Reduction of IL-6, IL-8 and ß2-ADRENOCEPTOR mRNA levels in circulating polymorphonuclear leukocytes after adenotonsillectomy in children with Obstructive Sleep Apnea Syndrome.

BACKGROUND: Obstructive Sleep Apnea Syndrome (OSAS) affects approximately 1-5% of children and is linked to cardiovascular, metabolic, and neurobehavioral complications. Dysregulation of inflammatory process and sympathetic nervous system overstimulation leading to increased catecholamine production may contribute to OSAS pathogenesis. Polymorphonuclear Neutrophils (PMN), key cells in the inflammatory process, express adrenergic receptors, including ß2-adrenergic receptor (ADRB2), which modulate their functions through an autocrine/paracrine loop. In this pilot study, we aimed to investigate the relationship between OSAS severity, ADRB2 expression in PMN and patient's inflammatory profile before and after adenotonsillectomy. PATIENTS/METHODS: In this pilot study we enrolled OSAS pediatric patients in which ADRB2, IL-6 and IL-8 mRNA expression levels were evaluated in circulating PMN by RT-PCR. RESULTS: 9 OSAS pediatric patients, ranged from 3 to 8 years of age, were enrolled in the study. We found that adenotonsillectomy significantly reduced ADRB2 as well as IL-6, IL-8 mRNA expression levels in PMN. CONCLUSIONS: These findings offer valuable insights into the underlying immune and inflammatory mechanisms of OSAS and open the way for the development of novel therapeutic approaches.

The PROB-PD trial: a pilot, randomised, placebo-controlled study protocol to evaluate the feasibility and potential efficacy of probiotics in modulating peripheral immunity in subjects with Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disease. No disease-modifying treatment is available, and therapy is symptomatic. The histopathologic hallmark is the loss of dopaminergic neurons and accumulation of α-synuclein (α-syn) in surviving neurons, but the underlying pathophysiology is unclear. Inflammatory mechanisms seem to play a prominent role, with an imbalance of immune functions and neurotoxicity caused by reactive oxygen species (ROS). Involvement of peripheral adaptive immunity, with an imbalance in T cell subpopulations and in the expression of transcriptional factors in CD4+ T cells, has also been reported. Although clinical presentation is defined by motor symptoms, patients also report non-motor symptoms, often before the onset of a clinically established disease. Etiopathogenesis of PD is unknown, but an initial aggregation of α-syn in the gut, with subsequent propagation along the vagus nerve to the brain has been hypothesised. Interestingly, in an α-syn overexpressing murine model, the absence of gut microbiota prevented both microglia activation and motor impairment, thus pointing to a fundamental role of microbiota in the development of PD. Magistrelli et al. showed that in peripheral blood mononuclear cells of PD patients, probiotics modulate the in vitro production of cytokines toward an anti-inflammatory profile and reduce the production of ROS. METHODS: This is a pilot randomised placebo-controlled clinical trial protocol for a 12-week treatment with probiotics. At least 80 patients affected by PD will be recruited and randomly allocated to either the treatment or placebo group in a 1:1 ratio. General inclusion criteria will be the onset of PD 2 to 5 years before the trial and absence of autoimmune comorbidities or immunomodulating therapy. Our primary endpoint is the assessment of changes in extracellular cytokine levels (Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-4, and IL-10) and ROS production. Secondary outcomes include changes in lymphocyte subpopulations and transcriptional factors mRNA levels. DISCUSSION: This study is designed to highlight the potential beneficial role of probiotics administration on peripheral immunity through the modulation of gut microbiota. Explorative outcomes will be evaluated to assess variations in motor and non-motor symptoms and the possible correlation with probiotics administration. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT05173701. Registered 08 November 2021.

Effects of Cannabidiol on Innate Immunity: Experimental Evidence and Clinical Relevance.

