Extracellular vesicles as human therapeutics: A scoping review of the literature.

Extracellular vesicles (EVs) are released by all cells and contribute to cell-to-cell communication. The capacity of EVs to target specific cells and to efficiently deliver a composite profile of functional molecules have led researchers around the world to hypothesize their potential as therapeutics. While studies of EV treatment in animal models are numerous, their actual clinical benefit in humans has more slowly started to be tested. In this scoping review, we searched PubMed and other databases up to 31 December 2023 and, starting from 13,567 records, we selected 40 pertinent published studies testing EVs as therapeutics in humans. The analysis of those 40 studies shows that they are all small pilot trials with a large heterogeneity in terms of administration route and target disease. Moreover, the absence of a placebo control in most of the studies, the predominant local application of EV formulations and the inconsistent administration dose metric still impede comparison across studies and firm conclusions about EV safety and efficacy. On the other hand, the recording of some promising outcomes strongly calls out for well-designed larger studies to test EVs as an alternative approach to treat human diseases with no or few therapeutic options.

Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

Cellular and molecular signaling towards T cell immunological self-tolerance.

The binding of a cognate antigen to T cell receptor (TCR) complex triggers a series of intracellular events controlling T cell activation, proliferation, and differentiation. Upon TCR engagement, different negative regulatory feedback mechanisms are rapidly activated to counterbalance T cell activation, thus preventing excessive signal propagation and promoting the induction of immunological self-tolerance. Both positive and negative regulatory processes are tightly controlled to ensure the effective elimination of foreign antigens while limiting surrounding tissue damage and autoimmunity. In this context, signals deriving from co-stimulatory molecules (i.e., CD80, CD86), co-inhibitory receptors (PD-1, CTLA-4), the tyrosine phosphatase CD45 and cytokines such as IL-2 synergize with TCR-derived signals to guide T cell fate and differentiation. The balance of these mechanisms is also crucial for the generation of CD4+ Foxp3+ regulatory T cells, a cellular subset involved in the control of immunological self-tolerance. This review provides an overview of the most relevant pathways induced by TCR activation combined with those derived from co-stimulatory and co-inhibitory molecules implicated in the cell-intrinsic modulation of T cell activation. In addition to the latter, we dissected mechanisms responsible for T cell-mediated suppression of immune cell activation through regulatory T cell generation, homeostasis, and effector functions. We also discuss how imbalanced signaling derived from TCR and accessory molecules can contribute to autoimmune disease pathogenesis.

Progression of type 1 diabetes is associated with high levels of soluble PD-1 in islet autoantibody-positive children.

AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disorder that is characterised by destruction of pancreatic beta cells by autoreactive T lymphocytes. Although islet autoantibodies (AAb) are an indicator of disease progression, specific immune biomarkers that can be used as target molecules to halt development of type 1 diabetes have not been discovered. Soluble immune checkpoint molecules (sICM) play a pivotal role in counteracting excessive lymphocyte responses, but their role in type 1 diabetes is unexplored. In this longitudinal study, we measured sICM levels in AAb-positive (AAb+) children to identify molecules related to type 1 diabetes progression. METHODS: We measured the levels of 14 sICM in the sera of AAb+ children (n=57) compared to those with recent-onset type 1 diabetes (n=79) and healthy children (n=44), obtained from two cohorts. AAb+ children were followed up and divided based on their progression to type 1 diabetes (AAbP) or not (AAbNP) (if they lost islet autoimmunity and did not develop disease in subsequent years). sICM were also measured in the sample taken at the visit closest to disease onset in AAbP children. RESULTS: We found that AAb+ children had a distinct sICM profile compared with healthy children and those with recent-onset type 1 diabetes. In addition, AAb+ children who progressed to type 1 diabetes (AAbP) had higher sICM concentrations than non-progressors (AAbNP). Further, sICM levels decreased in AAbP children close to disease onset. Application of Cox regression models highlighted that high concentrations of soluble programmed cell death protein 1 (sPD-1) are associated with type 1 diabetes progression (HR 1.71; 95% CI 1.16, 2.51; p=0.007). CONCLUSIONS/INTERPRETATION: This study reveals an sICM profile that is dysregulated during the preclinical stage of type 1 diabetes, and identifies sPD-1 as a pathophysiologically-relevant molecule that is associated with disease progression, offering a potential target for early interventions in autoimmune diabetes.

