work indicate the potential use of SpHL-GEVPG:DOX 1:1 as a new anticancer formulation.

Comparison of high-intensity laser therapy and combination of ultrasound treatment and transcutaneous nerve stimulation in patients with cervical spondylosis: a randomized controlled trial.

The aim of the study was to compare the effect of high-intensity laser therapy (HILT) and a combination of ultrasound (US) treatment and transcutaneous nerve stimulation (TENS) on pain, range of motion (ROM), and functional activity in patients with cervical spondylosis (CS). A total of 84 patients with a mean age of 51.54 years (52 women and 32 men) affected by CS were enrolled in this study. Patients were randomly divided into two groups. In group A (42 subjects), patients received 12 sessions of HILT plus exercise, while in group B (42 subjects), they received a combination of US, TENS, and exercise. The outcomes measured were cervical segment ROM, pain level measured by visual analogue scale (VAS), and functional activity measured by neck disability index (NDI) at the end of the therapy. The level of statistical significance was set as p < 0.05. In the two groups, cervical ROM, VAS, and functional scores showed significant changes. Both HILT plus exercise and US/TENS plus exercise effectively increased cervical ROM and reduced pain (with a significant greater decrease in group A). Statistically significant differences in NDI scores were observed after treatment sessions with better results for participants enrolled in group A (HILT plus exercise) Both therapeutic modalities demonstrated analgesic efficacy and improved function in patients affected by cervical spondylosis 4 weeks after the therapy. HILT plus exercise was more effective than US/TENS plus exercise. HILT can be promoted and used in this pathology with positive outcomes. However, further studies are needed to optimize the dose and duration of HILT therapy.

Long-circulating and fusogenic liposomes loaded with a glucoevatromonoside derivative induce potent antitumor response.

Cancer is an important public health problem, being one of the leading causes of death worldwide. Most antineoplastic agents cause severe toxic effects and some types of cancer do not respond or are resistant to the existing pharmacotherapy, necessitating the research and development of new therapeutic strategies. Cardenolides have shown significant antitumor activity due to their ability to inhibit the Na+K+ATPase enzyme, and the expression of this enzyme is increased in tumor cells. Glucoevatromonoside containing peracetylated glucose hydroxyl groups (GEVPG) is a cardenolide derivative that has low solubility in aqueous media, which constitutes a barrier to its potential biological applications. In this context, the use of liposomes represents a promising strategy to deliver GEVPG, thus allowing its intravenous administration. In this study, long-circulating and fusogenic liposomes containing GEVPG (SpHL-GEVPG) were developed, and their chemical and physicochemical properties were evaluated. SpHL-GEVPG presented adequate properties, including a mean diameter of 182.2 ± 2.7 nm, a polydispersity index equal to 0.36 ± 0.03, a zeta potential of -2.37 ± 0.31 mV, and a GEVPG entrapment of 0.38 ± 0.04 mg/mL. Moreover, this formulation showed a good stability after having been stored for 30 days at 4 °C. The cytotoxic studies against breast (MDA-MB-231, MCF-7, and SKBR-3) and lung (A549) cancer cell lines demonstrated that SpHL-GEVPG treatment significantly reduced the cell viability. In addition, the SpHL-GEVPG formulation presented a good selectivity toward these cancer cells. The evaluation of the therapeutic efficacy of the treatment with SpHL-GEVPG showed a potent anticancer effect in an A549 human lung cancer xenograft model. SpHL-GEVPG administered at doses of 1.0 and 2.0 mg/kg (i.v.) induced antitumor effect comparable to paclitaxel given at dose of 10 mg/kg (i.v.) to mice. Therefore, the results of the present work indicate the potential applicability of SpHL-GEVPG as a new anticancer formulation.

Strychnos pseudoquina A. St. Hil.: a Brazilian medicinal plant with promising in vitro antiherpes activity.

