Sonia Radice

On the relationship between human papilloma virus vaccine and autoimmune diseases

The human papilloma virus (HPV) vaccines were introduced to reduce the incidence of cervical cancer. The bivalent vaccine is effective against HPV-16, -18, -31, -33 and -45 while the quadrivalent vaccine is effective against HPV-16, 18, 31, 6 and 11 types. The immunisation, recommended for adolescent females, has led to high vaccine coverage in many countries. Along with the introduction of the HPV vaccines, several cases of onset or exacerbations of autoimmune diseases following the vaccine shot have been reported in the literature and pharmacovigilance databases, triggering concerns about its safety. This vaccination programme, however, has been introduced in a population that is at high risk for the onset of autoimmune diseases, making it difficult to assess the role of HPV vaccine in these cases and no conclusive studies have been reported thus far. We have thus analysed and reviewed comprehensively all case reports and studies dealing with either the onset of an autoimmune disease in vaccinated subject or the safety in patients with autoimmune diseases to define the role of the HPV vaccines in these diseases and hence its safety. A solid evidence of causal relationship was provided in few cases in the examined studies, and the risk vs. benefit of vaccination is still to be solved. The on-going vigilance for the safety of this vaccine remains thus of paramount importance.

Early oxidative damage in primary cultured trout hepatocytes: a time course study

The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.

Adaptation to oxidative stress: effects of vinclozolin and iprodione on the HepG2 cell line

It is well known that the dicarboximide fungicides, vinclozolin and iprodione, induce lipid peroxidation by means of oxygen activation in fungi, but their action on mammalian cells is not yet clear. We therefore investigated the effect of 1- and 24-h treatments with vinclozolin at concentrations of 25, 50, 100 microg/ml and iprodione at concentration of 62.5, 125, 250 microg/ml on malonaldehyde and free radical production and on reduced glutathione levels in the human HepG2 hepatoma cell line. The concentrations were chosen on the basis of neutral red cytotoxicity assays. One-hour treatment with the different concentrations of either vinclozolin or iprodione increased both malonaldehyde and free radical content, and decreased reduced glutathione levels, whereas 24-h treatment decreased malonaldehyde content and free radical production, and increased reduced glutathione concentration. These results suggest that the mammalian cells respond to the initial oxidative damage caused by the two dicarboximide fungicides by means of a characteristic adaptative phenomenon within 24 h. This hypothesis is supported by the antagonized effects caused by treatment with the two dicarboximide fungicides and buthionine sulfoximine 0.5 mM, a specific and irreversible inhibitor of reduced glutathione synthesis. The data confirm that the two dicarboximide fungicides maintain their specific action in mammalian cells, although this action is masked by adaptation.

Endothelial nitric oxide synthase overexpression by neuronal cells in neurodegeneration: a link between inflammation and neuroprotection

The roles of neuronal and inducible nitric oxide synthases in neurones have been extensively investigated; by contrast, the biological significance of endothelial nitric oxide synthase (eNOS) overexpression that occurs in several pathological conditions has not yet been studied. We have started addressing this issue in a cell model of neurodegeneration, i.e. human SKNBE neuroblastoma cells transfected with a mutant form of alsin, a protein causing an early‐onset type of amyotrophic lateral sclerosis, ALS2. We found that eNOS, which is endogenously expressed by these cells, was activated by tumour necrosis factor‐α (TNF‐α), a proinflammatory cytokine that plays important roles in ALS2 and several neurodegenerative diseases. The TNF‐α‐dependent eNOS activation occurred through generation, by sphingosine‐kinase‐1, of sphingosine‐1‐phosphate, stimulation of its membrane receptors and activation of Akt, as determined using small interference RNA and dominant negative constructs specific for the enzymes and receptors. eNOS activation by TNF‐α conferred cytoprotection from excitotoxicity and neurotoxic cues such as reactive oxygen species, endoplasmic reticulum stress, DNA damage, and mutated alsin itself. Our results suggest that overexpression of eNOS by neurones is a broad‐range protective mechanism activated during damage and establish a link of pathophysiological relevance between this enzyme and inflammation accompanying neurodegenerative diseases. These findings also question the concept that high NO output in the presence of oxidative stress leads always to peroxynitrite formation contributing to neurodegeneration.

