Lionello Ruggeri

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9ΔC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9ΔC bound to EGF(AB)^H306Y, a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)^H306Y-PCSK9 interaction is pH-independent, LDLR^H306Y binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9ΔC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

Background/Aims: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease.The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection.Methods: The clinical course of acute hepatitis C was determined in 34 consecutive patients.Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed.Results: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome.Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis.No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles.HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease.A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. Conclusion:The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus.Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

ABSTRACT Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8 + T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

<b>Background and aims:</b> Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4⁺ and CD8⁺ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. <b>Methods:</b> CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. <b>Results:</b> Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. <b>Conclusion:</b> Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Sporadic cases of cell-mediated immunity (CMI) in persons exposed to hepatitis C (HCV) but evidently uninfected have been reported. To further define this, we measured CMI in individuals without evidence of HCV infection, that is, negative for HCV-antibodies (anti-HCV) and RNA, residing in a rural Egyptian community where prevalence of anti-HCV was 24%. Cell-mediated immunity (CMI) measured by interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay, confirmed by intracellular staining using flow cytometry, against HCV peptides was measured in seronegative individuals with high-risk (HR) and low-risk (LR) exposures to HCV. Thirteen of 71 (18.3%) HR subjects but only 1 of 35 (2.9%) LR subjects had detectable CMI (P = 0.032). These data are compatible with the hypothesis that exposures to HCV may lead to development of HCV-specific CMI without anti-HCV and ongoing viral replication. We speculate induced CMI clears HCV sometimes when anti-HCV is not detectable, and HCV-specific CMI is a useful surrogate marker for exposure to HCV.

Summary. Some chronic hepatitis C (CHC) patients exhibit persistently normal alanine aminotransferase (ALT) levels (PNAL). Patients with PNAL experience significantly milder disease. In order to understand the differences between CHC patients with elevated ALT levels compared with those with PNAL better, we compared epidemiological, immunological and histological findings, in particular, the value of proliferating hepatocyte activity (PCNA) between the two groups of patients. We studied 40 chronic hepatitis C virus (HCV) carriers with increased ALT who underwent liver biopsy for histological diagnosis and determination of clinical prognosis, and 24 PNAL patients under follow‐up for 10 years. Immunological response to different HCV genomic epitopes was tested in both the control group and in PNAL subjects. PCNA values from liver specimens of all patients as well as liver biopsies of PNAL patients at time points 0 and 5 years were calculated according to Hall et al. Age, sex and body mass index (BMI) were not significantly different between the two groups. The median liver histology stage was significantly higher in HCV carriers vs the PNAL group (2.5, range = 2–6 vs 1.5, range = 1–2; P &lt; 0.01). Among PNAL patients, histological stage was not statistically different at the three time points considered. Interferon (IFN)‐gamma production was comparable in the two groups. PCNA was significantly higher in the group with elevated ALT levels vs the PNAL group (8%, range = 4–15% vs 5% range = 3–8%; P &lt; 0.05) and no statistically significant differences were found in PNAL patients at time points 0, 5 and 10 years. This study confirms that progression to cirrhosis is slow or absent in PNAL patients after 10 years of follow‐up. Accordingly, the hepatic proliferative activity index is low and seems to be stable over time.

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute-phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I-restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8(+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi-species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute-phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.

The ability of HCV to mutate in response to cytotoxic T lymphocyte (CTL) pressure is increasingly recognized, but the influence of such a mechanism in viral persistence and final disease outcome has not been ascertained. In this study, we performed a detailed longitudinal analysis of cell mediated immunity and HCV evolution in two self limiting and two chronically evolving HCV acutely infected patients, one of whom transiently controlled viremia. Aminoacid mutations in immunodominant regions of viruses were observed in all patients, although they conferred viral escape from CTL responses only in chronically infected individuals. Resurgence of viremia coincided with the replacement of the original virus quasispecies with mutant viruses that had escaped recognition by primary CD8+ T cell responses and infection persisted in the presence of variant viruses which were less efficiently recognized by preexisting and de novo induced T cell responses.