AIMS: To investigate the anti-HSV and anti-inflammatory effects of a standardized ethyl acetate extract (SEAE) prepared with the stem bark of Strychnos pseudoquina, along with two isolated compounds: quercetin 3-O-methyl ether (3MQ) and strychnobiflavone (SBF). METHODS AND RESULTS: The mechanisms of action were evaluated by different methodological strategies. SEAE and SBF affected the early stages of viral infection and reduced HSV-1 protein expression. Both flavonoids elicited a concentration-dependent inhibition of monocyte chemoattractant protein-1 (MCP-1), whereas 3MQ reduced the chemokine release more significantly than SBF. Conversely, both compounds stimulated the production of the cytokines TNF-α and IL-1-ß in LPS-stimulated cells, especially at the intermediate and the highest tested concentrations. CONCLUSIONS: SEAE and SBF interfered with various steps of HSV replication cycle, mainly adsorption, postadsorption and penetration, as well as with ß and γ viral proteins expression; moreover, a direct inactivation of viral particles was observed. Besides, both flavonoids inhibited MCP-1 selectively, a feature that may be beneficial for the development of new anti-HSV agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the samples present anti-HSV and anti-inflammatory activities, at different levels, which is an interesting feature since cold and genital sores are accompanied by an inflammation process.

Rationale and efficacy of proteasome inhibitor combined with arsenic trioxide in the treatment of acute promyelocytic leukemia.

Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia-retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL.

Lithium chloride antileukemic activity in acute promyelocytic leukemia is GSK-3 and MEK/ERK dependent.

We recently identified that the MEK/ERK1/2 pathway synergized with retinoic acid (RA) to restore both transcriptional activity and RA-induced differentiation in RA-resistant acute promyelocytic leukemia (APL) cells. To target the MEK/ERK pathway, we identified glycogen synthase kinase-3ß (GSK-3ß) inhibitors including lithium chloride (LiCl) as activators of this pathway in APL cells. Using NB4 (RA-sensitive) and UF-1 (RA-resistant) APL cell lines, we observed that LiCl as well as synthetic GSK-3ß inhibitors decreased proliferation, induced apoptosis and restored, in RA-resistant cells, the expression of RA target genes and the RA-induced differentiation. Inhibition of the MEK/ERK1/2 pathway abolished these effects. These results were corroborated in primary APL patient cells and translated in vivo using an APL preclinical mouse model in which LiCl given alone was as efficient as RA in increasing survival of leukemic mice compared with untreated mice. When LiCl was combined with RA, we observed a significant survival advantage compared with mice treated by RA alone. In this work, we demonstrate that LiCl, a well-tolerated agent in humans, has antileukemic activity in APL and that it has the potential to restore RA-induced transcriptional activation and differentiation in RA-resistant APL cells in an MEK/ERK-dependent manner.

Sonographic diagnosis of anatomical variations associated with carpal tunnel syndrome.

Bifid median nerve is an anatomic variation that occurs in about 18% of patients with symptoms suggestive of carpal tunnel syndrome and in about 15% of symptom-free subjects. Reversed palmaris longus is a rare anatomic muscular variation. The simultaneous presence of a bifid median nerve and a reversed palmaris longus has been very rarely described, usually during surgical exploration or in cadavers. We present two cases where ultrasound showed the presence of both abnormalities, allowing a correct diagnosis and influencing the treatment plan.

Italian version of ASES questionnaire for shoulder assessment: cross-cultural adaptation and validation.

The patient-oriented measures, represented by self-administered questionnaire, have become an important aspect of clinical outcome assessment. To be used with different language groups and in different countries, questionnaires must be translated and adapted to new cultural characteristics and then validated by a widely accepted process to evaluate reliability and validity, fundamental characteristic for each measure. The aim of the study is to perform the cross-cultural adaptation and to assess the Italian version instrument reliability and validity. The study design is a cross-cultural adaptation and cross-sectional study of a sample of patients affected by shoulder disorder with a subsample followed prospectively for retest reliability. The American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form was culturally adapted for Italian-speaking people, following the simplified Guillemin criteria. Reliability and validity were assessed in a cross-sectional study of 50 consecutive patients affected by shoulder disorder. A sub-sample of 20 patients was followed prospectively for retest reliability. The results were compared with other validated patient-oriented measures. The ASES scales showed a high correlation with other patient-oriented measures, as hypothesized, and it also showed good values with regard to reproducibility, consistency and validity, to the original versions published in English. These findings suggest that the evaluation capacities of the Italian version of ASES are equivalent to those of English language version.

Health-related quality of life in patients with anterior cruciate ligament insufficiency undergoing arthroscopic reconstruction: a practice-based Italian normative group in comorbid-free patients.

