Optimization and validation of organochlorine compounds in adipose tissue by SPE-gas chromatography

Scientific evidence has shown an association between organochlorine compounds (OCC) exposure and human health hazards. Concerning this, OCC detection in human adipose samples has to be considered a public health priority. This study evaluated the efficacy of various solid-phase extraction (SPE) and cleanup methods for OCC determination in human adipose tissue. Octadecylsilyl endcapped (C18-E), benzenesulfonic acid modified silica cation exchanger (SA), poly (styrene-divinylbenzene (EN) and EN/RP18 SPE sorbents were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture detection (GC–ECD). The C18-E columns with strong homogenization were found to provide the most effective cleanup, removing the greatest amount of interfering substance, and simultaneously ensuring good analyte recoveries higher than 70%. Recoveries>70% with standard deviations (SD)<15% were obtained for all compounds under the selected conditions. Method detection limits were in the 0.003–0.009 mg/kg range. The positive samples were confirmed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The highest percentage found of the OCC in real samples corresponded to HCB, o,p′-DDT and methoxychlor, which were detected in 80 and 95% of samples analyzed respectively. Copyright © 2012 John Wiley & Sons, Ltd.

Macrophages as biomarkers in BCG treatment response in bladder cancer

Bladder cancer is a common urologic cancer and the majority has origin in the urothelium. Patients with intermediate and high risk of recurrence/progression bladder cancer are treated with intravesical instillation with Bacillus Calmette-Guérin, however, approximately 30% of patients do not respond to treatment. At the moment, there are no accepted biomarkers do predict treatment outcome and an early identification of patients better served by alternative therapeutics. The treatment initiates a cascade of cytokines responsible by recruiting macrophages to the tumor site that have been shown to influence treatment outcome. Effective BCG therapy needs precise activation of the Th1 immune pathway associated with M1 polarized macrophages. However, tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype, either immunosuppressive or angiogenic, that interfere in different ways with the BCG induced antitumor immune response. The M2 macrophage is influenced by different microenvironments in the stroma and the tumor. In particular, the degree of hypoxia in the tumors is responsible by the recruitment and differentiation of macrophages into the M2 angiogenic phenotype, suggested to be associated with the response to treatment. Nevertheless, neither the macrophage phenotypes present nor the influence of localization and hypoxia have been addressed in previous studies. Therefore, this work devoted to study the influence of TAMs, in particular of the M2 phenotype taking into account their localization (stroma or tumor) and the degree of hypoxia in the tumor (low or high) in BCG treatment outcome. The study included 99 bladder cancer patients treated with BCG. Tumors resected prior to treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. Tumor hypoxia was evaluated based on HIF-1α expression. As a main finding it was observed that a high predominance of CD163+ macrophage counts in the stroma of tumors under low hypoxia was associated with BCG immunotherapy failure, possibly due to its immunosuppressive phenotype. This study further reinforces the importance the tumor microenvironment in the modulation of BCG responses.

The predominance of M2-polarized macrophages in the stroma of low-hypoxic bladder tumors is associated with BCG immunotherapy failure

