SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

High TARC plasma levels confer protection to long living individuals by inducing M2 profile.

A way to delay aging and the related low-grade chronic inflammatory state is to study the model of positive physiology such as the Long-Living Individuals (LLIs). Our recent studies have shown higher levels of the host defense BPI Fold-Containing Family B Member 4 (BPIFB4) protein in the LLIs' blood. Notably, BPIFB4 has been shown to influence monocytes typesetting and M2 anti-inflammatory phenotype (CD206+CD163++) macrophages skewing. According to the role of a complex cytokine milieu in guiding the macrophage polarization, here we found that circulating concentrations of thymus and activation regulated chemokine (TARC)/CCL17 and small-inducible cytokine B10 (IP-10)/CXCL10) cytokines, were additionally associated with the LLIs' state. In a differentiation process in vitro, the addition of LLIs' plasma to the cell culture medium, enhanced the ability of monocytes, either from LLIs or controls, to acquire a M2 phenotype. Interestingly, a neutralizing antibody against TARC blunted the M2 skewing effect of the LLIs' plasma. Collectively, these data indicate that exceptional longevity may associate with a peculiar anti-inflammatory myeloid profile responsible for improved reparative processes and reduced inflammatory status mediated in part by TARC and M2 generation.

Butyrate as a bioactive human milk protective component against food allergy.

BACKGROUND: Food allergy (FA) is a growing health problem worldwide. Effective strategies are advocated to limit the disease burden. Human milk (HM) could be considered as a protective factor against FA, but its mechanisms remain unclear. Butyrate is a gut microbiota-derived metabolite able to exert several immunomodulatory functions. We aimed to define the butyrate concentration in HM, and to see whether the butyrate concentration detected in HM is able to modulate the mechanisms of immune tolerance. METHODS: HM butyrate concentration from 109 healthy women was assessed by GS-MS. The effect of HM butyrate on tolerogenic mechanisms was assessed in in vivo and in vitro models. RESULTS: The median butyrate concentration in mature HM was 0.75 mM. This butyrate concentration was responsible for the maximum modulatory effects observed in all experimental models evaluated in this study. Data from mouse model show that in basal condition, butyrate up-regulated the expression of several biomarkers of gut barrier integrity, and of tolerogenic cytokines. Pretreatment with butyrate significantly reduced allergic response in three animal models of FA, with a stimulation of tolerogenic cytokines, inhibition of Th2 cytokines production and a modulation of oxidative stress. Data from human cell models show that butyrate stimulated human beta defensin-3, mucus components and tight junctions expression in human enterocytes, and IL-10, IFN-γ and FoxP3 expression through epigenetic mechanisms in PBMCs from FA children. Furthermore, it promoted the precursors of M2 macrophages, DCs and regulatory T cells. CONCLUSION: The study's findings suggest the importance of butyrate as a pivotal HM compound able to protect against FA.

Single systemic transfer of a human gene associated with exceptional longevity halts the progression of atherosclerosis and inflammation in ApoE knockout mice through a CXCR4-mediated mechanism.

AIMS: Here, we aimed to determine the therapeutic effect of longevity-associated variant (LAV)-BPIFB4 gene therapy on atherosclerosis. METHODS AND RESULTS: ApoE knockout mice (ApoE-/-) fed a high-fat diet were randomly allocated to receive LAV-BPIFB4, wild-type (WT)-BPIFB4, or empty vector via adeno-associated viral vector injection. The primary endpoints of the study were to assess (i) vascular reactivity and (ii) atherosclerotic disease severity, by Echo-Doppler imaging, histology and ultrastructural analysis. Moreover, we assessed the capacity of the LAV-BPIFB4 protein to shift monocyte-derived macrophages of atherosclerotic mice and patients towards an anti-inflammatory phenotype. LAV-BPIFB4 gene therapy rescued endothelial function of mesenteric and femoral arteries from ApoE-/- mice; this effect was blunted by AMD3100, a CXC chemokine receptor type 4 (CXCR4) inhibitor. LAV-BPIFB4-treated mice showed a CXCR4-mediated shift in the balance between Ly6Chigh/Ly6Clow monocytes and M2/M1 macrophages, along with decreased T cell proliferation and elevated circulating levels of interleukins IL-23 and IL-27. In vitro conditioning with LAV-BPIFB4 protein of macrophages from atherosclerotic patients resulted in a CXCR4-dependent M2 polarization phenotype. Furthermore, LAV-BPIFB4 treatment of arteries explanted from atherosclerotic patients increased the release of atheroprotective IL-33, while inhibiting the release of pro-inflammatory IL-1ß, inducing endothelial nitric oxide synthase phosphorylation and restoring endothelial function. Finally, significantly lower plasma BPIFB4 was detected in patients with pathological carotid stenosis (>25%) and intima media thickness >2 mm. CONCLUSION: Transfer of the LAV of BPIFB4 reduces the atherogenic process and skews macrophages towards an M2-resolving phenotype through modulation of CXCR4, thus opening up novel therapeutic possibilities in cardiovascular disease.

Characterisation of HIV-1 molecular transmission clusters among newly diagnosed individuals infected with non-B subtypes in Italy.

OBJECTIVE: We evaluated the characteristics of HIV-1 molecular transmission clusters (MTCs) in 1890 newly diagnosed individuals infected with non-B subtypes between 2005 and 2017 in Italy. METHODS: Phylogenetic analyses were performed on pol sequences to characterise subtypes/circulating recombinant forms and identify MTCs. MTCs were divided into small (SMTCs, 2-3 sequences), medium (MMTCs, 4-9 sequences) and large (LMTCs, ≥10 sequences). Factors associated with MTCs were evaluated using logistic regression analysis. RESULTS: 145 MTCs were identified and involved 666 individuals (35.2%); 319 of them (16.9%) were included in 13 LMTCs, 111 (5.9%) in 20 MMTCs and 236 (12.5%) in 112 SMTCs. Compared with individuals out of MTCs, individuals involved in MTCs were prevalently Italian (72.7% vs 30.9%, p<0.001), male (82.9% vs 62.3%, p<0.001) and men who have sex with men (MSM) (43.5% vs 14.5%, p<0.001). Individuals in MTCs were also younger (median (IQR) years: 41 (35-49) vs 43 (36-51), p<0.001) and had higher CD4 cell count in comparison with individuals out of MTCs (median (IQR): 109/L: 0.4 (0.265-0.587) vs 0.246 (0.082-0.417), p<0.001). The viral load remained stable between the two groups (median (IQR) log10 copies/mL: 4.8 (4.2-5.5) vs 5.0 (4.3-5.5), p=0.87). Logistic regression confirmed that certain factors such as being MSM, of Italian origin, younger age and higher CD4 cell count were significantly associated with MTCs. CONCLUSIONS: Our findings show that HIV-1 newly diagnosed individuals infected with non-B subtypes are involved in several MTCs in Italy. These MTCs include mainly Italians and MSM and highlight the complex phenomenon characterising the HIV-1 spread. This is important especially in view of monitoring the HIV epidemic and guiding the public health response.

A Model of Evolutionary Selection: The Cardiovascular Protective Function of the Longevity Associated Variant of BPIFB4.

Evolutionary forces select genetic variants that allow adaptation to environmental stresses. The genomes of centenarian populations could recapitulate the evolutionary adaptation model and reveal the secrets of disease resistance shown by these individuals. Indeed, longevity phenotype is supposed to have a genetic background able to survive or escape to age-related diseases. Among these, cardiovascular diseases (CVDs) are the most lethal and their major risk factor is aging and the associated frailty status. One example of genetic evolution revealed by the study of centenarians genome is the four missense Single Nucleotide Polymorphisms (SNPs) haplotype in bactericidal/permeability-increasing fold-containing family B, member 4 (BPIFB4) locus that is enriched in long living individuals: the longevity associated variant (LAV). Indeed, LAV-BPIFB4 is able to improve endothelial function and revascularization through the increase of endothelial nitric oxide synthase (eNOS) dependent nitric oxide production. This review recapitulates the beneficial effects of LAV-BPIFB4 and its therapeutic potential for the treatment of CVDs.

Comparative Evaluation of Subtyping Tools for Surveillance of Newly Emerging HIV-1 Strains.

HIV-1 non-B subtypes/circulating recombinant forms (CRFs) are increasing worldwide. Since subtype identification can be clinically relevant, we assessed the added value in HIV-1 subtyping using updated molecular phylogeny (Mphy) and the performance of routinely used automated tools. Updated Mphy (2015 updated reference sequences), used as a gold standard, was performed to subtype 13,116 HIV-1 protease/reverse transcriptase sequences and then compared with previous Mphy (reference sequences until 2014) and with COMET, REGA, SCUEAL, and Stanford subtyping tools. Updated Mphy classified subtype B as the most prevalent (73.4%), followed by CRF02_AG (7.9%), C (4.6%), F1 (3.4%), A1 (2.2%), G (1.6%), CRF12_BF (1.2%), and other subtypes (5.7%). A 2.3% proportion of sequences were reassigned as different subtypes or CRFs because of misclassification by previous Mphy. Overall, the tool most concordant with updated Mphy was Stanford-v8.1 (95.4%), followed by COMET (93.8%), REGA-v3 (92.5%), Stanford-old (91.1%), and SCUEAL (85.9%). All the tools had a high sensitivity (≥98.0%) and specificity (≥95.7%) for subtype B. Regarding non-B subtypes, Stanford-v8.1 was the best tool for C, D, and F subtypes and for CRFs 01, 02, 06, 11, and 36 (sensitivity, ≥92.6%; specificity, ≥99.1%). A1 and G subtypes were better classified by COMET (92.3%) and REGA-v3 (98.6%), respectively. Our findings confirm Mphy as the gold standard for accurate HIV-1 subtyping, although Stanford-v8.1, occasionally combined with COMET or REGA-v3, represents an effective subtyping approach in clinical settings. Periodic updating of HIV-1 reference sequences is fundamental to improving subtype characterization in the context of an effective epidemiological surveillance of non-B strains.

Atazanavir/ritonavir monotherapy: 96 week efficacy, safety and bone mineral density from the MODAt randomized trial.

OBJECTIVES: To report the 96 week results on efficacy, safety and bone mineral density (BMD) in subjects with HIV-1 that were virologically suppressed and treated with atazanavir/ritonavir monotherapy versus atazanavir/ritonavir triple therapy. METHODS: MODAt is a prospective, multicentre, open-label, non-inferiority, randomized, 96 week trial (NCT01511809) comparing efficacy of atazanavir/ritonavir monotherapy versus atazanavir/ritonavir triple therapy. Treatment success was defined as no occurrence of confirmed viral rebound (two consecutive HIV-RNA >50 copies/mL) or discontinuation for any cause of the ongoing regimen. RESULTS: The 96 week treatment success was 64% in the atazanavir/ritonavir monotherapy arm and 63% in the triple-therapy arm (difference 1.3%, 95% CI: -17.5 to 20.1). In the atazanavir/ritonavir monotherapy arm, no PI- or NRTI-associated resistance mutations were observed at virological failure and all patients re-suppressed after re-intensification. In the monotherapy arm, treatment failure was more frequent in patients coinfected with hepatitis C virus [64% versus 28% (difference 35.4%, 95% CI: 3.7-67.2)]. Drug-related adverse events leading to discontinuation were 3 (6%) in the atazanavir/ritonavir monotherapy arm and 11 (21.5%) in the triple-therapy arm (P = 0.041). The 96 week adjusted mean percentage change in total proximal femur (not at lumbar spine) BMD was +1.16% and -1.64% in the atazanavir/ritonavir monotherapy arm and the triple-therapy arm, respectively (P = 0.012). CONCLUSIONS: The 96 week analyses suggested that long-term efficacy of atazanavir/ritonavir monotherapy was inferior as compared with atazanavir/ritonavir triple therapy, particularly when administered in subjects coinfected with hepatitis C virus. In the atazanavir/ritonavir monotherapy arm, reintroduction of nucleosides, as needed, was always effective with no new resistance mutation; monotherapy was also associated with a lower incidence of adverse events and improvement in femur BMD.

The problem of renal function monitoring in patients treated with the novel antiretroviral drugs.

