Mitochondria in tumor cells studied by laser scanning confocal microscopy

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be ssen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Oure results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity. © 2004 Society of Photo-Optical Instrumentation Engineer

Novel nontoxic mitochondrial probe for confocal fluorescence microscopy.

We propose a 2,5-Bis[1-(4-N-methylpyridinium)ethen-2-yl)]-N-methylpyrrole ditriflate (PEPEP) as a novel nontoxic, nonpotentiometric mitochondrial probe for confocal fluorescence microscopy. PEPEP is a representative chromophore of a large family of heterocyclic fluorescent dyes that show fluorescence emission in aqueous media and great DNA affinity. We check its cytotoxicity and intracellular localization in mammalian and yeast cell cultures. We demonstrate that PEPEP is a very efficient dye for fluorescence confocal microscopy and a valuable alternative to the most frequently used mitochondrial stains. © 2006 Society of Photo-Optical Instrumentation Engineers

Ethidium bromide as a marker of mtDNA replication in living cells.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE)

SURGICAL AND NON-SURGICAL THERAPY OF OBSTRUCTIVE SLEEP APNEA SYNDROME IN CHILDREN.

Interventions of paediatric obstructive sleep apnea syndrome are complex, varied and multidisciplinary. The goal of the treatment is to restore optimal breathing during the night and to relieve associated symptoms. Evidence suggests that the surgical intervention with removal of the tonsils and adenoids will lead to significant improvements in the most incomplicated cases, as recently reported from a meta-analysis. However, post-operative persistence of this syndrome in paediatric population is more frequent than expected, which supports the idea of the complexity of this syndrome. Adenotomy alone may not be sufficient in children with OSAS, because it does not address oropharyngeal obstruction secondary to tonsillar hyperplasia. Continuous positive airway pressure can effectively treat this syndrome in selected groups of children, improving both nocturnal and daytime symptoms, but poor adherence is a limiting factor. For this reason, CPAP is not recommended as first-line therapy for OSAS when adenotonsillectomy is an option. It is now being investigated the incorporation of nonsurgical approaches for milder forms and for residual OSAS after surgical intervention. Althought adeno­tonsillar hypertrophy is the most common for OSAS in children; obesity is emerging as an equally important etiological factor. Therefore an intensive weight reduction program and adequate sleep hygiene are also important lifestyle changes that may be very effective in mitigating the symptoms of this syndrome. Pharmacological therapy (leukotriene antagonists, topical nasal steroids) is usually use for mild forms of OSAS and in children with associated allergic diseases. Special orthodontic treatment and oropharyngeal exercises are a relatively new and promising alternative therapeutic modality used in selected groups of children with OSAS

Sleep Clinical Record application in Brazilian children and its comparison with Italian children

Abstract Objective To apply the Sleep Clinical Record (SCR) to a sample of Brazilian children with sleep complaints, to compare the results with Italian children, and to identify variables that influence phenotype. Methods Brazilian and Italian children, 4−11 years of age and matched for age, gender, obesity, and apnea−hypopnea index and who presented with complaints related to sleep, were selected. The instrument used was the SCR, and the procedure used was full-night cardiorespiratory monitoring. Results The sample consisted of 51 Brazilian children and 102 Italian children. Brazilian children presented with oral breathing (55%), tonsillar hypertrophy (69%), Friedman palate position (88%), malocclusion (84%), and OSAS score (Brouilette questionnaire) (55%). The SCR among obese Brazilian children was higher as compared to that in nonobese subjects (obese, 10.84 vs nonobese, 9.13; p = 0.03). In the comparison between Brazilian and Italian children, the total Brazilian SCR was higher than the Italian SCR score (Brazilian SCR, 10.21 ± 7.56; Italian SCR, 8.95 ± 2.55; p = 0.002). The Italian SCR score was influenced by obesity, whereas the Brazilian SCR was influenced by others symptoms (daytime sleepiness, enuresis, nocturnal choking, headache, limb movements). Conclusion Brazilian children with sleep-disordered breathing show a higher SCR score as compared to Italian children. Obesity and tonsillar hypertrophy, Friedman palate position alteration, and dental malocclusion further influenced the total SCR score among Brazilian children. This may be due to access difficulties in Brazil where children should have more assistance to obtain medical care

