Early Diagnosis and Linkage to Care: an Experience over 14 years of Point of Care Rapid HIV Testing.

Point-of-care rapid testing is one of the strategies to increase HIV screening. We present data on over 14 years of the "EASY Test Program", an ongoing cross-sectional collaborative project that provides free and anonymous rapid HIV testing in the metropolitan city of Milan, Italy. Overall, 22,186 HIV tests were performed, with a 0.52% prevalence of HIV infection; 100% of those diagnosed with HIV were linked to care. The "EASY Test Program" is an appropriate test-and-treat strategy, allowing a fast HIV assessment (24 hours). Motivated clinicians, in partnership with community associations, can perform an easy HIV screening out of hospitals in alternative settings, among individuals who in the majority of cases had never tested for HIV, ultimately providing an effective linkage to care.

Persistent but dysfunctional mucosal SARS-CoV-2-specific IgA and low lung IL-1ß associate with COVID-19 fatal outcome: A cross-sectional analysis.

The role of the mucosal pulmonary antibody response in coronavirus disease 2019 (COVID-19) outcome remains unclear. Here, we found that in bronchoalveolar lavage (BAL) samples from 48 patients with severe COVID-19-infected with the ancestral Wuhan virus, mucosal IgG and IgA specific for S1, receptor-binding domain (RBD), S2, and nucleocapsid protein (NP) emerged in BAL containing viruses early in infection and persist after virus elimination, with more IgA than IgG for all antigens tested. Furthermore, spike-IgA and spike-IgG immune complexes were detected in BAL, especially when the lung virus has been cleared. BAL IgG and IgA recognized the four main RBD variants. BAL neutralizing titers were higher early in COVID-19 when virus replicates in the lung than later in infection after viral clearance. Patients with fatal COVID-19, in contrast to survivors, developed higher levels of mucosal spike-specific IgA than IgG but lost neutralizing activities over time and had reduced IL-1ß in the lung. Altogether, mucosal spike and NP-specific IgG and S1-specific IgA persisting after lung severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance and low pulmonary IL-1ß correlate with COVID-19 fatal outcome. Thus, mucosal SARS-CoV-2-specific antibodies may have adverse functions in addition to protective neutralization. Highlights: Mucosal pulmonary antibody response in COVID-19 outcome remains unclear. We show that in severe COVID-19 patients, mucosal pulmonary non-neutralizing SARS-CoV-2 IgA persit after viral clearance in the lung. Furthermore, low lung IL-1ß correlate with fatal COVID-19. Altogether, mucosal IgA may exert harmful functions beside protective neutralization.

SARS-CoV-2 vaccination elicits unconventional IgM specific responses in naïve and previously COVID-19-infected individuals.

BACKGROUND: Currently, evaluation of the IgG antibodies specific for the SARS-CoV-2 Spike protein following vaccination is used worldwide to estimate vaccine response. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. METHODS: We performed a longitudinal study to quantify anti-S SARS-CoV-2 IgG and IgM (IgG-S and IgM-S) in health care worker (HCW) recipients of the BNT162b2 vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after T1 (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 with history of previous infection (PI). FINDINGS: IN showed three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing at T1 (37.4%) and (c) IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titres. In IgM-S positive PI, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose, respectively. IgM-S positive sera had higher pseudovirus neutralization titres compared to the IgM-S negative. INTERPRETATION: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody levels and virus-neutralizing activity. The unconventional IgG-S positive/IgM-S negative responses may suggest a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. FUNDING: Italian Ministry of Health under "Fondi Ricerca Corrente"- L1P5 and "Progetto Ricerca Finalizzata COVID-2020-12371675"; FUR 2020 Department of Excellence 2018-2022, MIUR, Italy; The Brain Research Foundation Verona.

Different decay of antibody response and VOC sensitivity in naïve and previously infected subjects at 15 weeks following vaccination with BNT162b2.

BACKGROUND: COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. METHODS: Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and the SARS-CoV-2 variants of concern emerged later, respectively. The Wilcoxon matched pair signed rank test and the Kruskal-Wallis test with Dunn's correction for multiple comparison were applied when needed. RESULTS: Although after 15 weeks from vaccination IgG-RBD dropped in all participants, naïve subjects experienced a more dramatic decline than those with previous SARS-CoV-2 infection. Neutralizing antibodies remained higher in subjects with SARS-CoV-2 history and conferred broad-spectrum protection. CONCLUSIONS: These data suggest that hybrid immunity to SARS-CoV-2 has a relevant impact on the development of IgG-RBD upon vaccination. However, the rapid decay of vaccination-elicited antibodies highlights that the administration of a third dose is expected to boost the response and acquire high levels of cross-neutralizing antibodies.