Mycobacterium tuberculosis Antigen 85B modifies BCG-induced anti-tuberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis (Mtb) has developed strategies not only to evade from host immunity but also to manipulate it for its survival. We investigated whether Mtb exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host anti-TB immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin (BCG) impaired the protection elicited by vaccination causing a more severe infection when mice were challenged with Mtb. Ag85B administration reduced BCG-induced CD4 T cell activation and IFN-γ, CCL-4 and IL-22 production in response to Mtb-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses and activation of regulatory and exhaustion pathways, including programmed death-ligand 1 expression on macrophages. All this resulted in impaired intracellular Mtb growth control by systemic immunity, both at pre- and post-Mtb challenge. Interestingly, Mtb infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mtb in both unvaccinated and BCG-vaccinated mice. Our data suggest that Mtb can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and it is commonly found in the lungs of the Mtb-infected host, our findings may advance the understanding on the mechanisms of Mtb pathogenesis and immune evasion.

Caloric restriction for the immunometabolic control of human health.

Nutrition affects all physiological processes occurring in our body, including those related to the function of the immune system; indeed, metabolism has been closely associated with the differentiation and activity of both innate and adaptive immune cells. While excessive energy intake and adiposity have been demonstrated to cause systemic inflammation, several clinical and experimental evidence show that calorie restriction (CR), not leading to malnutrition, is able to delay aging and exert potent anti-inflammatory effects in different pathological conditions. This review provides an overview of the ability of different CR-related nutritional strategies to control autoimmune, cardiovascular, and infectious diseases, as tested by preclinical studies and human clinical trials, with a specific focus on the immunological aspects of these interventions. In particular, we recapitulate the state of the art on the cellular and molecular mechanisms pertaining to immune cell metabolic rewiring, regulatory T cell expansion, and gut microbiota composition, which possibly underline the beneficial effects of CR. Although studies are still needed to fully evaluate the feasibility and efficacy of the nutritional intervention in clinical practice, the experimental observations discussed here suggest a relevant role of CR in lowering the inflammatory state in a plethora of different pathologies, thus representing a promising therapeutic strategy for the control of human health.

Immunobiology of pregnancy: from basic science to translational medicine.

Embryo implantation failure and spontaneous abortions represent the main causes of infertility in developed countries. Unfortunately, incomplete knowledge of the multiple factors involved in implantation and fetal development keeps the success rate of medically assisted procreation techniques relatively low. According to recent literature, cellular and molecular mechanisms of 'immunogenic tolerance' towards the embryo are crucial to establish an 'anti-inflammatory' state permissive of a healthy pregnancy. In this review we dissect the role played by the immune system in the endometrial-embryo crosstalk, with a particular emphasis towards the fork-head-box-p3 (Foxp3+) CD4+CD25+ regulatory T (Treg) cells and discuss the most recent therapeutic advances in the context of early immune-mediated pregnancy loss.

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.

The link between obesity and autoimmunity.

Overnutrition could lead to loss of self-tolerance by impinging on immune regulation.

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

BACKGROUND: In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. METHODS: CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). RESULTS: We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. CONCLUSIONS: The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. TRIAL REGISTRATION: ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . IMPACT: Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

Prevaccination Glucose Time in Range Correlates With Antibody Response to SARS-CoV-2 Vaccine in Type 1 Diabetes.

CONTEXT: Poor glucose control has been associated with increased mortality in COVID-19 patients with type 1 diabetes (T1D). OBJECTIVE: This work aimed to assess the effect of prevaccination glucose control on antibody response to the SARS-CoV-2 vaccine BNT162b2 in T1D. METHODS: We studied 26 patients with T1D scheduled to receive 2 doses, 21 days apart, of BNT162b2, followed prospectively for 6 months with regular evaluation of SARS-CoV-2 antibodies and glucose control. Immunoglobulin G (IgG) to spike glycoprotein were assessed by enzyme-linked immunosorbent assay, and serum neutralization by a live SARS-CoV-2 assay (Vero E6 cells system). Glycated hemoglobin A1c (HbA1c) and continuous glucose monitoring (CGM), including time in range (TIR) and above range (TAR), were collected. The primary exposure and outcome measures were prevaccination glucose control, and antibody response after vaccination, respectively. RESULTS: Prevaccination HbA1c was unrelated to postvaccine spike IgG (r = -0.33; P = .14). Of note, the CGM profile collected during the 2 weeks preceding BNT162b2 administration correlated with postvaccine IgG response (TIR: r = 0.75; P = .02; TAR: r = -0.81; P = .008). Patients meeting the recommended prevaccination glucose targets of TIR (≥ 70%) and TAR (≤ 25%) developed stronger neutralizing antibody titers (P < .0001 and P = .008, respectively), regardless of HbA1c. Glucose control along the study time frame was also associated with IgG response during follow-up (TIR: r = 0.93; P < .0001; TAR: r = -0.84; P < .0001). CONCLUSION: In T1D, glucose profile during the 2 weeks preceding vaccination is associated with stronger spike antibody binding and neutralization, highlighting a role for well-controlled blood glucose in vaccination efficacy.