Drug resistance outcomes of long-term ART with tenofovir disoproxil fumarate in the absence of virological monitoring

Objectives: The resistance profiles of patients receiving long-term ART in sub-Saharan Africa have been poorly described. This study obtained a sensitive assessment of the resistance patterns associated with long-term tenofovir-based ART in a programmatic setting where virological monitoring is yet to become part of routine care. Methods: We studied subjects who, after a median of 4.2 years of ART, replaced zidovudine or stavudine with tenofovir disoproxil fumarate while continuing lamivudine and an NNRTI. Using deep sequencing, resistance-associated mutations (RAMs) were detected in stored samples collected at tenofovir introduction (T0) and after a median of 4.0 years (T1). Results: At T0, 19/87 (21.8%) subjects showed a detectable viral load and 8/87 (9.2%) had one or more major NNRTI RAMs, whereas 82/87 (94.3%) retained full tenofovir susceptibility. At T1, 79/87 (90.8%) subjects remained on NNRTI-based ART, 5/87 (5.7%) had introduced lopinavir/ritonavir due to immunological failure, and 3/87 (3.4%) had interrupted ART. Whilst 68/87 (78.2%) subjects maintained or achieved virological suppression between T0 and T1, a detectable viral load with NNRTI RAMs at T0 predicted lack of virological suppression at T1. Each treatment interruption, usually reflecting unavailability of the dispensary, doubled the risk of T1 viraemia. Tenofovir, lamivudine and efavirenz selected for K65R, K70E/T, L74I/V and Y115F, alongside M184V and multiple NNRTI RAMs; this resistance profile was accompanied by high viral loads and low CD4 cell counts. Conclusions: Viraemia on tenofovir, lamivudine and efavirenz led to complex resistance patterns with implications for continued drug activity and risk of onward transmission

Obesity Is a Marker of Reduction in QoL and Disability

The purpose of this paper is to verify the association between outcome measures of health-related quality of life (HRQoL) and disability, BMI, gender, and age. Adult obese patients were clustered using HRQoL (IWQoL-Lite) and disability (WHO-DAS II) scores into three groups: mild, moderate, and high. One-way ANOVA with Bonferroni post hoc test was used to evaluate differences in age and BMI between subjects from different clusters, contingency coefficient to test the relationship between cluster groups and gender. In total, 117 patients were enrolled: subjects with higher disability and HRQoL decrement were older and had higher BMI. Women were more likely to present moderate disability and reduction in HRQoL, while men more likely presented mild disability and HRQoL reduction. Our data further confirm the connection between disability and HRQoL, high BMI and older age. These data obtained with outcomes measures might better address rehabilitation programs

Health professionals' lived experience of the presence of family members during resuscitation: an observational study