Acute Disseminated Encephalomyelitis Onset: Evaluation Based on Vaccine Adverse Events Reporting Systems

Objective To evaluate epidemiological features of post vaccine acute disseminated encephalomyelitis (ADEM) by considering data from different pharmacovigilance surveillance systems. Methods The Vaccine Adverse Event Reporting System (VAERS) database and the EudraVigilance post-authorisation module (EVPM) were searched to identify post vaccine ADEM cases. Epidemiological features including sex and related vaccines were analysed. Results We retrieved 205 and 236 ADEM cases from the EVPM and VAERS databases, respectively, of which 404 were considered for epidemiological analysis following verification and causality assessment. Half of the patients had less than 18 years and with a slight male predominance. The time interval from vaccination to ADEM onset was 2-30 days in 61% of the cases. Vaccine against seasonal flu and human papilloma virus vaccine were those most frequently associated with ADEM, accounting for almost 30% of the total cases. Mean number of reports per year between 2005 and 2012 in VAERS database was 40±21.7, decreasing after 2010 mainly because of a reduction of reports associated with human papilloma virus and Diphtheria, Pertussis, Tetanus, Polio and Haemophilus Influentiae type B vaccines. Conclusions This study has a high epidemiological power as it is based on information on adverse events having occurred in over one billion people. It suffers from lack of rigorous case verification due to the weakness intrinsic to the surveillance databases used. At variance with previous reports on a prevalence of ADEM in childhood we demonstrate that it may occur at any age when post vaccination. This study also shows that the diminishing trend in post vaccine ADEM reporting related to Diphtheria, Pertussis, Tetanus, Polio and Haemophilus Influentiae type B and human papilloma virus vaccine groups is most likely due to a decline in vaccine coverage indicative of a reduced attention to this adverse drug reaction.

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.

Drinking water quality: An in vitro approach for the assessment of cytotoxic and genotoxic load in water sampled along distribution system

An in vitro approach was performed to assess the quality of drinking water collected at two treatment/distribution networks located near the source (Plant #1) and the mouth of River Po (Plant #2). The water was sampled at different points of each distribution network, before (raw water) and after the chlorine dioxide disinfection, and in two points of the pipeline system to evaluate the influence of the distribution system on the amount and quality of the disinfection by-product. Cytotoxicity and genotoxicity of water extracts were evaluated in human peripheral lymphocytes and Hep-G2 cells by the use of the micronucleus (MN) test and Comet assay. Raw water samples of both plants induced cytotoxic effects, but not the increases of MN frequency in Hep-G2 cells and in human lymphocytes. Increases of DNA damage in human leukocytes was detected by Comet assay for raw water of Plant #2 at concentration > or = 0.25 Leq/mL. The disinfection process generally has reduced the toxicity of water samples, even if potential direct DNA-damaging compounds have been detectable in drinking water samples. The proposal approach, if currently used together with chemical analysis, can contribute to improve the monitoring drinking water.

The importance of monitoring adverse drug reactions in pediatric patients: the results of a national surveillance program in Italy

Objective: To gain information on safety of drugs used in pediatrics through a 4-year post-marketing active pharmacovigilance program. The program sampled the Italian population and was termed ‘Monitoring of the Adverse Effects in Pediatric population’ (MEAP). Research design and methods: Adverse drug reactions (ADRs) were collected for individuals aged 0 – 17 years treated in hospitals and territorial health services in Lombardy, Tuscany, Apulia and Campania; located to gain an appropriate sampling of the population. ADRs were evaluated using the Adverse Drug Reaction Probability Scale (Naranjo) and analyzed with respect to time, age, sex, category of ADR, seriousness, suspected medicines, type of reporter and off-label use. Results: We collected and analyzed reports from 3539 ADRs. Vaccines, antineoplastic and psychotropic drugs were the most frequently pharmacotherapeutic subgroups involved. Seventeen percent of reported ADRs were serious; of them fever, vomiting and angioedema were the most frequently reported. Eight percent of ADRs were associated with off-label use, and 10% were unknown ADRs. Analysis of these revealed possible strategies of therapy optimization. Conclusions: The MEAP project demonstrated that active post-marketing pharmacovigilance programs are a valid strategy to increase awareness on pediatric pharmacology, reduce underreporting and provide information on drug actions in pediatrics. This information enhances drug therapy optimization in the pediatric patients.