Osteopontin Is Associated with Multiple Sclerosis Relapses.

BACKGROUND: Osteopontin, an extracellular matrix protein involved in bone remodeling, tissue repair and inflammation, has previously been associated with increased inflammation and neurodegeneration in multiple sclerosis (MS), promoting a worse disease course. Osteopontin is also likely involved in acute MS relapses. METHODS: In 47 patients with relapsing-remitting MS, we explored the correlation between the time elapsed between the last clinical relapse and lumbar puncture, and the cerebrospinal fluid (CSF) levels of osteopontin and a group of inflammatory cytokines and adipokines such as resistin, plasminogen activator inhibitor-1, osteoprotegerin, interleukin (IL)-1ß, IL-2, IL-6 and IL-1 receptor antagonist (IL-1ra). We also analyzed the correlations between CSF levels of osteopontin and the other CSF molecules considered. RESULTS: Osteopontin CSF concentrations were higher in patients with a shorter time interval between the last clinical relapse and CSF withdrawal. In addition, CSF levels of osteopontin were positively correlated with the proinflammatory cytokines IL-2 and IL-6 and negatively correlated with the anti-inflammatory molecule IL-1ra. CONCLUSIONS: Our results further suggest the role of osteopontin in acute MS relapses showing that, in proximity to relapses, osteopontin expression in CSF may be increased along with other proinflammatory mediators and correlated with decreased concentrations of anti-inflammatory molecules.

BACE1 influences clinical manifestations and central inflammation in relapsing remitting multiple sclerosis.

Neurodegenerative and inflammatory processes influence the clinical course of multiple sclerosis (MS). The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) has been associated with cognitive dysfunction, amyloid deposition and neuroinflammation in Alzheimer's disease. We explored in a group of 50 patients with relapsing-remitting MS the association between the cerebrospinal fluid (CSF) levels of BACE1, clinical characteristics at the time of diagnosis and prospective disability after three-years follow-up. In addition, we assessed the correlations between the CSF levels of BACE 1, amyloid ß (Aß) 1-40 and 1-42, phosphorylated tau (pTau), lactate, and a set of inflammatory and anti-inflammatory molecules. BACE1 CSF levels were correlated positively with depression as measured with Beck Depression Inventory-Second Edition scale, and negatively with visuospatial memory performance evaluated by the Brief Visuospatial Memory Test-Revised. In addition, BACE CSF levels were positively correlated with Bayesian Risk Estimate for MS at onset, and with Expanded Disability Status Scale score assessed three years after diagnosis. Furthermore, a positive correlation was found between BACE1, amyloid ß 42/40 ratio (Spearman's r = 0.334, p = 0.018, n = 50), pTau (Spearman's r = 0.304, p = 0.032, n = 50) and lactate concentrations (Spearman's r = 0.361, p = 0.01, n = 50). Finally, an association emerged between BACE1 CSF levels and a group of pro and anti-inflammatory molecules, including interleukin (IL)-4, IL-17, IL-13, IL-9 and interferon-γ. BACE1 may have a role in different key mechanisms such as neurodegeneration, oxidative stress and inflammation, influencing mood, cognitive disorders and disability progression in MS.

Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

Regulatory T cells as metabolic sensors.

Compelling experimental evidence links immunity and metabolism. In this perspective, we propose forkhead-box-P3 (FoxP3)+CD4+CD25+ regulatory T (Treg) cells as key metabolic sensors controlling the immunological state in response to their intrinsic capacity to perceive nutritional changes. Treg cell high anabolic state in vivo, residency in metabolically crucial districts, and recirculation between lymphoid and non-lymphoid sites enable them to recognize the metabolic cues and adapt their intracellular metabolism and anti-inflammatory function at the paracrine and systemic levels. As privileged regulators at the interface between neuroendocrine and immune systems, the role of Treg cells in maintaining metabolic homeostasis makes these cells promising targets of therapeutic strategies aimed at restoring organismal homeostasis not only in autoimmune but also metabolic disorders.

A rapid and inexpensive genotyping method using dried blood spots for mutational analysis in a mutant mouse model: an update.

BACKGROUND: Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a non-invasive sampling followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). RESULTS: We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the specificity and feasibility of this novel procedure. CONCLUSIONS: Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a tiny amount of sample.

High levels of blood circulating immune checkpoint molecules in children with new-onset type 1 diabetes are associated with the risk of developing an additional autoimmune disease.

AIMS/HYPOTHESIS: We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. METHODS: Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. RESULTS: We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD- children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. CONCLUSIONS/INTERPRETATION: Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time.