A Thermoresponsive injectable drug delivery system of chitosan/ß-glycerophosphate with gellan gum/alginate microparticles.

The development of new Drug Delivery Systems (DDS) by incorporating microparticles within hydrogels can prolong the release rate of drugs and/or other bioactive agents. In this study, we combined gellan gum/alginate microparticles within a thermoresponsive chitosan (Ch) hydrogel with ß-Glycerophosphate (ß-GP), designing the system to be in the sol state at 21 °C and in the gel state at 37 °C to enable the injectability of the system. The system was in the sol state between 10 °C and 21 °C. Higher concentrations of ß-GP (0, 2, 3, 4, 5 w/v%) and microparticles (0, 2 and 5 w/v%) allowed a faster sol-gel transition with higher mechanical strength at 37 °C. However, the sol-gel transition was not instantaneous. The release profile of methylene blue (MB) from the microparticles was significantly affected by their incorporation in Ch/ß-GP hydrogels, only allowing the release of 60-70 % of MB for 6 days, while the microparticles alone released all the MB in 48 h. The proposed system did not present cytotoxicity to VERO cell lines as a preliminary assay, with the Ch/ß-GP/GG:Alg having >90 % of cellular viability. The proposed Ch/ß-GP system proved to have a delaying effect on drug release and biocompatible properties, being a promising future DDS.

Purification and Characterization of Murine MZ and T2-MZP Cells.

The spleen is the second major reservoir of B cells in the adult. In the spleen, cells, generated in the bone marrow, are selected, mature, and become part of the peripheral B-cell pool. Murine spleen comprises several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized into two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone (MZ) and contains the MZ B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and of other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and fluorescence-activated cell sorting (FACS).Here, we will focus on MZ B cells and on their precursors, giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize MZ B cells in cell suspensions and in tissue sections, respectively. We will also give some clues to analyze B-cell repertoire on isolated MZ-B cells.

Evaluation of smart gelatin matrices for the development of scaffolds via 3D bioprinting.

Throughout history, different techniques have been used for the development of scaffolds for Tissue Engineering. Among them, three-dimensional (3D) printing for this application has been recently enhanced due to its ease in defining the structure of the material. In this sense, a novel potential alternative could be the development of a three-part device whose leading utility is to improve the introduction of the scaffold in a bioreactor. Thus, the device consists of a polycaprolactone support on which smart gelatin (GE) matrix, and finally, on top, a collagen (C) scaffold. This gelatin matrix is included to integrate the scaffold into the support, but once both are assembled, it must be removed, leaving only the support and the scaffold. Thus, in the present work, a small gelatin matrix has been evaluated. To this end, matrices with different gelatin percentages were studied, evaluating their mechanical and morphological properties at different temperatures (22 and 37 °C) to control their deposition and elimination. The results show the high application of this smart matrix for the development of scaffolds via 3D bioprinting for Tissue Engineering.

Effect of p53 activation through targeting MDM2/MDM4 heterodimer on T regulatory and effector cells in the peripheral blood of Type 1 diabetes patients.

Various immunotherapies for the treatment of type 1 diabetes are currently under investigation. Some of these aim to rescue the remaining beta cells from autoimmune attack caused by the disease. Among the strategies employed, p53 has been envisaged as a possible target for immunomodulation. We studied the possible effect of p53 activation on Treg subsets and Treg/Teff balance in type 1 diabetes patients' PBMC. Upon p53 activation, we observed an increase in CD8+ Treg and activated CD8+ Teff whilst CD8+ Teff cells significantly decreased in healthy PBMC when stimulated with anti-CD3/CD28. No effect was detected on percentages of CD4+ Treg, while a reduction was seen in CD4+ Teff cells and an increase in activated CD4+ Teff cells. In patients' PBMC, upon p53 activation followed by 6 days of anti-CD3/CD28 stimulation, CD8+ Treg and activated CD8+ Teff were increased while CD8+ Teff were decreased. No differences were detected in the CD4+ counterparts. CD8+ Teff PD1+, CD8+ Teff PD1low were increased upon p53 activation in type 1 diabetics compared to controls while CD8+ Teff PD1high were increased in both groups. The same increased percentages were detected for CD4+ counterparts. CD4+ Treg PD1high cells were decreased in diabetics upon p53 activation at day 6 of anti-CD3/CD28 stimulation. In conclusion, a Teff dysregulation is observed upon p53 activation suggesting that molecules promoting p53 cannot be used for therapy in type 1 diabetics.