OBJECTIVE: Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard treatment for superficial bladder tumors with intermediate/high risk of recurrence or progression. However, approximately 30% of patients fail to respond to the treatment. Effective BCG therapy needs precise activation of the type 1 helper cells immune pathway. Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype and may directly interfere with the BCG-induced antitumor immune response. Thus, we aim to clarify the influence of TAMs, in particular of the M2 phenotype in stroma and tumor areas, in BCG treatment outcome. PATIENTS AND METHODS: The study included 99 patients with bladder cancer treated with BCG. Tumors resected before treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. CD68+ and CD163+ macrophages were evaluated within the stroma and tumor areas, and high density of infiltrating cells spots were selected for counting. Hypoxia, an event known to modulate macrophage phenotype, was also assessed through hypoxia induced factor (HIF)-1α expression. RESULTS: Patients in whom BCG failed had high stroma-predominant CD163+ macrophage counts (high stroma but low tumor CD163+ macrophages counts) when compared with the ones with a successful treatment (71% vs. 47%, P = 0.017). Furthermore, patients presenting this phenotype showed decreased recurrence-free survival (log rank, P = 0.008) and a clear 2-fold increased risk of BCG treatment failure was observed in univariate analysis (hazard ratio = 2.343; 95% CI: 1.197-4.587; P = 0.013). Even when adjusted for potential confounders, such as age and therapeutic scheme, multivariate analysis revealed 2.6-fold increased risk of recurrence (hazard ratio = 2.627; 95% CI: 1.340-5.150; P = 0.005). High stroma-predominant CD163+ macrophage counts were also associated with low expression of HIF-1α in tumor areas, whereas high counts of CD163+ in the tumor presented high expression of HIF-1α in tumor nests. CONCLUSIONS: TAMs evaluation using CD163 is a good indicator of BCG treatment failure. Moreover, elevated infiltration of CD163+ macrophages, predominantly in stroma areas but not in the tumor, may be a useful indicator of BCG treatment outcome, possibly owing to its immunosuppressive phenotype.

Efficacy of low-level laser therapy on scar tissue

Background: Physiotherapy has a very important role in the maintenance of the integumentary system integrity. There is very few evidence in humans. Nevertheless, there are some studies about tissue regeneration using low-level laser therapy (LLLT). Aim: To analyze the effectiveness of LLLT on scar tissue. Methods: Seventeen volunteers were stratified by age of their scars, and then randomly assigned to an experimental group (EG) — n = 9 – and a placebo group (PG) – n = 8. Fifteen sessions were conducted to both the groups thrice a week. However, in the PG, the laser device was switched off. Scars’ thickness, length, width, macroscopic aspect, pain threshold, pain perception, and itching were measured. Results: After 5 weeks, there were no statistically significant differences in any variable between both the groups. However, analyzing independently each group, EG showed a significant improvement in macroscopic aspect (p = 0.003) using LLLT. Taking into account the scars’ age, LLLT showed a tendency to decrease older scars’ thickness in EG. Conclusion: The intervention with LLLT appears to have a positive effect on the macroscopic scars’ appearance, and on old scars’ thickness, in the studied sample. However, it cannot be said for sure that LLLT has influence on scar tissue.

Biochemistry of adipose tissue: an endocrine organ

Adipose tissue is no longer considered to be an inert tissue that stores fat. This tissue is capable of expanding to accommodate increased lipids through hypertrophy of existing adipocytes and by initiating differentiation of pre-adipocytes. Adipose tissue metabolism exerts an impact on whole-body metabolism. As an endocrine organ, adipose tissue is responsible for the synthesis and secretion of several hormones. These are active in a range of processes, such as control of nutritional intake (leptin, angiotensin), control of sensitivity to insulin and inflammatory process mediators (tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), resistin, visfatin, adiponectin, among others) and pathways (plasminogen activator inhibitor 1 (PAI-1) and acylation stimulating protein (ASP) for example). This paper reviews some of the biochemical and metabolic aspects of adipose tissue and its relationship to inflammatory disease and insulin resistance.

Ibuprofen-loaded poly(trimethylene carbonate-co-ϵ- caprolactone) electrospun fibres for nerve regeneration