Chronic kidney disease (CKD) is currently considered a major comorbidity in patients affected by HIV infection. In addition, new generation antiretroviral drugs that interact with creatinine transporters were recently introduced. Rilpivirine, dolutegravir, and cobicistat, with different mechanisms, inhibit the amount of tubular secretion of creatinine causing a slight increase in serum creatinine levels and consensual eGFRcreat reduction. This will require an unprecedented attention to renal issues, because the new drugs can also be associated to old antiretroviral drugs that may exert renal toxic effects. Owing to the interference of these drugs with creatinine secretion, an alternative way of estimating GFR would be desirable. At the moment, methods of direct GFR measurement have a high impact on the patient, are not readily available, or are not reliable in HIV patients. Consequently, use of classic formulas to estimate GFR is still recommended, considering the apparent reduction of eGFRcreat due to these drugs. Tubular function needs to be carefully monitored with simple tests such as proteinuria, phosphatemia, urinary excretion of phosphate, normoglycemic glycosuria, and excretion of uric acid. More specific and sensitive markers of tubular damage are still not readily available in all clinical labs. HIV patients treated by the novel drugs need to be monitored on a monthly basis for the first 3 months. Subsequent monitoring should be performed on a quarterly basis or guided by comorbidities.

The longevity-associated BPIFB4 gene guarantees vascular homeostasis and immune protection through platelets.

Beyond their activity in hemostasis and thrombosis, recent advances attribute platelets a pro-youthful role capable to attenuate immune senescence and age-related neuroinflammation. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models has proved strategic to cope with frailty conditions, aging-related events, e.g., cardiovascular ones, and immune dysfunction mainly through a favorable conditioning of the immune system. However, whether platelets participate in LAV-BPIFB4 therapeutic action is currently unknown. Herein, we discovered that platelets were instrumental in boosting the favorable health outcomes of the systemic AAV-LAV-BPIFB4 gene transfer in vivo, as the α-CD42b platelet depletion completely abolished the vascular protective action of LAV-BPIFB4 and suppressed its pro-resolutive CD206 + anti-/CD86 + pro-inflammatory Ly6C + monocyte skewing to LPS stimulation. Of note, this is associated with a huge drop in the protective levels of BPIFB4 in the plasma of AAV-LAV-BPIFB4-injected C57BL/6 mice, indicating that plasma circulating platelets may be a reservoir of the BPIFB4 protein. Indeed, we noticed that BPIFB4 was released by human platelets, a process that is amplified in LAV-allele carrier donors. Accordingly, lentivirus-mediated overexpression of human LAV-BPIFB4 isoform, but not WT-BPIFB4 isoform was able in leading differentiated megakaryocytes to release more platelet-like-particles enriched for BPIFB4. In addition, in vitro, the M2 macrophage polarization increased when releasate from platelets, and even more from LAV pre-stimulated once, was added in monocyte cell culture. Our data suggest that platelet release of BPIFB4 and of yet-to-be-determined unidentified factors mediates the therapeutic efficacy of LAV-BPIFB4 treatment.

[Genetic and environmental contribution to rheumatoid arthritis: a family study]. / Studio di una famiglia con elevata concordanza per artrite reumatoide: ruolo dei fattori genetici, ambientali e costituzionali.

We report on the HLA typing of three brothers (A, B, C) with rheumatoid arthritis (RA) and their six sons. This family is interesting for the full concordance for RA between parents. The aim of this study was the discovery of genetic and/or enviromental cofactors determining this absolute concordance.

Gender Differences Associated with the Prognostic Value of BPIFB4 in COVID-19 Patients: A Single-Center Preliminary Study.

In the ongoing global COVID-19 pandemic, male sex is a risk factor for severe disease and death, and the reasons for these clinical discrepancies are largely unknown. The aim of this work is to study the influence of sex on the course of infection and the differences in prognostic markers between genders in COVID-19 patients. Our cohort consisted of 64 adult patients (n = 34 men and n = 30 women) with PCR-proven SARS-CoV-2 infection. Further, a group of patients was characterized by a different severity degree (n = 8 high- and n = 8 low-grade individuals for both male and female patients). As expected, the serum concentrations of LDH, fibrinogen, CRP, and leucocyte count in men were significantly higher than in females. When serum concentrations of the inflammatory cytokines, including IL-6, IL-2, IP-10 and IL-4 and chemokines like MCP-1, were measured with multiplex ELISA, no significant differences between male and female patients were found. In COVID-19 patients, we recently attributed a new prognostic value to BPIFB4, a natural defensin against dysregulation of the immune responses. Here, we clarify that BPIFB4 is inversely related to the disease degree in men but not in women. Indeed, higher levels of BPIFB4 characterized low-grade male patients compared to high-grade ones. On the contrary, no significant difference was reported between low-grade female patients and high-grade ones. In conclusion, the identification of BPIFB4 as a biomarker of mild/moderate disease and its sex-specific activity would open an interesting field for research to underpin gender-related susceptibility to the disease.

The Longevity-Associated Variant of BPIFB4 Reduces Senescence in Glioma Cells and in Patients' Lymphocytes Favoring Chemotherapy Efficacy.

Glioblastoma (GBM) is the most common primary brain cancer with the median age at diagnosis around 64 years, thus pointing to aging as an important risk factor. Indeed, aging, by increasing the senescence burden, is configured as a negative prognostic factor for GBM stage. Furthermore, several anti-GBM therapies exist, such as temozolomide (TMZ) and etoposide (ETP), that unfortunately trigger senescence and the secretion of proinflammatory senescence-associated secretory phenotype (SASP) factors that are responsible for the improper burst of (i) tumorigenesis, (ii) cancer metastasis, (iii) immunosuppression, and (iv) tissue dysfunction. Thus, adjuvant therapies that limit senescence are urgently needed. The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) gene previously demonstrated a modulatory activity in restoring age-related immune dysfunction and in balancing the low-grade inflammatory status of elderly people. Based on the above findings, we tested LAV-BPIFB4 senotherapeutic effects on senescent glioblastoma U87-MG cells and on T cells from GBM patients. We interrogated SA-ß-gal and HLA-E senescence markers, SASP factors, and proliferation and apoptosis assays. The results highlighted a LAV-BPIFB4 remodeling of the senescent phenotype of GBM cells, enhancement of their sensitivity to temozolomide and a selective reduction of the T cells' senescence from GBM patients. Overall, these findings candidate LAV-BPIFB4 as an adjuvant therapy for GBM.

Transfer of the longevity-associated variant of BPIFB4 gene rejuvenates immune system and vasculature by a reduction of CD38+ macrophages and NAD+ decline.

As we age, our body experiences chronic, systemic inflammation contributing to the morbidity and mortality of the elderly. The senescent immune system has been described to have a causal role in driving systemic aging and therefore may represent a key therapeutic target to prevent pathological consequences associated with aging and extend a healthy lifespan. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models halted the progression of cardiovascular diseases (CVDs) and frailty by counterbalancing chronic inflammation. In the present study, we aimed to delineate the action of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer (AAV-LAV-BPIFB4) on the deleterious age-related changes of the immune system and thereby the senescence-associated events occurring in C57BL/6J mice aged 26 months. Our in vivo data showed that 26-months-old mice had a higher frequency of CD45+SA-beta Gal+ immune cells in peripheral blood than young (4-months-old) C57BL/6J mice. Notably, AAV-LAV-BPIFB4 gene transfer in aged mice reduced the pool of peripheral immunosenescent cells that were shown to be enriched in the spleen. In addition, the proper tuning of the immune secretory phenotype (IL1ßlow, IL6low, IL10high) associated with a significant reduction in SA-beta Gal-positive area of aorta from AAV-LAV treated mice. At the functional level, the reduction of senescence-associated inflammation ensured sustained NAD+ levels in the plasma of AAV-LAV-BPIFB4 old mice by preventing the NADase CD38 increase in F4/80+ tissue-resident macrophages and Ly6Chigh pro-inflammatory monocytes of the spleen and bone marrow. Finally, to validate the clinical implication of our findings, we showed that Long-living-individuals (LLIs, >95 years), which delay CVDs onset, especially if LAV-carriers, were characterized by high NAD+ levels. In conclusion, the new senotherapeutic action of LAV-BPIFB4 may offer a valuable therapeutic tool to control aging and reduce the burden of its pathophysiological disorders, such as CVDs.

[Rheumatoid arthritis: a typical multifactorial genetic disease: review of the literature]. / L'artrite reumatoide come modello di malattia genetica multifattoriale: una rassegna della letteratura.

Environmental factors can be triggers for the clinical appearance of rheumatoid arthritis in subjects with genetic susceptibility. Genetic factors account for 60% of disease susceptibility. This review is focused on the genetic and environmental basis of the susceptibility to arthritis.

Reasoned therapeutic protocol in outpatients with COVID-19.