Cannabidiol (CBD) is the main non-psychotropic cannabinoid derived from cannabis (Cannabis sativa L., fam. Cannabaceae). CBD has received approval by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome. However, CBD also has prominent anti-inflammatory and immunomodulatory effects; evidence exists that it could be beneficial in chronic inflammation, and even in acute inflammatory conditions, such as those due to SARS-CoV-2 infection. In this work, we review available evidence concerning CBD's effects on the modulation of innate immunity. Despite the lack so far of clinical studies, extensive preclinical evidence in different models, including mice, rats, guinea pigs, and even ex vivo experiments on cells from human healthy subjects, shows that CBD exerts a wide range of inhibitory effects by decreasing cytokine production and tissue infiltration, and acting on a variety of other inflammation-related functions in several innate immune cells. Clinical studies are now warranted to establish the therapeutic role of CBD in diseases with a strong inflammatory component, such as multiple sclerosis and other autoimmune diseases, cancer, asthma, and cardiovascular diseases.

Pharmacological counseling in hepatotoxicity induced by macitentan and selexipag: a case report.

BACKGROUND: Pulmonary arterial hypertension is a progressive, debilitating condition characterized by increased resistance in the pulmonary arterial circulation. Current treatments for pulmonary arterial hypertension include endothelin receptor antagonists such as bosentan, sitaxentan, ambrisentan, macitentan, and oral prostacyclin receptor agonists such as selexipag. Endothelin receptor antagonists have been associated with liver injury, while hepatotoxicity was not reported for selexipag. Although genetic variability has been indisputably associated with variability in drug response, no study has been designed until now to assess its effects on the pharmacokinetics of endothelin receptor antagonists or selexipag. CASE PRESENTATION: We report the case of a 58-year-old female Caucasian patient with a dramatic increase in plasma levels of transaminases after treatment with macitentan and selexipag, drugs whose risk of causing liver injury has so far been considered limited. After therapy discontinuation, plasma levels of transaminases returned to baseline, thus suggesting a role of these drugs in the observed hepatotoxicity. After pharmacological counseling, we decided to introduce ambrisentan for the patient's treatment. After 7 months of treatment, no liver injury has been reported. To evaluate the role of genetic factors in the observed hepatotoxicity, we genotyped the patient for single-nucleotide polymorphisms previously associated with macitentan, ambrisentan, or selexipag metabolism. We found a genetic profile associated with a poor metabolizer (PM) phenotype for CYP2C8 and CYP2C9, key enzymes for elimination of both macitentan and selexipag. The reported results suggest that an allelic profile associated with low activity for CYP2C8 and CYP2C9 enzyme could be a potential risk factor for macitentan and selexipag-induced liver injury and could provide a possible marker for early identification of subjects at higher risk of developing hepatotoxicity. CONCLUSIONS: A multidisciplinary approach based on clinical evaluation, as well as pharmacological counseling and evaluation of the patient's genetic profile, might be useful for identification of patients with a high chance of drug-induced liver injury, avoiding unnecessary risks in therapy selection and prescription.

Inflammatory markers at baseline correlate with subsequent clinical response to quetiapine in patients with bipolar disorder.

OBJECTIVES: Recent studies proposed the existence of a correlation between patients' inflammatory status and therapy response in bipolar disorder (BD). Here we investigated the correlation between levels of inflammatory markers and quetiapine (QUE) effects in BD patients. METHODS: In 15 hospitalised BD patients, we investigated changes in inflammatory markers such as C-Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR) and cytokines after a 6-week treatment with QUE monotherapy. RESULTS: We found QUE treatment to significantly reduce CRP and IL-6 plasma levels. Moreover, we found higher CRP and IL-6 plasma levels at baseline correlated with better improvement of patients' clinical symptoms. CONCLUSION: The reported results, although preliminary, could be useful in clinical practice, providing not only markers for QUE response, but also allowing for identification of new targets and new therapies for the treatment of this condition.