Crosslinking of hybrid scaffolds produced from collagen and chitosan.

The development of biodegradable scaffolds able to support cell growth has recently become of great importance. Therefore, the main objective of this work was the development of hybrid scaffolds made from the mixture of two biopolymers (collagen and chitosan) and the comparison of the effect of glutaraldehyde as crosslinking agent with three different crosslinking methods (chemical: genipin; physical: temperature and enzymatic: transglutaminase) in order to look for a promising candidate to substitute it. To achieve this purpose, the mechanical properties, structure, porosity, degree of crosslinking and swelling of the different scaffolds were assessed. The best ratio of biopolymers (collagen:chitosan) to form hybrid scaffolds was 1:1, which improve their mechanical and morphological properties compared to unitary scaffolds (only collagen or chitosan). In addition, the incorporation of 10% w/w transglutaminase (crosslinking agent) with respect to the mass of biopolymers made these scaffolds a good structure for the growth and proliferation of cells.

Impact of the baseline study with penile doppler ultrasound in patients with prostate cancer before radical prostatectomy. / Impacto del estudio basal con ecografía doppler en pacientes con cáncer de próstata previo a prostatectomía radical.

INTRODUCTION: Given the high prevalence of erectile dysfunction in male population between 40-70 years old and the effect of radical prostatectomy on this domain, it is important to perform a baseline study. MATERIAL AND METHODS: Prior radical prostatectomy, erectile function has been assessed prospectively in 112 prostate cancer patients using the erectile function (EF) domain of the International Index of Erectile Function (EF-IIEF), Erectile Hardness Score (EHS) and a penile doppler ultrasound (PDUS). Comorbidities and Charlson index were collected. The EORTC QLQ C-30 and PR-25 tests were administered. RESULTS: According to EF-IIEF questionnaire, 50.9% of patients showed normal EF and EHS grade 3-4 erection was achieved in the 75.9%. PDUS was normal only in 28.6% of patients and 51.8% showed arterial insufficiency. We found a significant association (P<.0001) between categorized EF-IIEF (normal, mild/moderate/severe) and the EHS value. Between PDUS (normal vs. pathologic) and EHS (3-4 vs. 1-2) statistically significant association (P=.005) was found. Just 35.3% of patients with EHS 3-4 showed normal PDUS. Correlation between the PDUS and the EF-IIEF (≥26 vs.<26) was statistically significant (P=.043). Moreover, only 38.6% of patients with EF-IIEF≥26 had a normal PDUS. CONCLUSIONS: In order to predict EF recovery after surgery, global assessment is required. Solely self-administered tests are not enough. In this baseline study, PDUS can play an important role.

Spleen development is modulated by neonatal gut microbiota.

The full development of the mammalian immune system occurs after birth upon exposure to non self-antigens. The gut is the first site of bacterial colonization where it is crucial to create the appropriate microenvironment able to balance effector or tolerogenic responses to external stimuli. It is a well-established fact that at mucosal sites bacteria play a key role in developing the immune system but we ignore how colonising bacteria impact the maturation of the spleen. Here we addressed this issue. Taking advantage of the fact that milk SIgA regulates bacterial colonization of the newborn intestine, we generated immunocompetent mice born either from IgA pro-efficient or IgA deficient females. Having demonstrated that SIgA in maternal milk modulates neonatal gut microbiota by promoting an increased diversity of the colonizing species we also found that immunocompetent pups, not exposed to milk SIgA, fail to properly develop the FDC network and primary follicles in the spleen compromising the response to T-dependent antigens. The presence of a less diverse microbiota with a higher representation of pathogenic species leads to a fast replenishment of the marginal zone and the IgM plasma cell compartment of the spleen as well as IgA plasma cells in the gut.