BACKGROUND: Seventy percent of patients affected by SARS-COV-2 disease are asymptomatic or with symptoms that not required Hospitalization. A prodromal period lasting about 5 days can be identified. In this phase a patient with a positive swab for viral RNA may or may not evolve towards the phase of symptomatic disease. METHODS: In this paper we reviewed the literature related to COVID-19 therapy we propose a reasoned treatment protocols in outpatients according to the age and the comorbidity. RESULTS: The aim of this study was to reduce the impact of the virus by reducing its ability to attack cells and the inflammatory burden and the prothrombotic effects proposing two therapeutic schemes of proven efficacy according to the age of the patients and the comorbidities. CONCLUSIONS: We aimed to reduce worsening of clinical status and hospitalization while protecting patients at home.

The Role of BPIFB4 in Immune System and Cardiovascular Disease: The Lesson from Centenarians.

Recent discoveries have shed light on the participation of the immune system in the physio pathology of the cardiovascular system underpinning the importance of keeping the balance of the first to preserve the latter. Aging, along with other risk factors, can challenge such balance triggering the onset of cardiovascular diseases. Among several mediators ensuring the proper cross-talk between the two systems, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been shown to have a pivotal role, also by sustaining important signals such as eNOS and PKC-alpha. In addition, the Longevity-associated variant (LAV), which is an haplotype allele in BPIFB4 characterized by 4 missense polymorphisms, enriched in homozygosity in Long Living Individuals (LLIs), has been shown to be efficient, if administered systemically through gene therapy, in improving many aspects of cardiovascular diseases (CVDs). This occurs mainly through a fine immune system remodeling across: 1) a M2 macrophage polarizing effect, 2) a favorable redistribution of the circulating monocyte cell subsets and 3) the reduction of T-cell activation. Furthermore, LAV-BPIFB4 treatment induced a desirable recovery of the inflammatory balance by mitigating the pro-inflammatory factor levels and enhancing the anti-inflammatory boost through a mechanism that is partially dependent on SDF-1/CXCR4 axis. Importantly, the remarkable effects of LAV-BPIFB4 treatment, which translates in increased BPIFB4 circulating levels, mirror what occurs in long-living individuals (LLIs) in whom the high circulating levels of BPIFB4 are protective from age-related and CVDs and emphasize the reason why LLIs are considered a model of successful aging. Here, we review the mechanisms by which LAV-BPIFB4 exerts its immunomodulatory activity in improving the cardiovascular-immune system dialogue that might strengthen its role as a key mediator in CVDs.

BPIFB4 Circulating Levels and Its Prognostic Relevance in COVID-19.

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.

Circulating BPIFB4 Levels Associate With and Influence the Abundance of Reparative Monocytes and Macrophages in Long Living Individuals.

Long-Living Individuals (LLIs) delay aging and are less prone to chronic inflammatory reactions. Whether a distinct monocytes and macrophages repertoire is involved in such a characteristic remains unknown. Previous studies from our group have shown high levels of the host defense BPI Fold Containing Family B Member 4 (BPIFB4) protein in the peripheral blood of LLIs. Moreover, a polymorphic variant of the BPIFB4 gene associated with exceptional longevity (LAV-BPIFB4) confers protection from cardiovascular diseases underpinned by low-grade chronic inflammation, such as atherosclerosis. We hypothesize that BPIFB4 may influence monocytes pool and macrophages skewing, shifting the balance toward an anti-inflammatory phenotype. We profiled circulating monocytes in 52 LLIs (median-age 97) and 52 healthy volunteers (median-age 55) using flow cytometry. If the frequency of total monocyte did not change, the intermediate CD14++CD16+ monocytes counts were lower in LLIs compared to control adults. Conversely, non-classical CD14+CD16++ monocyte counts, which are M2 macrophage precursors with an immunomodulatory function, were found significantly associated with the LLIs' state. In a differentiation assay, supplementation of the LLIs' plasma enhanced the capacity of monocytes, either from LLIs or controls, to acquire a paracrine M2 phenotype. A neutralizing antibody against the phosphorylation site (ser 75) of BPIFB4 blunted the M2 skewing effect of the LLIs' plasma. These data indicate that LLIs carry a peculiar anti-inflammatory myeloid profile, which is associated with and possibly sustained by high circulating levels of BPIFB4. Supplementation of recombinant BPIFB4 may represent a novel means to attenuate inflammation-related conditions typical of unhealthy aging.