The Problem of Home Therapy during COVID-19 Pandemic in Italy: Government Guidelines versus Freedom of Cure?

After starting in late 2019, COVID-19 spread worldwide, and Italy was one of the first Western nations to be seriously affected. At that time, both the virus and the disease were little known and there were no Evidence-Based Medicine indications for treatment. The Italian Health Ministry guidelines claimed that, unless oxygen saturation fell to <92%, no pharmacological treatment was necessary during the first 72 hours, other than on a purely symptomatic basis, preferably with paracetamol. As later confirmed, that delay in therapeutic intervention may have been responsible for numerous hospital admissions and a very high lethality (3.5 %). To try to remedy this situation, several volunteer groups were formed, managing to promptlycure thousands of patients at home with non-steroidal anti-inflammatory drugs and a variety of re-purposed drugs (principally hydroxychloroquine, ivermectin) and supplements (such as antioxidants, polyphenols and vitamin D). Although not documented by any randomized controlled studies, these approaches were nonetheless based on the best available evidence, were aimed at addressing otherwise unmet major needs and produced a significant reduction of hospitalizations, of symptom duration, and a complete recovery from the disease compared with late treatment, according to some retrospective observational studies and the clinical experience of many physicians. A prompt discussion, with a clear and open exchange between healthcare Institutions and the said groups of voluntary physicians, could clarify the most effective approaches to reduce the number of hospitalizations and the lethality of this disease.

Cannabidiol for Viral Diseases: Hype or Hope?

Background: The possibility of cannabidiol (CBD) to be used as an antiviral or to treat viral diseases has received limited attention so far, despite the growing number of claims that CBD could be used for the treatment of viral infection-related conditions. Aim and Methods: Therefore, we systematically retrieved and critically evaluated the scientific literature available on PubMed and the claims on the Internet, to assess the current state of knowledge on the use of CBD in viral diseases, and to provide suggestions for future research directions. Results: PubMed search referenced two original articles supporting the use of CBD for the treatment of hepatitis C and Kaposi sarcoma and one article reporting the ability of CBD to reduce neuroinflammation in a virus-induced animal model of multiple sclerosis. Internet search found 25 websites claiming more indications for CBD. Remarkably, those claims were provided mostly by commercial websites and were not supported by appropriate scientific references. Conclusion: Although preclinical studies suggest the potential effectiveness of CBD in viral diseases such as hepatitis C and Kaposi sarcoma, clinical evidence is still lacking. Anecdotal experiences of CBD use retrieved on the Internet, on the other side, lack any support from sound scientific evidence, although they might in some cases provide suggestions for conditions associated with viral infections that may deserve proper assessment in well-designed clinical trials.

Immune profile of hyaluronic acid hydrogel polyethylene glycol crosslinked: An in vitro evaluation in human polymorphonuclear leukocytes.

Neauvia hydrogel (N-Gel) is a hyaluronic acid-based dermal filler, cross-linked with polyethylene glycol. This filler contains sodium hyaluronate at different concentrations, poly(ethylene glycol) diglycidyl ether cross-linked, glycine, and l-prolyne. Assessing any effects of N-Gel on immunity and inflammation is of crucial importance. The aim of the study was to characterize the ability of N-Gel to modulate human polymorphonuclear leukocyte (PMN) functions, including migration, oxidative metabolism, and production of proinflammatory mediators. N-Gel was tested on isolated human PMN. Spontaneous and N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated migration were examined using the Boyden Chamber technique, whereas the oxidative metabolism was assessed through spectrofluorometric measurement of reactive oxygen species (ROS) production under resting conditions and after stimulation with fMLP. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 mRNA levels were measured by real-time PCR after stimulation with fMLP or Escherichia coli lipopolysaccharide. This study showed that N-Gel reduced fMLP-induced migration and ROS production without affecting these functions in resting cells. In addition, incubation of PMN with N-Gel effectively reduced both TNF-α and IL-8 mRNA levels. N-Gel modulates critical functions of human PMN such as migration and oxidative metabolism, indicating its potential as an anti-inflammatory agent.