Background/Aims Several studies suggest that the evolutionary rate of HVR1 sequence in acute HCV hepatitis derives from the action of a continuous immune-driven positive selection. However, these studies have not been performed examining the relationship between HVR1 evolution and the development of specific immunity to autologous HVR1 sequences. Methods We performed a longitudinal analysis of HVR1 sequences and specific antibodies and CD4+ T cells in ten HCV acutely infected patients with different clinical outcomes (recovery versus persistence). Results We showed that although both recovered and chronically evolving individuals developed IFN-γ+ T cells specific for Core and NS sequences, HVR1-specific CD4+ T cells were detected only in patients clearing the virus. On the contrary, all patients displayed anti-HVR1 antibodies that recognized sequences exclusively carried by autologous viruses. Measurements of genetic diversity and the number of non-synonymous per synonymous substitutions within HVR1 sequences before and after antibody appearance showed an increase of these parameters only in concomitance with the appearance of anti-HVR1 antibodies. Conclusions The evidence that anti-HVR1 antibodies favor HVR1 variant selection suggests that viral complexity in chronically infected patients could represent a virus adaptive strategy to escape the continuous selective process mediated by anti-HVR1 antibodies. Several studies suggest that the evolutionary rate of HVR1 sequence in acute HCV hepatitis derives from the action of a continuous immune-driven positive selection. However, these studies have not been performed examining the relationship between HVR1 evolution and the development of specific immunity to autologous HVR1 sequences. We performed a longitudinal analysis of HVR1 sequences and specific antibodies and CD4+ T cells in ten HCV acutely infected patients with different clinical outcomes (recovery versus persistence). We showed that although both recovered and chronically evolving individuals developed IFN-γ+ T cells specific for Core and NS sequences, HVR1-specific CD4+ T cells were detected only in patients clearing the virus. On the contrary, all patients displayed anti-HVR1 antibodies that recognized sequences exclusively carried by autologous viruses. Measurements of genetic diversity and the number of non-synonymous per synonymous substitutions within HVR1 sequences before and after antibody appearance showed an increase of these parameters only in concomitance with the appearance of anti-HVR1 antibodies. The evidence that anti-HVR1 antibodies favor HVR1 variant selection suggests that viral complexity in chronically infected patients could represent a virus adaptive strategy to escape the continuous selective process mediated by anti-HVR1 antibodies.

Background: Cancer permanently marks body, leaving scars, mutilations and creating functional alterations. These wounds can persist a long time and leave signs both from physical and psychological . Body changes consequent to surgery and cancer treatments require a process of adaptation and, after many years they condition the sense of general health, personal care, social contacts and sexuality. These changes make patients loose self-esteem and reduce quality of life. Patients feel a sense of loss of physical integrity, they feel strangers in their body. Materials and methods: Since May 2013 our Department has joined the project “La Forza e il Sorriso” (Strenght and Smile), organizing workshops of beauty for women in cancer treatments.The aim is to give psychological support to patients in cancer treatment and in follow up. During project, we teach techniques to manage the effects of treatments. Through this project we want to help patients to rediscover important rituals such as cohoperation, confidences and alliance between “friends”, rediscovering the pleasure of embrace, makeup or simply meet and go out from home, home where often patients stay closed because of fear and shame. 5/6 women to each laboratory; they are followed by three beauty consultants volunteers, specially trained; during two and half hour session they help the participants to take care of their image and to cope with the secondary effects of cancer therapies. Psychologist is always present during these sessions. Women know about beauty laboratory through information materials and website. At the end of each session, every woman receives a beauty-bag donated by the association, to practice at home. At the end of each session we ask patients to complete an anonymous satisfaction questionnaire. Results: 18 workshops were conducted, involving 72 women. Average age 55 years (range 24-69). 73% breast cancer. 81% in treatment, 19% in follow-up, 26.5% metastatic. Women have expressed great enthusiasm for taking part to the initiative. Questionnaires showed that: 70% felt valued and beautiful, 55% more cheerful and serene, 45% inspirating and positive, 30% renewed; no woman reported to feel herself like before. Conclusion: Chemotherapy and its consequences will never stop to create fear, but certainly the women who were accompanied to the care of their appearance, are outputs from the meetings with a look and a new posture, different, more aware of their own femininity and with a positive attitude.