BACKGROUND: Health-related quality of life (HRQoL) in anterior cruciate ligament (ACL) insufficiency has not been assessed in comorbid-free patients to date. An observational study was therefore conducted on a practice-based sample to test the hypothesis that SF-36 scoring in patients with chronic ACL insufficiency differs from the age- and gender-matched Italian norm. MATERIALS AND METHODS: Chronically ACL-insufficient patients with or without meniscal and/or focal chondral lesions were enrolled in the study. Exclusion criteria were acute ACL tear, severe and diffuse chondral lesions, concomitant knee major ligamentous injuries and/or fractures requiring surgery, previous ACL surgery and infectious, neoplastic and inflammatory disease. Knee function was evaluated by International Knee Documentation Committee (IKDC) form, HRQoL with the SF-36 questionnaire, and associated medical comorbidities by a Self-Administered Comorbidity Questionnaire (SCQ). RESULTS: A total of 316 consecutive patients, 265 males and 51 females (median age 25 years, range 15-52 years) met the inclusion/exclusion criteria. SF-36 norm-based scoring showed that the Physical Functioning, Role Physical, Bodily Pain, and Social Functioning domains were significantly lower than the Italian norm; the Role Emotional domain was also lower than the norm, but the difference was not significant. Conversely, the General Health and Mental Health domains scored significantly higher than the norm; the Vitality domain also exceeded, albeit not significantly, the norm. CONCLUSIONS: The decision-making process leading to ACL reconstruction currently emphasises the evaluation of knee function and patients' level of activity. The findings in our study, by showing that chronic ACL insufficiency significantly affects HRQoL in otherwise healthy patients, suggest that a multidimensional evaluation including HRQoL in addition to knee function might be integrated into outcome assessment.

Treatment of acute grade III acromioclavicular dislocation: a lack of evidence.

BACKGROUND: Although nonoperative treatment is considered the standard of care for the treatment of grade I and II acromioclavicular joint injuries, the treatment of grade III injuries is controversial. There are as many methods of nonoperative treatment as there are for operative stabilization. That is why we conducted a literature research to find out the best evidence regarding the treatment of acute grade III acromioclavicular dislocation. METHOD: The research was limited to RCTs, systematic review and meta-analysis in the most representative databases. Even if research identifies more than 600 articles, only five were included in the study because there were RCTs, and systematic reviews, but no meta-analysis articles were found. Moreover, no meta-analysis was performed because of differences of data published in the three RCTs (different type of surgical treatments and different outcome measures). RESULTS: From the literature evaluation, clinical results seem to be comparable between the operative and the conservative treatments, but complications are more evident in the surgery group. Since there is not a preponderance of positive papers showing the benefits of a surgical technique over conservative therapy, the nonoperative treatment is still considered a valid procedure in the grade III acromioclavicular separation. CONCLUSION: More prospective randomized studies using validated outcome measures are needed to identify the suitable operation techniques for the acute injuries.

Range of motion correlates with patient perception of TKA outcome.

Despite the clinical success of total knee arthroplasty, little information can be found in the literature about the relationship between certain postoperative physical findings and the outcome. Specifically, is the range of motion related to patient perception of outcome? We performed a cohort prospective study on 48 patients assessed by patient-oriented evaluations (Short Form 36 Health Survey and Oxford Knee Questionnaire) and objective evaluations after total knee arthroplasty. Thirty-four patients were women and 14 were men. The mean age at followup was 71 years (range, 64-80 years) and the minimum followup was 20 months (mean, 28.5 months; range, 20-30 months). We found a positive correlation between range of motion and patient-oriented evaluations in some domains of the Short Form 36 and in the Oxford knee score.

Quality of life of patients operated on for lumbar stenosis: a long-term follow-up.

BACKGROUND: We evaluated a sample of 28 patients surgically treated for lumbar stenosis (LS) four years after the first evaluation (length of the first follow-up mean 44.6 months, range 15-88) in order to evaluate the long-term follow-up of Quality of Life (QoL) after surgical treatment as LS may greatly impair the patients' QoL. We previously assessed QoL in 30 patients operated on for LS four years before, by performing a retrospective follow-up through the Short-Form 36 Health Survey (SF-36). METHODS: In this current study we performed a phone call evaluation administering the SF-36 and the official Italian version of the North American Spine Society (NASS) lumbar spine outcome assessment instrument. FINDINGS: With regard to the SF-36 results at long term follow-up we observed a significant improvement of Physical Function, Bodily Pain, Mental Health and the Physical Composite Score with respect to the first follow-up. Conversely, Vitality worsened. Regarding the results of NASS an improvement of neurological symptoms was observed. Comparison of SF-36 mean scores in the current LS sample vs. the Italian normal population at the same age, showed similar QoL pattern. CONCLUSIONS: The long-term follow-up showed that patients operated on for LS continue to improve their QoL pattern even between the 4th and the 8th year after surgery.

Single-bundle versus double-bundle arthroscopic reconstruction of the anterior cruciate ligament: what does the available evidence suggest?

The introduction of the double-bundle technique as a surgical option for primary anterior cruciate ligament (ACL) surgery stems from the hypothesis that replicating the double-bundle anatomy of the native ACL improves knee kinematics by supplying better rotational control. We performed a systematic review of the literature comparing double-bundle with standard single-bundle reconstruction methods. One RCT and three quasi-RCTs with a one-to two-year follow-up were included in this review. On the basis of these studies, ACL reconstruction with a double-bundle technique leads to less residual pivot-shift as assessed on manual and instrumented tests. Conflicting results exist as to whether the double-bundle technique leads to less side-to-side anterior tibial translation, and no significant differences were found regarding proprioception, flexorextensor peak torque and knee function as assessed with the International Knee Documentation Committee score. On the other hand, better subjective knee functionwas found in one quasi-RCT. However, there is a lack of correlation between these kinematic differences and an as yet unproven clinical effect. Uncertainties also exist regarding the mid- and long-term performances of the ACL reconstructed with a double-bundle technique. Comparison between the single-bundle and double-bundle techniques should be expanded to cover unresolved issues such as the rate of complications from a more challenging surgical technique, the risk of complicating revision surgery due to the presence of two tunnels, and the cost-effectiveness of a procedure with a higher consumption of fixation devices. The doublebundle technique should be further investigated by experienced knee surgeons in studies with higher methodological quality.

Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography.

Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.

Quality of life, clinical and neurophysiological picture in patients operated on for lumbar stenosis.

BACKGROUND: In lumbar stenosis (LS) patients, clinical, neuroradiological and neurophysiological findings were not related to validated measurements of the outcomes that are more relevant to patients such as functional status and symptoms. METHOD: We have retrospectively studied 30 patients surgically treated for LS. We have evaluated the patients by means of self-administered questionnaires (SF-36), clinical examination, and neuroradiological and neurophysiological measurements and we have registered preoperative and follow-up clinical and neurophysiological findings. Finally we evaluated the relations between patient-oriented data and validated conventional clinical and neurophysiological measurements. FINDINGS: The comparison between pre- and post-operative clinical picture showed an improvement of most parameters tested. The comparison between pre- and post-operative neurophysiological picture revealed worsening of most tested parameters. The comparison between the current sample and the Italian normative data for the SF-36 showed a worsening of physical aspects of health related quality of life; conversely there was an improvement of some mental domains. CONCLUSIONS: In our sample of LS patients the most compromised SF-36 domain was Role-Physical that measures the difficulty in every-day activities due to physical problems. Conversely, the clinical findings showed a significant improvement after surgery: patients reported in particular lower sciatica after surgical treatment, but the neurophysiological evaluation did not show any improvement.

Back pain in pregnancy: 1-year follow-up of untreated cases.

Back Pain (BP) is one of the most frequent symptoms during the last period of pregnancy, and high incidence has been described in several studies. Until now no wide, multicenter and prospective clinical studies on the natural course of BP after pregnancy have been available. We performed a multicenter follow-up study in a sample of pregnant women using the Italian validated version of the Roland questionnaire to assess the evolution of BP after pregnancy and identify prognostic factors. Each center had to re-evaluate at least 75% of the initially enrolled women, with latency of 1 year after delivery. At the follow-up, we acquired substantial clinical data concerning the post-delivery period. The evaluation of symptom evolution was based on the Roland questionnaire. At follow-up, 53% of re-evaluated women had no BP symptoms. Moreover, there was a significant improvement of patient-oriented assessment in women who suffered BP after delivery. With regard to the predictive factors, the presence of BP before pregnancy implied a 3.1-fold higher probability of improvement after delivery. In conclusion, women without history of BP before pregnancy and who complain of these symptoms during pregnancy require greater attention, because they have a lower possibility for improvement. Conversely, in women with a history of BP, pregnancy represents a transient period of worsening symptoms, probably due to the temporary para-physiological mechanical condition.