Effect of beta-blockers on survival of lung cancer patients: a systematic review and meta-analysis.

The recent interest in beta-blockers as possible agents for drug repurposing in oncology arises from many pre-clinical and epidemiologic studies suggesting a possible clinically relevant antitumour effect. In lung cancer, given the contradictory results obtained, it is crucial to further study its effects. A systematic review of the literature was planned to evaluate a possible beneficial effect of beta-blocker on overall survival in lung cancer patients. Medline and Embase databases were searched from inception until 1 May 2018 to identify published studies that assessed the effect beta-blocker use on overall survival in lung cancer patients. Risk of bias was evaluated by Newcastle-Ottawa scale. Hazard ratios and 95% confidence intervals for overall survival were estimated using a random-effects model. Of 920 studies, seven (all retrospective and observational, six cohort and one case-control), including 7448 patients, met the inclusion criteria. Beta-blocker users with lung cancer had no increased overall survival compared to non-users (hazard ratio = 1.00; 95% confidence interval = 0.91-1.10; I = 45%). Similarly, beta-blocker users with non-small cell lung cancer had no increased overall survival compared to beta-blocker non-users (hazard ratio = 0.96; 95% confidence interval = 0.80-1.17; I = 56%). Our findings do not suggest an overall survival advantage in patients with lung cancer using beta-blocker therapy when compared to non-users. Further prospective cohort studies, designed to overcome the intrinsic limitations of retrospective observational studies are warranted to definitively clarify any possible beneficial effect of beta-blockers on lung cancer overall survival.

Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.

Assessment of Beta-AR protein expression on tumour tissues might be a plausible strategy to select cancer patients who can benefit from Beta-blockers therapy. The aim of this study is to evaluate the differences between resected tissue specimens from primary lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SCC)) in terms of expression pattern of Beta1- and Beta2-AR in both tumour and adjacent surrounding non-tumour tissue. This retrospective study was based on the analysis of 80 patients with histologically confirmed diagnosis of primary Non-Small Cell Lung Cancer (NSCLC) who received surgical treatment. The cases were carefully selected in order to obtain the most homogeneous sample in terms of histologic subtype (40 ADCs and 40 SCCs) and clinical stage (10 each). Beta1- and Beta2-AR expression was determined by immunohistochemistry and the staining evaluated by semi-quantitative scoring using the H-score method. In our NSCLC series, Beta1- and Beta2-AR are differentially expressed. Beta1-AR expression is present at low levels in both SCC and ADC. Likewise, when compared with the matched surrounding non-tumour tissues, Beta1-AR expression level was significantly lower in both histologic subtypes. Conversely, Beta2-AR is highly expressed in both histologic subtypes, but clearly highly expressed in ADC when compared with SCC and with their matched surrounding non-tumour tissue. Overall, this clinicopathological study highlights the differential expression of Beta1- and Beta2-AR in ADC and SCC. Repurposing non-selective Beta-blockers in oncologic setting might be a suitable therapeutic strategy for lung ADC. Graphical abstract.

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors.

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing Cα,α-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity in vitro at nanomolar concentrations and were resistant to proteolytic degradation.

A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.