Background: The first oncological visit is a central moment in the treatment and in the establishment of the therapeutic relationship; the meeting with the oncologist involves psychological aspects and has a strong emotional impact on patients (pts). First visit is the time of diagnosis communication and therapeuthic condivision. Pts arrive at the first visit with expectations, fears, fantasies, ideas not only about their health conditions, but also about the doctor and the relationship with him. Materials and methods: In our Oncology Unit we give an important value to the first visit. We recognize that disease has both an organic and a psychological component. That is the reason why we choose to plan the majority of first visits both with doctor and psychologist. This is an important legitimation of emotional impact of cancer and the possibility that the experiences related to that event can then find spaces of expression, listening and processing. Cohoperation between psychologist and oncologist can improve a positive exchange between two visions to face pts that are often different. Steps of the first visit are: - Preliminary view of the medical oncologist and psychologist - Presentation to the patient and the caregivers of the Psycho-Oncology Service - Drafting of brief psychological report that will be included in the medical record - Possible first date for a psychological support if necessary The aims: - Increase the therapeutic alliance and adherence to treatment path - Focus on patients and family members considered ‘at risk’ for an early taking care, to decrease the risk of psychopathological manifestations - Facilitate the comparison between different professionals. Results: In 2014 we made 210 joint visits (65% of total), 52 pts with breast cancer, 47 colon, 35 with gastric cancer, 21 with gynecological cancer, 25 lung, 15 melanoma and 15 more. 28 pts and eight caregivers were taken into care after the first visit, while in other situations it is active a surveillance in cohoperation with the team of care. Conclusions: The presence of the psychologist in the Unit, the cohoperation and the communication with health staff has improved early detection of psychological suffering situations. Psychoncological service integrated with oncological treatment is a valuable resource for patients and caregiver that are facing a state of vulnerability.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) binds to LDL receptor (LDLR) protein, which ultimately leads to LDLR degradation and inhibition of plasma LDL cholesterol (LDL-C) clearanc...

Introduction the early neonatal period is characterized by a rapid maturation of kidney function. Moreover, a variety of therapeutic interventions and the administration drugs may have harmful renal consequences. Goal We tested the hypothesis that drugs or therapeutics in the neonatal period could increase the risk for ARF in preterm newborns. Methods All preterm infants (261) with a G.A. < 36 weeks admitted in the NICUs of 7 Italian University hospitals (March 2000-March 2003) were involved. Data were collected in detailed questionnaires, and entered into a computerized relational database. B.W., G.A., Apgar scores, blood pressure, respiratory status, lab assays, radiological exams, clinical outcomes and all therapeutical interventions (catheters, intubation, phototherapy, drugs) were recorded. Statistical analysis was performed using the Statistical Package for Social Sciences for Windows (SPSS Inc., Chicago, IL).246 subjects enrolled in the study are divided into four groups according to G.A.: group A, 22–25; group B, 26–28; group C, 29–32; and group D, 33–36 weeks. Results All the diseases diagnosed and the therapeutic interventions applied to these preterm infants resulted inversely correlated with the G.A. (p<0.001 for all examined parameters, except as regards NEC: p=0.019). No differences at birth were observed among the 4 groups as regards either BUN or Crs values, while statistical significant differences were evident from the 3rd to the 21st day of life. The diagnosis was made on the basis of high Crs concentrations at 60th hour of life or later (values > 1.3 mg/dl or > 1 mg/dl respectively in subjects with a G.A. < or > 33 weeks) and/or presence of oliguria. In neonates with impaired renal function, an important increase in Crs values was observed in all groups from the 3rd to the 10th day of life, with statistical significant differences among groups on day 7 (p=0.03) and day 10 (p=0.006). Then, values decreased but remained higher, compared to subject with normal renal function, in all neonates at 28th day of life. An inverse correlation is evident between G.A. And the percentage of neonates with impaired renal function (?2= 25.707, p<0.001): 56% of neonates in group A resulted with impaired renal function, while in group D only 15% had Crs pathological values. Conclusion in more than 50% of patients of group A have renal problems.Instead, in neonates of group D the percentage of subjects with renal impairment is considerably lower (15% vs 56%): It is evident the role played by some drugs, therapeutic interventions and diseases.

A wide range of topics can be included under the heading of recurrent infections in children. This discussion focuses on 1) the definition of recurrent infection and physiopathogenetic mechanisms predisposing to; 2) controversies in the management of upper respiratory tract infections; 3) recurrent upper and lower respiratory infections in immunocompromised hosts, emphasizing advances in diagnosis and treatment of "mild" immunodeficiencies such as IgG subclass deficiency or antibody deficiency in normogammaglobulimia, trying to define an operative flow chart.