Efficacy of vaccination against influenza in patients with multiple sclerosis: The role of concomitant therapies

Multiple sclerosis is a chronic progressive demyelinating disease affecting over 2.1 million patients worldwide. Patients affected by MS are exposed to an increased risk of infection from communicable diseases, which may lead to severe disease relapses. Studies have analysed the issue of vaccination of MS-affected patients. These studies, however, deal mostly with safety-related issues documenting that most vaccines have been proven to be safe in MS patients and that vaccination is not associated with an increased risk of relapses. By contrast, evidence on the efficacy is comparatively scant and not yet systematised in a comprehensive picture. This aspect is however important, as both MS and its treatment alter the immune responses, a situation that may be associated with a reduced vaccine efficacy. We have now reviewed the literature and assessed the effects of the therapy for MS on vaccine efficacy; we focused on the vaccine against influenza as for the other vaccines the information is still too scant. The majority of drugs appear not associated with a reduced response to vaccination against influenza, with the notable exception of mitoxantrone and glatiramer acetate. For a few drugs, among which natalizumab, information is not sufficiently clear and additional studies are needed to draw a definite conclusion. These results highlight the importance to evaluate the efficacy of vaccination in patients treated with immunosuppressant drugs.

Can HPV immunisation cause ADEM? Two case reports and literature review

Human papilloma virus (HPV) vaccination is widely used in order to decrease the incidence of cervical cancer cases worldwide.1 The safety profile of these vaccines, assessed during phase III studies, showed that both vaccines have a good profile of tolerability. Recently, however, Schäffer et al. provided the first report regarding the onset of acute disseminated encephalomyelitis (ADEM) 23 days following HPV vaccination in a 15-year-old woman.2 Further reports provided similar observations,3,4 indicating a possible relationship between HPV vaccination and ADEM. ADEM is an uncommon condition, usually preceded by an acute infection.5 In about 5% of ADEM cases, however, a precedent immunisation was described as the only risk factor.3 We report on two additional cases of patients, two girls aged 13 and 12 respectively, who developed ADEM following one primary and two booster administrations of HPV vaccine. In both patients’ medical history, blood and cerebrospinal fluid (CSF) analyses ruled out a possible infectious aetiology and autoantibody testing was negative for ANA, ANCA and AQP4-Ab. The 13-year-old Caucasian girl was hospitalised for a unilateral deficit of the second and third cranial nerves and objective signs of meningo-encephalitis 6 months after receiving the third dose of HPV vaccination (unknown manufacturer). Magnetic resonance imaging (MRI) of the brain revealed the presence of multiple ring-enhancing lesions located primarily in thalamic, pons and insula white matter. CSF was normal and we did not observe the presence of oligoclonal bands (OCBs). The long time interval between vaccine and disease onset is unusual in ADEM developed after vaccination, and does not necessarily argue for a causal relationship; however, we did not identify any other significant risk factor. Her clinical condition improved following steroids therapy and she was discharged fully recovered. The 12-year-old girl was hospitalised due to the onset of numbness of the left foot, ambulation difficulty, and speech disorder. Parents reported that she had been vaccinated with the third dose of vaccine against HPV (Quadrivalent) 15 days before. At the admission, both computed axial tomography (CAT) and MRI were negative. During hospitalisation, patient developed enuresis, faecal incontinence and dysphagia. MRI of the brain revealed the presence of multiple ring-enhancing lesions at subcortical levels, pons, trigeminal nuclei and mesencephalon. Spinal cord MRI revealed swelling from C4 to C6 without contrast enhancement. After 1 month from hospitalisation, a reduction in visual acuity was observed and MRI confirmed the previously observed lesion and identified new lesions in the subcortical white matter. MRI of the spinal cord showed extensive enhancing lesion from C1/2 to D1/2. Analysis of CSF revealed lymphocytic pleocytosis and excluded the presence of OCBs. A therapy with high dose steroids was started that led to improvement of the patient’s conditions. For a better evaluation of the relationship between HPV immunisation and ADEM, we analysed data from the Vaccine Adverse Event Reporting System (VAERS), a passive surveillance system that collects vaccine adverse drug reaction data from the USA and other countries. After causality assessment with World Heath Organisation criteria, we were able to identify 12 ADEM reports that could be classified as related to qHPV vaccination between 1 June 2006 and 30 July 2012. By considering these reports and the number of qHPV doses distributed within this period (46 million doses), the reporting rate was estimated to be 0.26/106 (CI 95%: 0.16/106–0.37/106). Despite known limitation of VAERS, these data strengthen our hypothesis of correlation between HPV immunisation and ADEM. These cases, taking together with pharmacovigilance data and literature findings, indicate the presence of a relationship between ADEM and HPV vaccination.