Cannabis and cannabinoids offer significant therapeutic benefits for a wide scope of pathological conditions. Among them, the clinical issues rooted in inflammation stand out, nonetheless, the underlying mechanisms are not yet plainly understood. Circumstantial evidence points to polymorphonuclear leukocytes (PMN) as targets for the anti-inflammatory effects of cannabis. Therefore, we conducted this study to assess the effects of CM5, a novel Cannabis sativa L. extract standardized in 5% cannabidiol (CBD), on human PMN functions, including cell migration, oxidative metabolism and production of tumour necrosis factor (TNF)-α. We then sought to investigate whether such effects could be ascribed to its content in CBD. Cell migration was assessed by the Boyden chamber assay, oxidative metabolism by means of spectrofluorimetric measurement of reactive oxygen species (ROS) production, and TNF-α was measured by real time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results show that both CM5 and CBD inhibit PMN migration, ROS and TNF-α production, indicating that CBD may be the main item responsible for the effects of CM5. CM5 is however more potent than CBD on cell migration and TNF-α production, and less effective on ROS production, suggesting that beyond CBD, other components of the cannabis plant may contribute to the biological effects of the extract. As a whole, such results support the use of cannabis standardized extract and CBD to stem inflammation; however, they also warrant in-depth investigation of the underlying cellular and molecular mechanisms to better exploit their therapeutic potential.

Dopamine inhibits human CD8+ Treg function through D1-like dopaminergic receptors.

CD8+ T regulatory/suppressor cells (Treg) affect peripheral tolerance and may be involved in autoimmune diseases as well as in cancer. In view of our previous data showing the ability of DA to affect adaptive immune responses, we investigated the dopaminergic phenotype of human CD8+ Treg as well as the ability of DA to affect their generation and activity. Results show that CD8+ T cells express both D1-like and D2-like dopaminergic receptors (DR), tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, and vesicular monoamine transporter (VMAT) 2 and contain high levels of intracellular DA. Preferential upregulation of DR mRNA levels in the CD8+CD28- T cell compartment occurs during generation of CD8+ Treg, which is reduced by DA and by the D1-like DR agonist SKF-38393. DA and SKF-38393 also reduce the suppressive activity of CD8+ Treg on human peripheral blood mononuclear cells. Treg are crucial for tumor escape from the host immune system, thus the ability of DA to inhibits Treg function supports dopaminergic pathways as a druggable targets to develop original and innovative antitumor strategies.

Improved solubility and increased biological activity of NeoSol™RCL40, a novel Red Clover Isoflavone Aglycones extract preparation.

Red clover (Trifolium pratense L., Fabaceae; RCL), a perennial plant rich in isoflavones, is a natural alternative for menopausal symptoms, as well as antiaging and antioxidant. Isoflavone preparations usually contain aglycones and ß-glycosides. Aglycones, the active moieties, are absorbed slowly and unevenly due to reduced water solubility and biotransformation from ß-glycosides. NeoSol™RCL40 is a novel RCL isoflavone aglycones preparation based on active solubilization technologies. In the present study, NeoSol™RCL40 was shown to induce solubilization of isoflavones and to increase estrogenic and antioxidative effects in comparison to a standard RCL extract (RCLE). NeoSol™RCL40 was prepared from RCLE using as host molecules either 2-pyrrolidone, 1-ethenyl homopolymer (PVP), γ-cyclodextrin, or maltodextrin. Solubilisation assays, performed by means of HPLC-UV, showed that solubilization of isoflavone aglycones was highest with RCLE processed with PVP, which was therefore selected for functional assays. In comparison to RCLE, NeoSol™RCL40 containing the same amount of isoflavone aglycones displayed 3.4 times higher estrogenicity in MCF-7 cell, 1.9-2.0 higher antioxidant activity in the DPPH and in the FRAP assay, and was cytoprotective in PC12 cells. As a whole, results support the ability of NeoSol™RCL40 to promote isoflavones solubilization leading to increased biological activity. NeoSol™RCL40 is therefore an interesting novel preparation providing improved availability of active isoflavones aglycones.

Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients.

BACKGROUND: Parkinson's disease (PD) affects an estimated 7 to 10 million people worldwide, and only symptomatic treatments are presently available to relieve the consequences of brain dopaminergic neurons loss. Neuronal degeneration in PD is the consequence of neuroinflammation in turn influenced by peripheral adaptive immunity, with CD4+ T lymphocytes playing a key role. CD4+ T cells may however acquire proinflammatory phenotypes, such as T helper (Th) 1 and Th17, as well as anti-inflammatory phenotypes, such as Th2 and the T regulatory (Treg) one, and to what extent the different CD4+ T cell subsets are imbalanced and their functions dysregulated in PD remains largely an unresolved issue. METHODS: We performed two cross-sectional studies in antiparkinson drug-treated and drug-naïve PD patients, and in age- and sex-matched healthy subjects. In the first one, we examined circulating Th1, Th2, Th17, and in the second one circulating Treg. Number and frequency of CD4+ T cell subsets in peripheral blood were assessed by flow cytometry and their functions were studied in ex vivo assays. In both studies, complete clinical assessment, blood count and lineage-specific transcription factors mRNA levels in CD4+ T cells were independently assessed and thereafter compared for their consistency. RESULTS: PD patients have reduced circulating CD4+ T lymphocytes, due to reduced Th2, Th17, and Treg. Naïve CD4+ T cells from peripheral blood of PD patients preferentially differentiate towards the Th1 lineage. Production of interferon-γ and tumor necrosis factor-α by CD4+ T cells from PD patients is increased and maintained in the presence of homologous Treg. This Th1-biased immune signature occurs in both drug-naïve patients and in patients on dopaminergic drugs, suggesting that current antiparkinson drugs do not affect peripheral adaptive immunity. CONCLUSIONS: The complex phenotypic and functional profile of CD4+ T cell subsets in PD patients strengthen the evidence that peripheral adaptive immunity is involved in PD, and represents a target for the preclinical and clinical assessment of novel immunomodulating therapeutics.

cAMP levels in lymphocytes and CD4+ regulatory T-cell functions are affected by dopamine receptor gene polymorphisms.

The neurotransmitter dopamine (DA) has prominent effects in the immune system and between the immune cells, CD4+ regulatory T (Treg) lymphocytes, a specialized T-cell subset crucial for the control of immune homeostasis, are especially sensitive to DA. Dopaminergic receptors (DR) are grouped into two families according to their pharmacological profile and main second messenger coupling: the D1 -like (D1 and D5 ), which activate adenylate cyclase, and the D2 -like (D2 , D3 and D4 ), which inhibit adenylate cyclase and exist in several variants that have been associated to clinical conditions such as schizophrenia, bipolar disorder, substance abuse and addiction. We aimed to examine, in venous blood samples from healthy volunteers, the relationship between the arbitrary DR score and DR functional responses in human lymphocytes. All the samples were genotyped for selected DR gene variants (DRD1: rs4532 and rs686; DRD2: rs1800497 and rs6277; DRD3: rs6280; DRD4: rs747302 and seven 48-base pair variable number tandem repeat (VNTR)) and a DR score was attributed to each participant. We have also tested whether DR gene polymorphisms might affect Treg cell ability to suppress effector T-cell function. To our knowledge, this is the first study showing a correlation between DR gene variants and human T lymphocyte function. The main results are that both D1 -like and D2 -like DR are functionally active in human lymphocytes, although the D1 -like DR stimulation results in stronger effects in comparison to the D2 -like DR stimulation. In addition, it seems that the DR genetic profile may affect the ability of lymphocytes to respond to dopaminergic agents. More investigations are needed about the possible clinical relevance of such findings.

Dopaminergic Regulation of Innate Immunity: a Review.