Laparoscopic partial nephrectomy: Comparative study of the transperitoneal pathway and the retroperitoneal pathway. / Nefrectomía parcial laparoscópica: estudio comparativo entre la vía transperitoneal y la vía retroperitoneal.

INTRODUCTION: Laparoscopic partial nephrectomy is the recommended treatment for tumours smaller than 4cm in cases where it is feasible. Depending on the location of the tumour, the transabdominal or direct retroperitoneal pathway may be considered. OBJECTIVE: To compare the transperitoneal (TPPN) and direct retroperitoneal (RPPN) partial nephrectomies performed between 2007 and 2016. MATERIAL AND METHODS: A retrospective study was conducted on 71 patients who underwent TPPN (42) or direct RPPN (29) partial nephrectomy. We evaluated the characteristics of the patients and tumours, including tumour complexity (PADUA, RENAL, C-index). We compared perioperational variables, including the complications between the 2 pathways. RESULTS: We found no differences in terms of age, sex, Charlson's score and BMI. A larger proportion of patients in the direct RPPN group had prior major abdominal surgery (7.1 vs. 24.1%; P=.043). There were no differences in tumour size, laterality, polarity or complexity in any of the assessed scores. There were significant differences in tumour location (anterior/middle/posterior) between the TPPN and RPPN groups (54.8/31/14.3 vs. 3.4/13.8/82.8%; P<.001). There were no differences in the surgical time or length of stay. The TPPN group had a smaller urinary tract opening (4.8 vs. 27.6%; P=.007) and a higher percentage of haemostatic renorrhaphy (47.6 vs. 17.2%; P=.008). There were no differences in the need for warm ischaemia, in the changes in haemoglobin levels or in the glomerular filtration rate. The complication rates were similar for the two series. CONCLUSION: The two pathways show similar results in terms of renal function preservation, complications and oncological results. However, we recommend understanding both techniques and adapting the access type to the clinical case.

Microbiota and chronic inflammatory arthritis: an interwoven link.

BACKGROUND: Only recently, the scientific community gained insights on the importance of the intestinal resident flora for the host's health and disease. Gut microbiota in fact plays a crucial role in modulating innate and acquired immune responses and thus interferes with the fragile balance inflammation versus tolerance. MAIN BODY: Correlations between gut bacteria composition and the severity of inflammation have been studied in inflammatory bowel diseases. More recently similar alterations in the gut microbiota have been reported in patients with spondyloarthritis, whereas in rheumatoid arthritis an accumulating body of evidence evokes a pathogenic role for the altered oral microbiota in disease development and course. In the context of dysbiosis it is also important to remember that different environmental factors like stress, smoke and dietary components can induce strong bacterial changes and consequent exposure of the intestinal epithelium to a variety of different metabolites, many of which have an unknown function. In this perspective, and in complex disorders like autoimmune diseases, not only the genetic makeup, sex and immunologic context of the individual but also the structure of his microbial community should be taken into account. CONCLUSIONS: Here we provide a review of the role of the microbiota in the onset, severity and progression of chronic inflammatory arthritis as well as its impact on the therapeutic management of these patients. Furthermore we point-out the complex interwoven link between gut-joint-brain and immune system by reviewing the most recent data on the literature on the importance of environmental factors such as diet, smoke and stress.

Increased serum IgM, immunodeficiency, and autoimmunity: A clinical series.

BACKGROUND: Primary immunodeficiencies (PIDs) are generally characterized by recurrent infections; however they may be complicated by other clinical disorders such as allergy, autoimmunity, and lymphoproliferation. In particular, autoimmunity may be the first manifestation of the disease in patients with low serum immunoglobulins (Ig) levels. Here we describe a group of patients that share features of immunodeficiency and autoimmunity. MATERIALS AND METHODS: All patients went through a complete T and B cell subset characterization and a B cell function analysis in the peripheral blood by flow-cytometry. B cell proliferation and plasma cell differentiation was measured, in vitro, after CpG stimulation for 7 days as previously described. Semi-quantitative PCR analysis for AID and UNG expression as well as serum levels of BAFF were carried out in order to better define the diagnosis. RESULTS: Immunological and molecular analysis did not lead to the identification of known molecular defect typical of Hyper IgM syndrome. A comparative study of the peripheral blood B cell subsets between patients and healthy donors showed that in patients with autoimmune manifestations all circulating B cells expressed high amounts of surface IgM. CONCLUSIONS: These results suggest that the increased IgM expression on circulating B cells, reflecting B cell activation, might identify a clinical condition characterized by hyper IgM serum levels of unknown molecular defects, associated with susceptibility to infections and autoimmunity.

Repeated vaccinations do not improve specific immune defenses against Hepatitis B in non-responder health care workers.

Hepatitis B is a major infectious occupational hazard for health care workers and can be prevented with a safe and effective vaccine. The serum titer of anti-HBsAg antibodies is the most commonly used correlate of protection and post-vaccination anti-HBsAg concentrations of ≥ 10 mIU/ml are considered protective. Subjects with post-vaccination anti-HBsAg titers of <10 mIU/ml 1-6 months post-vaccination, who tested negative for HBsAg and anti-HBc, are defined as non-responders. The question of whether non-responders should be repeatedly vaccinated is still open. The aim of the study was to (i) evaluate the distribution of lymphocyte subpopulations and the percentage of HBsAg-specific memory B cells in responders and non-responders (ii) assess whether non-responders can be induced to produce antibodies after administration of a booster dose of vaccine (iii) determine whether booster vaccination increases the number of specific memory B cells in non-responders. Combining flow-cytometry, ELISPOT and serology we tested the integrity and function of the immune system in 24 health care workers, confirmed to be non-responders after at least three vaccine injections. We compared the results with those obtained in 21 responders working in the same institution. We found that the great majority of the non-responders had a functional immune system and a preserved ability to respond to other conventional antigens. Our most important findings are that the frequency of HBsAg-specific memory B cells is comparable in non-responders and controls and that booster immunization does not lead either to antibody production or memory B cell increase in non-responders.

Altered B cell homeostasis and toll-like receptor 9-driven response in type 1 diabetes carriers of the C1858T PTPN22 allelic variant: implications in the disease pathogenesis.

Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM- memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.

Purification and immunophenotypic characterization of murine MZ and T2-MZP cells.

B cells are generated every day in the bone marrow, but only a small fraction integrates the peripheral B-cell pool. In the murine spleen, we can find several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized in two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone and contains the marginal zone B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and cell sorting (FACS).Here, we will focus on marginal zone B cells and their precursors giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize marginal zone B cells in cell suspension and tissue sections, respectively.

Plasma cells in the mucosa of patients with inflammatory bowel disease produce granzyme B and possess cytotoxic activities.

In both Crohn's disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19(+) and IgA(+) cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19(+) and IgA(+) cells expressed perforin with no difference between IBD and controls. GrB-producing CD19(+) cells expressed CD27 and were CD38(high) and CD20 negative. CD19(+) B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19(+) B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19(+) and IgA(+) cells, suggesting a role for these cells in IBD-associated epithelial damage.

Abatacept (cytotoxic T lymphocyte antigen 4-immunoglobulin) improves B cell function and regulatory T cell inhibitory capacity in rheumatoid arthritis patients non-responding to anti-tumour necrosis factor-α agents.