The development of scaffolds that combine the delivery of drugs with the physical support provided by electrospun fibres holds great potential in the field of nerve regeneration. Here it is proposed the incorporation of ibuprofen, a well-known non-steroidal anti-inflammatory drug, in electrospun fibres of the statistical copolymer poly(trimethylene carbonate-co-ε-caprolactone) [P(TMC-CL)] to serve as a drug delivery system to enhance axonal regeneration in the context of a spinal cord lesion, by limiting the inflammatory response. P(TMC-CL) fibres were electrospun from mixtures of dichloromethane (DCM) and dimethylformamide (DMF). The solvent mixture applied influenced fibre morphology, as well as mean fibre diameter, which decreased as the DMF content in solution increased. Ibuprofen-loaded fibres were prepared from P(TMC-CL) solutions containing 5% ibuprofen (w/w of polymer). Increasing drug content to 10% led to jet instability, resulting in the formation of a less homogeneous fibrous mesh. Under the optimized conditions, drug-loading efficiency was above 80%. Confocal Raman mapping showed no preferential distribution of ibuprofen in P(TMC-CL) fibres. Under physiological conditions ibuprofen was released in 24h. The release process being diffusion-dependent for fibres prepared from DCM solutions, in contrast to fibres prepared from DCM-DMF mixtures where burst release occurred. The biological activity of the drug released was demonstrated using human-derived macrophages. The release of prostaglandin E2 to the cell culture medium was reduced when cells were incubated with ibuprofen-loaded P(TMC-CL) fibres, confirming the biological significance of the drug delivery strategy presented. Overall, this study constitutes an important contribution to the design of a P(TMC-CL)-based nerve conduit with anti-inflammatory properties.

Persistent organic pollutant levels in human visceral and subcutaneous adipose tissue in obese individuals - Depot differences and dysmetabolism implications

Background: The role of persistent organic pollutants (POPs) with endocrine disrupting activity in the aetiology of obesity and other metabolic dysfunctions has been recently highlighted. Adipose tissue (AT) is a common site of POPs accumulation where they can induce adverse effects on human health. Objectives: To evaluate the presence of POPs in human visceral (vAT) and subcutaneous (scAT) adipose tissue in a sample of Portuguese obese patients that underwent bariatric surgery, and assess their putative association with metabolic disruption preoperatively, as well as with subsequent body mass index (BMI) reduction. Methods: AT samples (n=189) from obese patients (BMI ≥35) were collected and the levels of 13 POPs were determined by gas chromatography with electron-capture detection (GC-ECD). Anthropometric and biochemical data were collected at the time of surgery. BMI variation was evaluated after 12 months and adipocyte size was measured in AT samples. Results: Our data confirm that POPs are pervasive in this obese population (96.3% of detection on both tissues), their abundance increasing with age (RS=0.310, p<0.01) and duration of obesity (RS=0.170, p<0.05). We observed a difference in AT depot POPs storage capability, with higher levels of ΣPOPs in vAT (213.9±204.2 compared to 155.1±147.4 ng/g of fat, p<0.001), extremely relevant when evaluating their metabolic impact. Furthermore, there was a positive correlation between POP levels and the presence of metabolic syndrome components, namely dysglycaemia and hypertension, and more importantly with cardiovascular risk (RS=0.277, p<0.01), with relevance for vAT (RS=0.315, p<0.01). Finally, we observed an interesting relation of higher POP levels with lower weight loss in older patients. Conclusion: Our sample of obese subjects allowed us to highlight the importance of POPs stored in AT on the development of metabolic dysfunction in a context of obesity, shifting the focus to their metabolic effects and not only for their recognition as environmental obesogens.

The characteristic time of glucose diffusion measured for muscle tissue at optical clearing

The study of agent diffusion in biological tissues is very important to understand and characterize the optical clearing effects and mechanisms involved: tissue dehydration and refractive index matching. From measurements made to study the optical clearing, it is obvious that light scattering is reduced and that the optical properties of the tissue are controlled in the process. On the other hand, optical measurements do not allow direct determination of the diffusion properties of the agent in the tissue and some calculations are necessary to estimate those properties. This fact is imposed by the occurrence of two fluxes at optical clearing: water typically directed out of and agent directed into the tissue. When the water content in the immersion solution is approximately the same as the free water content of the tissue, a balance is established for water and the agent flux dominates. To prove this concept experimentally, we have measured the collimated transmittance of skeletal muscle samples under treatment with aqueous solutions containing different concentrations of glucose. After estimating the mean diffusion time values for each of the treatments we have represented those values as a function of glucose concentration in solution. Such a representation presents a maximum diffusion time for a water content in solution equal to the tissue free water content. Such a maximum represents the real diffusion time of glucose in the muscle and with this value we could calculate the corresponding diffusion coefficient.