Refining Stereotaxic Neurosurgery Techniques and Welfare Assessment for Long-Term Intracerebroventricular Device Implantation in Rodents.

Stereotaxic surgeries enable precise access to specific brain regions, being of particular interest for chronic intracerebroventricular drug delivery. However, the challenge of long-term studies at this level is to allow the implantation of drug storage devices and their correct intrathecal connection while guaranteeing animal welfare during the entire study period. In this study, we propose an optimized method for safe intrathecal device implantation, focusing on preoperative, intraoperative, and postoperative procedures, following the 3Rs principle and animal welfare regulations. Our optimized protocol introduces three main refinements. Firstly, we modify the dimensions of the implantable devices, notably diminishing the device-to-mouse weight ratio. Secondly, we use a combination of cyanoacrylate tissue adhesive and UV light-curing resin, which decreases surgery time, improves healing, and notably minimizes cannula detachment or adverse effects. Thirdly, we develop a customized welfare assessment scoresheet to accurately monitor animal well-being during long-term implantations. Taken together, these refinements positively impacted animal welfare by minimizing the negative effects on body weight, surgery-related complications, and anxiety-like behaviors. Overall, the proposed refinements have the potential to reduce animal use, enhance experimental data quality, and improve reproducibility. Additionally, these improvements can be extended to other neurosurgical techniques, thereby advancing neuroscience research, and benefiting the scientific community.

Development and evaluation of an electrochemical biosensor for creatinine quantification in a drop of whole human blood.

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.

Performance of a New Al2O3/Ce-TZP Ceramic Nanocomposite Dental Implant: A Pilot Study in Dogs.

Although titanium remains as the prevalent material in dental implant manufacturing new zirconia-based materials that overcome the major drawbacks of the standard 3Y-yttria partially-stabilized zirconia (Y-TZP) are now emerging. In this study, a new ceramic nanocomposite made of alumina and ceria-stabilized TZP (ZCe-A) has been used to produce dental implants with the mechanic and topographic characteristics of a pilot implant design to evaluate bone and soft tissue integration in a dog model (n = 5). Histological cross-section analysis of the implanted ceramic fixations (n = 15) showed not only perfect biocompatibility, but also a high rate of osseous integration (defined as the percentage of bone to implant contact) and soft tissue attachment. This clinical success, in combination with the superior mechanical properties achieved by this Al2O3/Ce-TZP nanocomposite, may place this material as an improved alternative of traditional 3Y-TZP dental implants.

Antibacterial and Antifungal Activity of ZnO Containing Glasses.

A new family of non-toxic biocides based on low melting point (1250°C) transparent glasses with high content of ZnO (15-40wt%) belonging to the miscibility region of the B2O3-SiO2-Na2O-ZnO system has been developed. These glasses have shown an excellent biocide activity (logarithmic reduction >3) against Gram- (E. coli), Gram+ (S. aureus) and yeast (C. krusei); they are chemically stable in different media (distilled water, sea-like water, LB and DMEN media) as well as biocompatible. The cytotoxicity was evaluated by the Neutral Red Uptake using NIH-3T3 (mouse embryonic fibroblast cells) and the cell viability was >80%. These new glasses can be considered in several and important applications in the field of inorganic non-toxic biocide agents such as medical implants, surgical equipment, protective apparels in hospitals, water purifications systems, food packaging, food storages or textiles.

Calcium and zinc containing bactericidal glass coatings for biomedical metallic substrates.

The present work presents new bactericidal coatings, based on two families of non-toxic, antimicrobial glasses belonging to B2O3-SiO2-Na2O-ZnO and SiO2-Na2O-Al2O3-CaO-B2O3 systems. Free of cracking, single layer direct coatings on different biomedical metallic substrates (titanium alloy, Nb, Ta, and stainless steel) have been developed. Thermal expansion mismatch was adjusted by changing glass composition of the glass type, as well as the firing atmosphere (air or Ar) according to the biomedical metallic substrates. Formation of bubbles in some of the glassy coatings has been rationalized considering the reactions that take place at the different metal/coating interfaces. All the obtained coatings were proven to be strongly antibacterial versus Escherichia coli (>4 log).

IFNα serum levels are associated with endothelial progenitor cells imbalance and disease features in rheumatoid arthritis patients.

INTRODUCTION: IFNα has been largely implicated in the ethiopathogenesis of autoimmune diseases but only recently it has been linked to endothelial damage and accelerated atherosclerosis in autoimmunity. In addition, proinflammatory conditions are supposed to be implicated in the cardiovascular status of these patients. Since a role for IFNα in endothelial damage and impaired Endothelial Progenitor Cell (EPC) number and function has been reported in other diseases, we aimed to evaluate the potential associations of IFNα serum levels on EPC populations and cytokine profiles in Rheumatoid Arthritis (RA) patients. METHODS: pre-EPC, EPC and mature EPC (mEPC) populations were quantified by flow cytometry analyzing their differential CD34, CD133 and VEGFR2 expression in blood samples from 120 RA patients, 52 healthy controls (HC), and 83 systemic lupus erythematosus (SLE) patients as disease control. Cytokine serum levels were measured by immunoassays and clinical and immunological data, including cardiovascular (CV) events and CV risk factors, were retrospectively obtained by reviewing clinical records. RESULTS: Long-standing, but not recent onset RA patients displayed a significant depletion of all endothelial progenitor populations, unless high IFNα levels were present. In fact, the IFN(high) RA patient group (n = 40, 33%), showed increased EPC levels, comparable to SLE patients. In addition, high IFNα serum levels were associated with higher disease activity (DAS28), presence of autoantibodies, higher levels of IL-1ß, IL-6, IL-10 and MIP-1α, lower amounts of TGF-ß, and increased mEPC/EPC ratio, thus suggesting higher rates of endothelial damage and an endothelial repair failure. Finally, the relationship between high IFNα levels and occurrence of CV events observed in RA patients seems to support this hypothesis. CONCLUSIONS: IFNα serum marker could be used to identify a group of RA patients with increased disease activity, EPC imbalance, enhanced proinflammatory profile and higher cardiovascular risk, probably due, at least in part, to an impaired endothelial repair.

Relationship between FOXP3 positive populations and cytokine production in systemic lupus erythematosus.

In this work we studied CD4+FOXP3+ populations in systemic lupus erythematosus (SLE) and the relationship with Th cytokine production. We found an increment in CD25-FOXP3+ population in SLE associated with CD4+ downregulation and disease progression. CD25low cells were also upregulated and showed increased percentages of FOXP3+ and CD127-/low cells, supporting the activated status of SLE lymphocytes. Despite the normal levels of CD25highFOXP3+ cells, the negative correlations observed in controls with the frequency of IFNγ, TNFα and IL-10 secreting cells were disrupted in patients, supporting a defective Treg function. Also, CD25high cells showed an altered balance in the production of these cytokines. In addition, CD25highFOXP3+ cells correlated directly with IL-17A and IL-8 but not with TGFß in SLE. The increased proportion of IL-17+ cells among the CD25high subset and the positive correlation between IL-17 levels and Treg cells suggest a trans-differentiation of Treg into Th17 cells in SLE.

Circulating endothelial cells and their progenitors in systemic lupus erythematosus and early rheumatoid arthritis patients.

OBJECTIVE: The aim of this study was to investigate the endothelial progenitor cell population in SLE and early RA patients and its potential relationships with disease features and cytokine serum levels. METHODS: Endothelial progenitor cells (EPCs), mature EPCs (mEPCs) and endothelial cells (ECs) were measured in peripheral blood samples from 83 SLE and 85 early RA patients and 39 healthy controls by flow cytometry on the basis of CD34, VEGF receptor 2 and CD133 expression. Serum levels of IL-1ß, IL-6, IL-8, IL-17, VEGF-A, IFN-α, TGF-ß and GM-CSF were quantified by immunoassays. Clinical and immunological data were obtained by reviewing clinical histories. RESULTS: Circulating EPCs were increased in SLE but not in early RA patients associated with an enhanced CD34(+) bone marrow-progenitor cell release but unrelated to disease features. The amount of mEPCs, however, was significantly higher in SLE patients presenting anti-SSA/SSB antibodies and/or malar rash, whereas the presence of specific autoantibodies was associated with EC counts in early RA and SLE patients. As expected, most cytokines tested were altered in both diseases but, interestingly, IFN-α levels, and to a lesser extent IL-6 and IL-1ß, were associated with CD133 loss and increased mEPC number, whereas VEGF and TGF-ß seem to exert an opposite effect. CONCLUSION: Our results show that high IFN-α levels and/or the presence of disease-specific antibodies may identify a group of SLE patients with increased mEPC and EC counts, and consequently probably defective endothelial repair, thus supporting their use as surrogate biomarkers of endothelial damage and high cardiovascular risk.

Effects of glucocorticoid treatment on CD25⁻FOXP3⁺ population and cytokine-producing cells in rheumatoid arthritis.

OBJECTIVES: To investigate CD25(-)FOXP3(+) cells in RA patients and their possible relationship with disease features and response to glucocorticoids (GCs). METHODS: Peripheral blood mononuclear cells were collected from 147 RA patients, 29 healthy controls and 75 SLE patients as disease controls. The proportion of CD4(+)FOXP3(+) cells with negative, low or high CD25 expression and the levels of IL-10-, TNF-α-, IL-17- and IFNγ-producing cells were assessed by flow cytometry. The presence of the high IL-10 genotype (-1082GG), associated with good response to GC, was determined by PCR amplification and hybridization with allele-specific fluorescently labelled probes. Data were related to treatment and clinical parameters. RESULTS: The CD25(-)FOXP3(+) population was significantly increased in RA patients and negatively correlated with DAS-28 and other disease parameters. The IL-10 genotype did not influence the frequency of these cells in controls or the entire RA group; however, GC-treated patient carriers of the high IL-10 genotype presented significantly higher levels of this population in addition to an increased percentage of IL-10-secreting cells and relatively low amounts of TNF-α-, IFN-γ- and IL-17-positive cells. Finally, a prospective study confirmed that genetically high IL-10 producers significantly increase CD25(-)FOXP3(+) cells after 6 months of GC treatment. CONCLUSION: The present study provides the first evidence of increased CD25(-)FOXP3(+) cells in RA patients, which were associated with disease activity and with GC treatment in carriers of the high IL-10 genotype, suggesting that this population plays a role in the clinical response to prednisone in RA.

Glucocorticoids enhance Th17/Th1 imbalance and signal transducer and activator of transcription 3 expression in systemic lupus erythematosus patients.

OBJECTIVE: To investigate the relative amounts of Th17 and Th1 cells present in SLE patients and the possible effects of treatments or disease features on these populations. METHODS: Peripheral blood mononuclear cells were collected from 75 SLE patients and 19 healthy controls and the proportion of Th17 and Th1 populations were assessed by flow cytometry measuring the amount of IL-17 and IFN-γ-producing cells. Gene expression of signal transducers and activators of transcription 3 (STAT3), STAT4, IL-6R and IL-12R were determined in 30 patients and 8 healthy individuals by real-time RT-PCR. Data were related to clinical and immunological parameters and to the treatment followed during the past 3 months. RESULTS: Th17 cells and the Th17/Th1 ratio were significantly increased in SLE patients treated with glucocorticoids compared with healthy individuals, untreated patients or those under other treatments. No association was detected with clinical parameters, but patients with anti-ENA antibodies also displayed increased Th17 responses. Disease activity (SLEDAI) is associated with the Th17/Th1 index only in glucocorticoid-treated patients. In line with these results, gene expression of STAT3 and IL-6R was up-regulated in patients taking these drugs. Accordingly, we found a positive correlation between the Th17/Th1 ratio and STAT3 levels. CONCLUSIONS: The present work provides the first evidence that aberrant Th17/Th1 balance in SLE is linked to the use of glucocorticoids and suggests that the up-regulatory effect of these drugs on the Th17 population could be associated with their ability to increase STAT3 and IL-6R expression. Additionally, anti-ENA positivity could represent a potential biomarker for Th17 bias.

Dexamethasone upregulates FOXP3 expression without increasing regulatory activity.

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are "truly" Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4(+)CD25(high) cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25(high) cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4(+)CD25(high) cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.

Cytokines and regulatory T cells in rheumatoid arthritis and their relationship with response to corticosteroids.

OBJECTIVE: To analyze circulating cytokines and regulatory T cells (Treg) in patients with rheumatoid arthritis (RA) of different durations, and their association with functional interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) genotypes in patients treated with corticosteroids. METHODS: Serum levels of IL-6, IL-10, IL-17, IL-18, TNF-α, and transforming growth factor-ß (TGF-ß) were quantified in 196 patients and 61 healthy controls. Percentage of CD4+CD25high cells was determined by flow cytometry and Foxp3 expression by real-time reverse-transcription polymerase chain reaction. Data were related to clinical measurements and presence of the genotype -1082GG IL-10/-308GG TNF-α, previously associated with good response to corticosteroids. RESULTS: Levels of TNF-α, IL-6, and IL-18 were significantly higher in patients compared to controls, while TGF-ß and IL-10 were lower. Serum samples of patients at disease onset (n = 32) had increased IL-6 and decreased TGF-ß, but there were no differences in other cytokines. These patients also presented a higher percentage of CD4+CD25high cells than those with established disease, although no significant differences were detected in Foxp3. Patients under corticosteroid treatment who were carriers of the good responder genotype had higher levels of TGF-ß, Foxp3, and Treg compared to patients with other genotypes, while relatively lower levels of TNF-α and IL-17 were observed. CONCLUSION: Patients at onset of RA present fewer alterations in cytokine levels and Treg than those with longer disease duration, supporting the role of disease progression in subsequent changes. The antiinflammatory balance observed in high IL-10/low TNF-α patients treated with prednisone supports the use of these genetic polymorphisms as predictors of response to corticosteroid therapy.

Interleukin 10 and tumor necrosis factor-alpha genotypes in rheumatoid arthritis--association with clinical response to glucocorticoids.

OBJECTIVE: There are dysregulated levels of interleukin 10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) in rheumatoid arthritis (RA), and their role in the disease is controversial. We analyzed the association of functional polymorphisms of IL-10 and TNF-alpha with susceptibility and disease characteristics at the time of diagnosis, and we also evaluated their possible use as predictors of clinical response to treatments. METHODS: Patients with recent-onset RA (n = 162) and healthy controls (n = 373) were genotyped for -1082 IL-10 and -308 TNF-alpha polymorphisms and data were related to clinical and immunological measurements of patients at the time of diagnosis. Response to treatment after 6 months was determined in 125 patients by the absolute change in Disease Activity Score (DAS28) and the American College of Rheumatology criteria for improvement. RESULTS: We found a reduced frequency of the low IL-10 producer genotype (-1082AA) in patients with RA compared to controls (26.5% vs 38.9%; p = 0.006), while it is a risk factor for anticyclic citrullinated peptide antibodies (anti-CCP) positivity (p = 0.028). Evaluation of clinical response to treatments indicated that carriage of the high IL-10 genotype was associated with a favorable outcome (p = 0.009), specifically to prednisone therapy (p = 0.0003). No significant effects were observed with TNF-alpha polymorphism alone; however, in combination with the IL-10 genotype, it increased the strength of these associations. CONCLUSION: Results show an association between the low IL-10 producer genotype and protection from RA; nevertheless, when other specific genetic and/or environmental factors trigger onset of RA, this genotype may predispose to development of anti-CCP+ RA disease with reduced response to prednisone treatment.

Conserved anti-proliferative effect and poor inhibition of TNFalpha secretion by regulatory CD4+CD25+ T cells in patients with systemic lupus erythematosus.

A role of natural regulatory CD4+CD25+ T cells in peripheral tolerance has been established, but a causative role of these cells has not been proved in human autoimmune diseases. Functional alterations have been reported in arthritis and contradictory results have been published in systemic lupus erythematosus (SLE). In this study, the CD4+CD25+ T cell function was studied in 12 SLE patients (6 with only antimalarials and 6 also with corticoids treatment) in parallel to 12 healthy controls. CD4+CD25- T cells were cocultured with allogenic monocyte derived dendritic cells (MDDC) and CD4+CD25+ T cells were added. Proliferation was measured by [3H]-thymidine incorporation and TNFalpha secretion was assessed by ELISA. A better anti-proliferative function of CD4+CD25+ T cells was found in patients than in controls (p=0.009), whereas higher TNFalpha secretion was found in patients (p=0.028). There were no significant differences regarding treatment. In summary, CD4+CD25+ T cells from SLE patients showed an undamaged anti-proliferative function. Our results and other authors' have not proved a SLE causative role for CD4+CD25+ T cells. The reduced inhibitive function on TNFalpha secretion found in patients could have some implications in lupus pathogenesis.

Influence of functional interleukin 10/tumor necrosis factor-alpha polymorphisms on interferon-alpha, IL-10, and regulatory T cell population in patients with systemic lupus erythematosus receiving antimalarial treatment.

OBJECTIVE: As interleukin 10 (IL-10)/tumor necrosis factor-alpha (TNF-alpha) polymorphisms have been shown to influence TNF-alpha inhibition and clinical response to antimalarial treatment in patients with systemic lupus erythematosus (SLE), we investigated involvement of these variants in antimalarial effects on cytokine serum levels and regulatory T cell population (Treg). METHODS: The alleles present at -308 TNF-alpha and -1082 IL-10 genes; serum concentrations of interferon-alpha (IFN-alpha), IL-10 and TNF-alpha; and size and function of CD4+CD25(high) (Treg) population were determined in SLE patients and in healthy controls. These data were related to treatment and clinical manifestations. RESULTS: Patients were observed to have increased IFN-alpha serum levels that did not correlate with any treatment. Among patients receiving antimalarial drugs, high IL-10/low TNF-alpha producers presented higher levels of IFN-alpha and IL-10 than carriers of other genotypes. In contrast, patients with the converse, low IL-10/high TNF-alpha genotype who were receiving antimalarial treatment presented increased size and function of Treg population. The percentage of CD4+CD25(high) cells was inversely correlated to TNF-alpha levels. CONCLUSION: Our findings suggest that the beneficial effect of antimalarials in low IL-10/high TNF-alpha patients with SLE may be partially attributable to the increase in Treg activity, whereas patients with the converse genotype did not show this phenomenon, yet did have significantly upregulated levels of IFN-alpha and IL-10, 2 cytokines that have been associated with SLE activity.