Analysis of Antibody Neutralisation Activity against SARS-CoV-2 Variants and Seasonal Human Coronaviruses NL63, HKU1, and 229E Induced by Three Different COVID-19 Vaccine Platforms.

Coronaviruses infections, culminating in the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic beginning in 2019, have highlighted the importance of effective vaccines to induce an antibody response with cross-neutralizing activity. COVID-19 vaccines have been rapidly developed to reduce the burden of SARS-CoV-2 infections and disease severity. Cross-protection from seasonal human coronaviruses (hCoVs) infections has been hypothesized but is still controversial. Here, we investigated the neutralizing activity against ancestral SARS-CoV-2 and the variants of concern (VOCs) in individuals vaccinated with two doses of either BNT162b2, mRNA-1273, or AZD1222, with or without a history of SARS-CoV-2 infection. Antibody neutralizing activity to SARS-CoV-2 and the VOCs was higher in BNT162b2-vaccinated subjects who were previously infected with SARS-CoV-2 and conferred broad-spectrum protection. The Omicron BA.1 variant was the most resistant among the VOCs. COVID-19 vaccination did not confer protection against hCoV-HKU1. Conversely, antibodies induced by mRNA-1273 vaccination displayed a boosting in their neutralizing activity against hCoV-NL63, whereas AZD1222 vaccination increased antibody neutralization against hCoV-229E, suggesting potential differences in antigenicity and immunogenicity of the different spike constructs used between various vaccination platforms. These data would suggest that there may be shared epitopes between the HCoVs and SARS-CoV-2 spike proteins.

Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission.

Background: The vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive. Methods: We tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo. Results: A single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells. Conclusion: Our results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.

Profiling Antibody Response Patterns in COVID-19: Spike S1-Reactive IgA Signature in the Evolution of SARS-CoV-2 Infection.

This contribution explores in a new statistical perspective the antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 141 coronavirus disease 2019 (COVID-19) patients exhibiting a broad range of clinical manifestations. This cohort accurately reflects the characteristics of the first wave of the SARS-CoV-2 pandemic in Italy. We determined the IgM, IgA, and IgG levels towards SARS-CoV-2 S1, S2, and NP antigens, evaluating their neutralizing activity and relationship with clinical signatures. Moreover, we longitudinally followed 72 patients up to 9 months postsymptoms onset to study the persistence of the levels of antibodies. Our results showed that the majority of COVID-19 patients developed an early virus-specific antibody response. The magnitude and the neutralizing properties of the response were heterogeneous regardless of the severity of the disease. Antibody levels dropped over time, even though spike reactive IgG and IgA were still detectable up to 9 months. Early baseline antibody levels were key drivers of the subsequent antibody production and the long-lasting protection against SARS-CoV-2. Importantly, we identified anti-S1 IgA as a good surrogate marker to predict the clinical course of COVID-19. Characterizing the antibody response after SARS-CoV-2 infection is relevant for the early clinical management of patients as soon as they are diagnosed and for implementing the current vaccination strategies.

HIV-1 Env Does Not Enable the Development of Protective Broadly Neutralizing Antibodies in an Experimental Autoimmune Encephalomyelitis Mouse Model.

Recent studies showed that immunological tolerance may restrict the development of Env-specific autoreactive broadly neutralizing antibodies. This evidence is consistent with the finding that Env immunization of a systemic lupus erythematosus (SLE) murine model produced antibodies that neutralize tier 2 HIV-1 strains. In this study, we address the possibility of eliciting neutralizing anti-Env antibodies in other autoimmune diseases such as multiple sclerosis (MS). While, as reported for SLE, we showed for the first time that a small number of HIV-1 negative, relapsing remitting MS patients exhibited antibodies with neutralizing properties, our attempts at inducing those antibodies in a EAE mouse model of MS failed. The success in eliciting Env-specific neutralizing antibodies might be related to the specific characteristics of the autoimmune disease, or it might rely in improving the vaccination design. Studies using mouse models are useful to gain insight in how HIV-specific neutralizing antibody responses are regulated in order to develop a protective HIV-1 vaccine.

Serum IgG1 and IgG4 could contribute to partial control of viral rebound in chronically HIV-1-infected patients.