BACKGROUND: The opportunity for family members to assist during cardiopulmonary resuscitation (CPR) is controversial among healthcare professionals. Despite supporting evidence, this presence in emergency departments is not widely practiced.   AIM: To measure the perception of physicians and nurses among the presence of family members in the Emergency Department (ED), Intensive Care Unit (ICU), Coronary Care Unit (CCU), and 118 of a Milan hospital.   METHODS: A questionnaire was administered with questions relating to the possibility of having relatives’ assistance, the effects of this decision, and the opinion of operators regarding this specific approach. The answers were compared based on the profession (physicians, nurses), the Department (118, ED, ICU, CCU), and the length of service (≤ 5 years, 6-14 years, ≥ 15 years).   RESULTS: The sample analyzed consisted of 200 forms (79% nurses, 21% physicians; 52.5% ED workers, 27% from ICU, 15.5% from CCU and 5% from 118; 18,5% ≤ years of service, 31,5% 6-14 years and 50% ≥ years). The prevailing opinion is that relatives should not assist to CPR. The medical staff is less opposed than the nursing staff, although there are clear differences between the ICU and the CCU staff. Health workers with less length of service consider the presence of family members as an obstacle and fear incurring legal repercussions.   CONCLUSIONS: It is helpful to delineate protocols to support health professionals in dealing with the request of relatives to assist during the CPR of their loved ones.BACKGROUND: La possibilità per i familiari di assistere durante la rianimazione cardiopolmonare (RCP) suscita controversie tra i professionisti sanitari. Nonostante esistano evidenze a supporto, tale presenza nei dipartimenti di emergenza-urgenza è poco praticata. OBIETTIVI: Misurare la percezione di medici e infermieri rispetto alla presenza dei familiari nelle UO di Pronto Soccorso (PS), Rianimazione (RIA), Unità coronarica (UTIC) e 118 di una ASST milanese. METODI: È stato somministrato un questionario agli operatori sanitari con quesiti relativi alla possibilità di far assistere i parenti, agli effetti di tale decisione e all’opinione degli operatori rispetto a questi temi. Si sono confrontate le risposte in base alla professione (medici, infermieri), all’UO di appartenenza (118, PS, RIA, UTIC) e all’anzianità professionale (≤ 5 anni, 6-14 anni, ≥ 15 anni). RISULTATI: Il campione analizzato è costituito da 200 schede (79% infermieri, 21% medici; 52.5% operatori di PS, 27% di RIA, 15.5% di UTIC e 5% di 118; 18,5% sanitari con ≤ 5 anni di esperienza, 31,5% 6-14 anni e 50% ≥ 15 anni). Prevale l’opinione che non sia possibile far assistere i parenti a manovre rianimatorie. I medici sono meno contrari degli infermieri, mentre si evidenziano differenze tra gli operatori di RIA e quelli di UTIC. Gli operatori con meno esperienza considerano maggiormente i famigliari di intralcio e temono ripercussioni legali. CONCLUSIONI: Nasce l’esigenza di delineare protocolli per supportare i professionisti sanitari su come comportarsi con la richiesta dei parenti di poter assistere a manovre rianimatorie sui propri cari.

Innovative strategies to treat skin wounds with mangiferin: fabrication of transferosomes modified with glycols and mucin

im: The moisturizing properties of glycerol, the penetration enhancing capability of propylene glycol and the bioadhesive properties of mucin were combined to improve the carrier capabilities of transfersomes and the efficacy of mangiferin in the treatment of skin lesions. Materials & methods: Mangiferin was incorporated in transfersomes and glycoltransfersomes, which were also modified with mucin. The physico-chemical features were assessed, along with the efficacy against oxidative stress and skin wounds in vitro and in vivo. Results: Glycoltransfersomes promoted the deposition of mangiferin in epidermis and dermis, protected fibroblasts from oxidative stress and stimulated their proliferation. The wound healing and anti-inflammatory efficacy of glycoltransfersomes were confirmed in vivo. Conclusion: Results confirmed the potential of glycoltransfersomes in preventing/treating of skin lesions

Methylprednisolone-induced toxic hepatitis after intravenous pulsed therapy for multiple sclerosis relapses

High-dose, intravenous methylprednisolone (MP) is the only recommended first-line treatment for multiple sclerosis relapses. However, there are increasing reports on liver toxicity induced by this treatment regimen. We report of 4 multiple sclerosis patients with no history of viral/metabolic liver disorders or alcohol/hepatotoxic drug intake, who developed hypertransaminasaemia following intravenous MP. In 2 of the patients, liver biopsy showed periportal fibrosis, piecemeal necrosis, and inflammatory cell infiltrates. A rechallenge test confirmed a causal association in 1 case. MP-induced liver toxicity may be more frequent than commonly thought and it is important to report this adverse reaction, which is potentially lethal, and to raise awareness on the potential hepatotoxicity of corticosteroid pulses