The use of biological agents combined with methotrexate (MTX) in rheumatoid arthritis (RA) patients has strongly improved disease outcome. In this study, the effects of abatacept on the size and function of circulating B and T cells in RA patients not responding to anti-tumour necrosis factor (TNF)-α have been analysed, with the aim of identifying immunological parameters helpful to choosing suitable tailored therapies. We analysed the frequency of peripheral B and T cell subsets, B cell function and T regulatory cell (Treg ) inhibitory function in 20 moderate/severe RA patients, according to the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) criteria, primary non-responders to one TNF-α blocking agent, who received abatacept + MTX. Patients were studied before and 6 months after therapy. We found that abatacept therapy significantly reduced disease activity score on 44 joints (DAS)/erythrocyte sedimentation rate (ESR) values without causing severe side effects. The size of the circulating B and T cell compartments in RA patients was not significantly different from healthy donors, but B cell proliferation and plasma cell differentiation was impaired before therapy and restored by abatacept. While Treg cell frequency was normal, its inhibitory function was absent before therapy and was partially recovered 6 months after abatacept. B and Treg cell function is impaired in RA patients not responding to the first anti-TNF-α agent. Abatacept therapy was able to rescue immune function and led to an effective and safe clinical outcome, suggesting that RA patients, in whom anti-TNF-α failed, are immunologically prone to benefit from an agent targeting a different pathway.

Body composition and body fat distribution are related to cardiac autonomic control in non-alcoholic fatty liver disease patients.

BACKGROUND/OBJECTIVES: Heart rate recovery (HRR), a cardiac autonomic control marker, was shown to be related to body composition (BC), yet this was not tested in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to determine if, and to what extent, markers of BC and body fat (BF) distribution are related to cardiac autonomic control in NAFLD patients. SUBJECTS/METHODS: BC was assessed with dual-energy X-ray absorptiometry in 28 NAFLD patients (19 men, 51±13 years, and 9 women, 47±13 years). BF depots ratios were calculated to assess BF distribution. Subjects' HRR was recorded 1 (HRR1) and 2 min (HRR2) immediately after a maximum graded exercise test. RESULTS: BC and BF distribution were related to HRR; particularly weight, trunk BF and trunk BF-to-appendicular BF ratio showed a negative relation with HRR1 (r=-0.613, r=-0.597 and r=-0.547, respectively, P<0.01) and HRR2 (r=-0.484, r=-0.446, P<0.05, and r=-0.590, P<0.01, respectively). Age seems to be related to both HRR1 and HRR2 except when controlled for BF distribution. The preferred model in multiple regression should include trunk BF-to-appendicular BF ratio and BF to predict HRR1 (r2=0.549; P<0.05), and trunk BF-to-appendicular BF ratio alone to predict HRR2 (r2=0.430; P<0.001). CONCLUSIONS: BC and BF distribution were related to HRR in NAFLD patients. Trunk BF-to-appendicular BF ratio was the best independent predictor of HRR and therefore may be best related to cardiovascular increased risk, and possibly act as a mediator in age-related cardiac autonomic control variation.

Effects of PARP-1 deficiency on Th1 and Th2 cell differentiation.

T cell differentiation to effector Th cells such as Th1 and Th2 requires the integration of multiple synergic and antagonist signals. Poly(ADP-ribosy)lation is a posttranslational modification of proteins catalyzed by Poly(ADP-ribose) polymerases (PARPs). Recently, many reports showed that PARP-1, the prototypical member of the PARP family, plays a role in immune/inflammatory responses. Consistently, its enzymatic inhibition confers protection in several models of immune-mediated diseases, mainly through an inhibitory effect on NF-κB (and NFAT) activation. PARP-1 regulates cell functions in many types of immune cells, including dendritic cells, macrophages, and T and B lymphocytes. Our results show that PARP-1KO cells displayed a reduced ability to differentiate in Th2 cells. Under both nonskewing and Th2-polarizing conditions, naïve CD4 cells from PARP-1KO mice generated a reduced frequency of IL-4(+) cells, produced less IL-5, and expressed GATA-3 at lower levels compared with cells from wild type mice. Conversely, PARP-1 deficiency did not substantially affect differentiation to Th1 cells. Indeed, the frequency of IFN-γ (+) cells as well as IFN-γ production, in nonskewing and Th1-polarizing conditions, was not affected by PARP-1 gene ablation. These findings demonstrate that PARP-1 plays a relevant role in Th2 cell differentiation and it might be a target to be exploited for the modulation of Th2-dependent immune-mediated diseases.