OBJECTIVES: Few studies have investigated chronically infected individuals after antiretroviral therapy (ART) interruption (ATI, analytical therapy interruption); thus, we investigated the association between some HIV-specific antibodies and viral control. DESIGN: All enrolled patients were previously described in the APACHE study. Briefly, the study was conducted on HIV-1 chronically infected patients, with HIV-RNA less than 50 copies/ml for at least 10 years, CD4+ cell count greater than 500 cells/µl and HIV-DNA less than 100 copies/106 PBMC. The ART regimen in use at the time of ATI was resumed at confirmed viral rebound (CVR, defined as two consecutive HIV-RNA >50 copies/ml). METHODS: Collection of sera and analysis of both binding antibodies (BAbs) and neutralizing antibodies (NAbs) was performed at three different time points: ATI, CVR and time of viral re-suppression after ART resumption. RESULTS: IgG subclasses (IgG1, IgG2, IgG3 and IgG4) from the four patients with highest levels of neutralization were found to block viral infection. All patients had CVR after ATI at a median time of 21 days (14-56). After ART resumption, all the enrolled patients achieved HIV-RNA less than 50 copies/ml in 42 days (21-98). We observed a strong increase of either BAbs and NAbs titers from ATI to viral re-suppression in one patient, who showed the longest period of virus undetectability during ATI. In this patient, BAbs and NAbs specifically belonged to both IgG1 and IgG4 subclasses, directed to env antigen. CONCLUSION: env-specific NAbs and BAbs belonging to IgG1, IgG4 subclasses could be helpful to monitor long-term responses able to control virus replication and eradicate HIV infection.

Immunotherapy with Cell-Based Biological Drugs to Cure HIV-1 Infection.

Since its discovery 35 years ago, there have been no therapeutic interventions shown to enable full HIV-1 remission. Combined antiretroviral therapy (cART) has achieved the sustained control of HIV-1 replication, however, the life-long treatment does not eradicate long-lived latently infected reservoirs and can result in multiple side effects including the development of multidrug-resistant escape mutants. Antibody-based treatments have emerged as alternative approaches for a HIV-1 cure. Here, we will review clinical advances in coreceptor-targeting antibodies, with respect to anti-CCR5 antibodies in particular, which are currently being generated to target the early stages of infection. Among the Env-specific antibodies widely accepted as relevant in cure strategies, the potential role of those targeting CD4-induced (CD4i) epitopes of the CD4-binding site (CD4bs) in eliminating HIV-1 infected cells has gained increasing interest and will be presented. Together, with approaches targeting the HIV-1 replication cycle, we will discuss the strategies aimed at boosting and modulating specific HIV-1 immune responses, highlighting the harnessing of TLR agonists for their dual role as latency reverting agents (LRAs) and immune-modulatory compounds. The synergistic combinations of different approaches have shown promising results to ultimately enable a HIV-1 cure.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-ß induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Virological and Serological Discordant Profiles in COVID-19 Pneumonia: Two Atypical Clinical Cases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is primarily diagnosed through viral RNA positivity in nasopharyngeal swabs, and it is associated with the early detection of specific immunoglobulins to SARS-CoV-2 proteins. We describe two moderate coronavirus disease 2019 (COVID-19) patients with WHO score 4/5 at the time of hospitalization, pneumonia, and oxygen saturation <94% and with a strong discrepancy between viral RNA and antibodies to SARS-CoV-2. One patient was positive for viral RNA but completely negative for binding and neutralizing antibodies, whereas the second patient was negative for viral RNA but with high levels of both neutralizing and binding antibodies. This observation is relevant to better understand the pathogenesis of this novel infection.

Humoral Immune Responses in COVID-19 Patients: A Window on the State of the Art.

The novel SARS-CoV-2 is a recently emerging virus causing a human pandemic. A great variety of symptoms associated with COVID-19 disease, ranging from mild to severe symptoms, eventually leading to death. Specific SARS-CoV-2 RT-PCR is the standard method to screen symptomatic people; however, asymptomatic subjects and subjects with undetectable viral load escape from the screening, contributing to viral spread. Currently, the lock down imposed by many governments is an important measure to contain the spread, as there is no specific antiviral therapy or a vaccine and the main treatments are supportive. Therefore, there is urgent need to characterize the virus and the viral-mediated responses, in order to develop specific diagnostic and therapeutic tools to prevent viral transmission and efficiently cure COVID-19 patients. Here, we review the current studies on two viral mediated-responses, specifically the cytokine storm occurring in a subset of patients and the antibody response triggered by the infection. Further studies are needed to explore both the dynamics and the mechanisms of the humoral immune response in COVID-19 patients, in order to guide future vaccine design and antibody-based therapies for the management of the disease.