Dopamine (DA) is a neurotransmitter in the central nervous system as well as in peripheral tissues. Emerging evidence however points to DA also as a key transmitter between the nervous system and the immune system as well as a mediator produced and released by immune cells themselves. Dopaminergic pathways have received so far extensive attention in the adaptive branch of the immune system, where they play a role in health and disease such as multiple sclerosis, rheumatoid arthritis, cancer, and Parkinson's disease. Comparatively little is known about DA and the innate immune response, although DA may affect innate immune system cells such as dendritic cells, macrophages, microglia, and neutrophils. The present review aims at providing a complete and exhaustive summary of currently available evidence about DA and innate immunity, and to become a reference for anyone potentially interested in the fields of immunology, neurosciences and pharmacology. A wide array of dopaminergic drugs is used in therapeutics for non-immune indications, such as Parkinson's disease, hyperprolactinemia, shock, hypertension, with a usually favorable therapeutic index, and they might be relatively easily repurposed for immune-mediated disease, thus leading to innovative treatments at low price, with benefit for patients as well as for the healthcare systems.

Complex Changes in the Innate and Adaptive Immunity Accompany Progressive Degeneration of the Nigrostriatal Pathway Induced by Intrastriatal Injection of 6-Hydroxydopamine in the Rat.

We investigated changes in innate and adaptive immunity paralleling the progressive nigrostriatal damage occurring in a neurotoxic model of Parkinson's disease (PD) based on unilateral infusion of 6-hydroxydopamine (6-OHDA) into the rat striatum. A time-course analysis was conducted to assess changes in morphology (activation) and cell density of microglia and astrocytes, microglia polarization (M1 vs. M2 phenotype), lymphocyte infiltration in the lesioned substantia nigra pars compacta (SNc), and modifications of CD8+ and subsets of CD4+ T cell in peripheral blood accompanying nigrostriatal degeneration. Confirming previous results, we observed slightly different profiles of activation for astrocytes and microglia paralleling nigral neuronal loss. For astrocytes, morphological changes and cell density increases were mostly evident at the latest time points (14 and 28 days post-surgery), while moderate microglia activation was present since the earliest time point. For the first time, in this model, we described the time-dependent profile of microglia polarization. Activated microglia clearly expressed the M2 phenotype in the earlier phase of the experiment, before cell death became manifest, gradually shifting to the M1 phenotype as SNc cell death started. In parallel, a reduction in the percentage of circulating CD4+ T regulatory (Treg) cells, starting as early as day 3 post-6-OHDA injection, was detected in 6-OHDA-injected rats. Our data show that nigrostriatal degeneration is associated with complex changes in central and peripheral immunity. Microglia activation and polarization, Treg cells, and the factors involved in their cross-talk should be further investigated as targets for the development of therapeutic strategies for disease modification in PD.

Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods.

OBJECTIVE: Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS: By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. RESULTS: HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1µg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α. CONCLUSION: Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.

ß-Adrenergic modulation of cancer cell proliferation: available evidence and clinical perspectives.

PURPOSE: In this review, we aimed to present and discuss the available preclinical and epidemiological evidences regarding the modulation of cancer cell proliferation by ß-adrenoceptors (ß-AR), with a specific focus on the putative effects of ß-blockers according to their pharmacological properties. METHODS: A comprehensive review of the published literature was conducted, and the evidences concerning the involvement of ß-AR in cancer as well as the possible role of ß-blockers were selected and discussed. RESULTS: The majority of reviewed studies show that: (1) All the cancer types express both ß1- and ß2-AR, with the exception of neuroblastoma only seeming to express ß2-AR; (2) adrenergic agonists are able to increase proliferation of several types of cancers; (3) the proliferative effect seems to be mediated by both ß1- and ß2-AR; (4) binding to ß-AR results in a cAMP transient flux which activates two major downstream effector systems: protein kinase A and EPAC and (5) ß-blockers might be putative adjuvants for cancer treatment. CONCLUSIONS: Overall, the reviewed studies show strong evidences that ß-AR activation, through several intracellular mechanisms, modulate tumor cell proliferation suggesting ß-blockers can be a feasible therapeutic approach to antagonize ß-adrenergic response or have a protective effect per se. This review highlight the need for intensifying the research not only on the molecular mechanisms underlying the ß-adrenergic influence in cancer, but also on the implications of biased agonism of ß-blockers as potential antitumor agents.