Cell Surface Proteins in Hepatocellular Carcinoma: From Bench to Bedside.

Cell surface proteins act as the go-between in carrying the information from the extracellular environment to the intracellular signaling proteins. However, these proteins are often deregulated in neoplastic diseases, including hepatocellular carcinoma. This review discusses several recent studies that have investigated the role of cell surface proteins in the occurrence and progression of HCC, highlighting the possibility to use them as biomarkers of the disease and/or targets for vaccines and therapeutics.

Short Communication: Decreased Plasma Calcitonin Gene-Related Peptide as a Novel Biomarker for HIV-1 Disease Progression.

HIV-1 mucosal transmission in genital epithelia occurs through infection of Langerhans cells and subsequent transinfection of CD4+ T cells. We previously reported that the vasodilator neuropeptide calcitonin gene-related peptide (CGRP), secreted upon activation of sensory peripheral neurons that innervate all mucosal epithelia, significantly inhibits transinfection. To investigate the association between CGRP and HIV-1 during infection, we evaluated circulating CGRP levels in HIV-1-infected patients. Plasma was obtained from combination antiretroviral therapy (cART)-naive or cART-treated patients with primary/acute (PHI) or chronic (CHI) HIV-1 infection, as well as from individuals who naturally control HIV-1 infection, namely exposed seronegatives (ESNs), elite controllers (ECs), and long-term nonprogressors (LTNPs). CGRP plasma levels were measured using an enzyme immunoassay. Compared with healthy HIV-1-negative controls, CGRP plasma levels significantly decreased in PHI patients and even further in CHI patients, but remained unchanged in ESNs, ECs, and LTNPs. Moreover, CGRP plasma levels were restored to baseline upon cART in both PHI and CHI. Finally, CGRP plasma levels directly correlated with CD4+ T cell counts and inversely with viral loads. Altogether, CGRP could serve as a novel diagnostic plasma biomarker for progression of HIV-1 infection. Moreover, administration of CGRP to cART-naive HIV-1-infected patients, to compensate for CGRP decline, could help controlling on-going HIV-1 infection.

The Abrogation of Phosphorylation Plays a Relevant Role in the CCR5 Signalosome Formation with Natural Antibodies to CCR5.

The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B ß-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that ß-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that ß-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV.

Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells.

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.

Nanoassemblies based on non-ionic amphiphilic cyclodextrin hosting Zn(II)-phthalocyanine and docetaxel: Design, physicochemical properties and intracellular effects.

The combination of conventional anticancer therapy with other treatment modalities such as photodynamic therapy (PDT) is paving the way to novel more effective treatment of solid tumors via light exposure. With this idea in mind, in this paper, nanoparticles (NPs) based on Heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-ß-CD (SC16OH) for dual delivery of Zinc-Phthalocyanine (ZnPc) and Docetaxel (DTX) were developed pointing to their potential application as nanomedicine for the combined photodynamic and chemo-therapy of solid tumors. NPs prepared by the emulsion-solvent evaporation technique displayed a hydrodynamic diameter of ≅ 200nm, a negative zeta potential (≅ -27mV) and a satisfactory entrapment efficiency of both drugs at a specific mass ratio. On these bases, NPs containing DTX and ZnPc with theoretical loading of 5% and 0.2% respectively (ZnPc/DTX5-NPs) were selected for further investigations. The allocation of ZnPc and DTX into the colloid was investigated by complementary spectroscopic techniques. In particular, fluorescence emission studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self-aggregate and consequently a fairly high propensity to photogenerate singlet oxygen. The interaction of SC16OH with DTX, co-entrapped with ZnPc, was elucidated by (1)H NMR and 2D ROESY, which suggested the presence of the chemotherapeutic in the hydrophobic portion of SC16OH. ZnPc/DTX5-NPs were fairly stable in different biological relevant media within 24h. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cancer cells by cell viability exploring both effects of DTX and ZnPc. Overall, results suggest the suitability of NPs based on SC16OH for delivering a combination of DTX with ZnPc to cancer cells, thus inducing photodynamic and antimitotic effects.

ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies.

Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60-90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein-dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs.