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Translating evidence-based guidelines into clinical practice is a complex challenge. This observational study aimed to assess the adherence to the Italian national guidelines on postpartum haemorrhage (PPH) and describe the clinical management of haemorrhagic events in a selection of maternity units (MUs) in six Italian regions, between January 2019 and October 2020. A twofold study design was adopted: (i) a before–after observational study was used to assess the adherence to national clinical and organisational key recommendations on PPH management, and (ii) a cross-sectional study enrolling prospectively 1100 women with PPH ≥ 1000 mL was used to verify the results of the before–after study. The post-test detected an improved adherence to 16/17 key recommendations of the guidelines, with clinical governance and communication with family members emerging as critical areas. Overall, PPH management emerged as appropriate except for three recommended procedures that emphasise different results between the practices adopted and the difference between what is considered acquired and what is actually practised in daily care. The methodology adopted by the MOVIE project and the adopted training materials and tools have proved effective in improving adherence to the recommended procedures for appropriate PPH management and could be adopted in similar care settings in order to move evidence into practice.

Ensuring drug safety for pregnant women through prescription drug monitoring is essential. The aim of this study was to describe the prescription pattern of medicines among pregnant immigrant women from countries with high migratory pressure (HMPCs) compared to pregnant Italian women. The prevalence of drug prescriptions among the two study populations was analysed through record linkage procedures applied to the administrative databases of eight Italian regions, from 2016 to 2018. The overall prevalence of drug prescription was calculated considering all women who received at least one prescription during the study period. Immigrants had a lower prevalence of drug prescriptions before (51.0% vs. 58.6%) and after pregnancy (55.1% vs. 60. 3%). Conversely, during pregnancy, they obtained a slightly higher number of prescriptions (74.9% vs. 72.8%). The most prescribed class of drugs was the blood and haematopoietic organs category (category ATC B) (56.4% vs. 45.9%, immigrants compared to Italians), followed by antimicrobials (31.3% vs. 33.7%). Most prescriptions were appropriate, while folic acid administration 3 months before conception was low for both study groups (3.9% immigrants and 6.2% Italians). Progesterone seemingly was prescribed against early pregnancy loss, more frequently among Italians (16.5% vs. 8.1% immigrants). Few inappropriate medications were prescribed among antihypertensives, statins and anti-inflammatory drugs in both study groups.

Introduction Peripartum hysterectomy is usually undertaken in cases of life-threatening obstetric hemorrhage to prevent the death of the mother. Near-miss events are still under-researched and inappropriate care continues to be a critical issue, even in countries with advanced obstetric surveillance systems. The aim of the present study was to estimate the prevalence, associated factors, management and intraoperative and postoperative complications of peripartum hysterectomy due to obstetric hemorrhage. Material and methods A prospective population-based study has been conducted in six Italian regions covering 49% of births in Italy. The study population comprised all women aged 11-59 years undergoing peripartum hysterectomy, from September 2014 to August 2016, due to obstetric hemorrhage within 7 days of delivery. In each maternity unit a trained reference person reported incident cases using electronic data collection forms. The background population comprised all women who delivered in the participating regions during the study period. Results The overall peripartum hysterectomy prevalence was 1.09 per 1000 maternities, with a large variability among regions, ranging from 0.52 to 1.60. Previous cesarean section (relative risk [RR] 4.97, 95% CI 4.13-5.96), assisted reproductive technology (RR 5.99, 95% CI 4.42-8.11) multiple pregnancy (RR 5.03, 95% CI 3.57-7.09) and maternal age ≥35 years (RR 2.69, 95% CI 2.25-3.21) were the main associated factors for hysterectomy. The most common causes of peripartum hysterectomy were uterine atony (45.1%) and abnormally invasive placentation (40.2%). Intensive care unit admission was reported in 49.9% of cases, 16.8% of women suffered severe morbidity and 5 women died. Conclusions The rate of peripartum hysterectomy in Italy was three times higher compared with the UK, the Netherlands and the Nordic countries. The wide difference may be associated with women's characteristics, such as age at delivery and previous cesarean section, and with different management options leading to peripartum hysterectomy.

OBIETTIVI: descrivere la metodologia di un sistema pilota di sorveglianza della mortalità perinatale (SPItOSS) volto a migliorare la qualità delle cure e a prevenire la grave morbosità e la mortalità perinatale evitabili in tre regioni italiane (Lombardia, Toscana e Sicilia). DISEGNO: un progetto di sorveglianza di popolazione per monitorare i casi di natimortalità dopo 28 settimane di gestazione e di mortalità neonatale entro 7 giorni dalla nascita. Per ogni decesso è stato organizzato un audit multidisciplinare per valutare la causa di morte presso la struttura dove si è verificato il caso. Una selezione dei decessi è stata analizzata da esperti attraverso indagini confidenziali regionali e nazionali per valutarne le cause e l’evitabilità. Per identificare le morti perinatali e codificarne le cause, sono stati adottati la definizione di mortalità perinatale dell’OMS e il suo sistema di classificazione ICD-PM. SETTING E PARTECIPANTI: professionisti sanitari di ogni unità di ostetricia e/o neonatologia nelle tre regioni coinvolte, che in totale registrano il 32,3% delle nascite in Italia. La raccolta dei dati è iniziata il 01.07.2017 e si è conclusa il 30.06.2019. PRINCIPALI MISURE DI OUTCOME: tasso di partecipazione delle unità ostetriche e neonatali nelle regioni aderenti al progetto, descrizione dell’organizzazione strutturale dei punti nascita, partecipazione degli operatori sanitari ai corsi di formazione sulle procedure della sorveglianza, stima dei tassi di mortalità perinatale nel periodo di studio, tasso degli audit clinici e delle indagini confidenziali eseguiti. RISULTATI: tutti i professionisti sanitari provenienti da tutte le unità di ostetricia e/o neonatologia (n. 138) hanno aderito al progetto pilota. Complessivamente, sono stati segnalati 830 decessi; 699 sono stati esaminati durante audit multiprofessionali presso le strutture dove si erano verificate le morti; 94 casi selezionati sono stati sottoposti a indagini confidenziali sia al livello regionale sia nazionale. Il 16% di questi ultimi sono stati valutati come casi di morti materne evitabili. Sono state identificate differenze interregionali nell’organizzazione strutturale dei punti nascita. CONCLUSIONI: la sorveglianza SPItOSS è stata efficiente nell’identificare e analizzare i casi di morte perinatale, individuare gli aspetti dell’assistenza suscettibili di miglioramento e le morti perinatali evitabili. Il prossimo obiettivo consiste nell’estendere la sorveglianza a livello nazionale prevedendo finanziamenti stabili e il coinvolgimento di un maggior numero di professionisti ed esperti. OBJECTIVES: to describe the methodology of a pilot perinatal mortality surveillance system (SPItOSS) aimed to improve quality of care and prevent avoidable perinatal severe morbidity and mortality in three Italian regions. DESIGN: population-based surveillance project monitoring incident cases of stillbirths, born dead ≥ 28 weeks of gestation and live births dying within 7 days. Local multidisciplinary audits in obstetric and neonatal units were conducted to assess causes of each death. A selection of deaths was also discussed by experts in regional and national Confidential Enquiries to assess causes and avoidability. The WHO perinatal mortality definition and the ICD-PM classification were adopted to codify perinatal deaths. SETTING AND PARTICIPANTS: health professionals working in any obstetric and neonatal unit in Lombardy (Northern Italy), Tuscany (Central Italy), and Sicily (Southern Italy), ac-counting for 32.3% of births in Italy. Data collection started on 01.07.2017 and ended on 30.06.2019. MAIN OUTCOME MEASURES: obstetric and neonatal units’ participation rate in the participating regions, facility struc-tural organisation description, health professionals’ training course participation, estimate of perinatal death rates, clinical audits and Confidential Enquiries rates. RESULTS: health professionals from all obstetric and/or neonatal units (N. 138) joined the pilot project. Overall, 830 incident perinatal deaths were reported; 699 underwent a faci-lity-based clinical audit, and 94 selected cases were analysed in detail through regional and national Confidential Enquiri-es. Among the latter, 16.0% were assessed as avoidable de-aths. Interregional differences related to the facility structu-ral organisation were identified. CONCLUSIONS: SPItOSS was efficient in identifying and analysing incident cases of perinatal deaths and detecting improvable aspects of care and avoidable perinatal deaths. The next objective is to extend the surveillance at the national level, considering that stable funding and a higher number of participating healthcare professionals and experts will be needed.

Introduction Distance learning efficacy on physician performances and patient health outcomes has been demonstrated. This study explored the participation and evaluation of CME e-learning courses for Italian health care professionals addressing leading causes of maternal mortality identified by the Italian Obstetric Surveillance System (ItOSS) at the Italian National Health Institute, namely postpartum hemorrhage and pregnancy hypertensive disorders. Methods A model for two online free 12-hour case-based training courses was used. Data on participants were collected, anonymized, and transferred to the Italian National Health Institute for later analysis. Participants were requested to sign an online informed consent form. Results Twenty-one thousand five hundred thirty-two health care professionals enrolled to the courses from 2014 to 2017 as follows: midwives (14,187, 65.9%); obstetricians (3,716, 17.2%); anesthesiologists (1,896, 8.8%); and other medical specialists (1,733, 8.0%). Overall, 85% of participants acquired CME credits. Participants' satisfaction on quality, efficacy, and relevance was very high. Discussion ItOSS courses were able to reach a substantial number of different professional profiles involved in perinatal care all over the country; ItOSS courses can be considered an effective way to spread evidence-based good clinical practices. Nevertheless, further studies are needed to verify the improvement in professional health care skills and patient outcomes.

Objectives To describe a cohort of Italian women with confirmed COVID-19 infection admitted during pregnancy for out or inpatient hospital care, in order to provide rapid feedback for clinicians and policymakers. Design National population-based cohort study. Setting 297 Italian public and private maternity units. Population Pregnant women with COVID-19 infection confirmed by RT-PCR testing through nasopharyngeal swab and/or chest RX or CT findings and/or antibody response from maternal blood. Exclusion criteria: age

OBJECTIVE: To describe the Italian Obstetric Surveillance System (ItOSS) investigating maternal death through incident case reporting and confidential enquiries. METHODS: All maternal deaths occurred in any public and private health facility in 8 Italian regions covering 73% of national births have been notified to the ItOSS. Every incident case is confidentially reviewed to assess quality of care and establish the cause and avoidability of the death. FINDINGS: A total of 106 maternal deaths among 1 455 545 live births have been notified to the surveillance system in 2013-17. Haemorrhage, sepsis and hypertensive disorders of pregnancy are the leading causes of direct maternal deaths due to obstetric causes. CONCLUSIONS: A maternal mortality surveillance system, including incidence reporting and confidential enquiries along with a retrospective analysis of administrative data sources, emerged as the best option for case ascertainment and for preventing avoidable maternal deaths

Introduction Accurate estimates and reliable classification of maternal deaths are imperative steps in the chain of actions targeted at reducing avoidable maternal mortality. The aims of this study were to estimate the maternal mortality ratio (MMR) in 10 Italian regions covering 77% of total national births and to identify the most suitable approach to classify the causes of death. Material and methods Deaths during and within 1 year after pregnancy have been identified through linkage between death registry and hospital discharge database. Regional and national data sources from 2006 to 2012 were used. The MMR has been estimated and deaths were classified as direct or indirect and according to their primary causes. Results A total of 277 maternal deaths within 42 days after pregnancy were identified: 149 direct, 102 indirect causes and 26 unclassified-resulting in a MMR of 9.18 per 100 000 live births. The under-reporting rate of official MMR figures in the participating regions is 60.3%. Hemorrhage (MMR 1.92), hypertensive disorders of pregnancy and cardiac diseases (MMR 1.06) were the leading causes of deaths occurring within 42 days after pregnancy, whereas malignancy (39%) and violent deaths (17%) were the most frequent of the 543 late maternal deaths. Conclusions Record-linkage is an efficient and reliable method to estimate maternal mortality and to identify causes of maternal deaths. Both the indirect/direct and the classification by primary cause have a role in countries where direct deaths exceed indirect maternal mortality. Building upon linkage data, confidential enquiries further increase the likelihood of reducing maternal mortality.

Suicide has been identified as one of the most common causes of death among women within 1 year after the end of pregnancy in several high-income countries. The aim of this study was to provide the first estimate of the maternal suicide ratio and a description of the characteristics of women who died by suicide during pregnancy or within 1 year after giving birth, induced abortion or miscarriage (i.e., maternal suicide) in 10 Italian regions, covering 77% of total national births. Maternal suicides were identified through the linkage between regional death registries and hospital discharge databases. Background population data was collected from the national hospital discharge, abortion and mortality databases. The previous psychiatric history of the women who died by maternal suicide was retrieved from the regionally available data sources. A total of 67 cases of maternal suicide were identified, corresponding to a maternal suicide ratio of 2.30 per 100,000 live births in 2006–2012. The suicide rate was 1.18 per 100,000 after giving birth (n = 2,876,193), 2.77 after an induced abortion (n = 650,549) and 2.90 after a miscarriage (n = 379,583). The majority of the women who died by maternal suicide (34/57) had a previous psychiatric history; 15/18 previously diagnosed mental disorders were not registered along with the index pregnancy obstetric records. Suicide is a relevant cause of maternal death in Italy. The continuity of care between primary, mental health and maternity care were found to be critical. Clinicians should be aware of the issue, as they may play an important role in preventing suicide in their patients.

The study aims to promote the reduction of preventable maternal and neonatal deaths related to sepsis worldwide. To achieve this goal, the study will test new WHO criteria for early identification of maternal sepsis across both low and high resource settings, describe and analyze the frequency and outcomes of suspected or confirmed maternal sepsis for mothers and infants. Additionally, in the European participating countries, the study will describe the patterns of anti-microbial usage amongst women with suspected maternal sepsis, explore migration status amongst women with possible severe maternal infection and describe any variations in their management.

Deficiency of folic acid (FA) has been identified as a risk factor for neural tube defects (NTDs) as well as other congenital anomalies. Thus, periconceptional folate supplementation is recommended for all women planning to get pregnant.We conducted a KAP (knowledge, attitude, practice) survey to investigate the use of FA and its appropriateness. The survey included a sample of 562 women who delivered in the Lazio region between 2013 and 2014. Two logistic regression analyses were performed to evaluate the association between the characteristics of participating women and both the information received on FA intake and its use.The prevalence of periconceptional FA assumption was 19.4% although 82.2% of the interviewed women had planned their pregnancies.It shows that more periconceptional counseling is needed to increase women's awareness on the opportunity of FA supplementation.

This paper reports on oocyte cryopreservation efficacy in Italy with respect to successful IVF from 2005 to 2007, presenting data from 193 centres collected by the Italian National Register. Post-thawing survival rates, number of transferred embryos, implantation rates and clinical pregnancy rates per transfer with respect to frozen/vitrified oocytes (FVO) were analysed. These numbers were compared with those obtained using frozen embryos or fresh oocytes. A total of 121,708 cycles were initiated, of which, 7.1% (8682) were FVO cycles and 2.4% (2952) were frozen embryo cycles. Of the 81,786 FVO, 52.5% (42,917) were thawed and 26.9% (22,005) inseminated. Of those inseminated, 68.0% (14,966) yielded good embryos. These numbers were significantly lower than those using fresh oocytes in which 77.9% (197,242; fresh oocytes versus FVO P < 0.001) of inseminated oocytes generated good embryos. Implantation rate using FVO was 6.9%, which was significantly lower than that using fresh oocytes (13.5%; P < 0.001) and frozen embryos (8.8%; P < 0.001). Pregnancy rate per transfer using FVO was 12.5% and significantly lower than that using fresh oocytes (24.9%; P < 0.001) or frozen embryos (16.4%; P < 0.001). There were 505 deliveries after IVF with FVO and 582 babies.

Summary The HIV Nef protein mediates endocytosis of surface receptors that correlates with disease progression, but the link between this Nef function and HIV pathogenesis is not clear. Here, we report that Nef-mediated activation of membrane trafficking is bidirectional, connecting endocytosis with exocytosis as occurs in activated T cells. Nef expression induced an extensive secretory activity in infected and, surprisingly, also in noninfected T cells, leading to the massive release of microvesicle clusters, a phenotype observed in vitro and in 36%–87% of primary CD4 T cells from HIV-infected individuals. Consistent with exocytosis in noninfected cells, Nef is transferred to bystander cells upon cell-to-cell contact and subsequently induces secretion in an Erk1/2-dependent manner. Thus, HIV Nef alters membrane dynamics, mimicking those of activated T cells and causing a transfer of infected cell signaling (TOS) to bystander cells. This mechanism may help explain the detrimental effect on bystander cells seen in HIV infection.

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

OBJECTIVE: To compare mature human oocytes cryopreservation with slow freezing (SF) and vitrification (VT) in infertile couples. DESIGN: Retrospective study of national Italian data submitted during the period 2007-2011. SETTING: National ART registry. PATIENT(S): Infertile patients with supernumerary oocytes. INTERVENTION(S): Thawing or warming of cryopreserved oocytes and ICSI. MAIN OUTCOME MEASURE(S): oocyte survival, fertilization, implantation and clinical pregnancy rate between SF and VT. RESULT(S): A total of 14,328 cycles with 11,599 transfers, 1,850 pregnancies, 1,168 deliveries and 1,342 babies born were analyzed from 146 reporting centers (range of cycles 1-1,255 per center). The SF oocytes' survival rate was lower than in VT (51.1% vs. 63.1%). Fertilization rate was significantly higher in SF than in VT (SF 71.6% vs. VT 70.1%). VT showed a significantly higher pregnancy rate, both per started cycle (14.4% vs. 12.0%) and per transfer (18.0% vs. 14.8%), and implantation rate (9.5% vs. 8.1%) than SF. However, the range and median pregnancy rate per started cycle were, respectively, 0%-50% and 7.7% in SF and 0%-100% and 6.7% in VT. CONCLUSION(S): VT showed a statistically significant higher performance than SF. As with other ART procedures, the results are not homogeneous among clinics and protocols, but the confirm the clinical value of oocyte cryopreservation in infertile patients.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression [1–3]. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC) [4]. The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Human immunodeficiency virus type 1 F12 (HIV-1 F12 ) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55 Gag precursor in the presence of Nef from HIV-1 F12 . Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1 NL4-3 also created a dominant-negative Nef protein. Effects of Nef from HIV-1 F12 on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.

We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag , vif , or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat , rev , or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr , pol , or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif -permissive and vif -nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif -expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.

The expression of the nonproducer F12-HIV-1 genome has been previously shown to protect the host cell from HIV superinfection. In order to estimate the efficacy of the F12-HIV genome as an anti-HIV reagent also in cells already infected, an HIV-1 chronically infected Hut-78 cell clone (D10) was superinfected with an amphotropic mouse/human pseudotype retrovirus whose genome expresses both the F12-HIV genome and the selection marker gene (i.e. the c-DNA of a truncated form of the nerve growth factor receptor, NGFr) under the control of F12-HIV 5'LTR. D10 cells homogenously expressing the F12-HIV genome (T-D10) released unaltered amounts of retrovirions whose infectivity was, however, dramatically impaired (from 9 x 10(3) in D10 to < 10(0.5). TCID50/ml in T-D10 supernatants). Electron microscopy showed that the morphology of retrovirions released by T-D10 cells was heavily altered, both in size and shape. Furthermore, no retrotranscription products were detectable in CD4 cells challenged with T-D10 retrovirions. For the first time, the block in the infectivity of HIV released from already infected cells through the expression of an anti-HIV retroviral vector was demonstrated. These data could have important implications both from a perspective of F12-HIV-based anti-HIV gene therapy and, in general, on the role that nonproducer and/or defective HIV could play 'in vivo' in HIV infection and AIDS pathogenesis.

A cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp 120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.

An Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus-1 (HIV) variant, and showing a complete resistance to HIV-1 or HIV-2 superinfection, was previously characterized. We demonstrated that the replication of the superinfecting HIVs is blocked at the retrotranscription step, despite the CD4 down-regulation, since HIVs are able to cross the Hut-78/F12 cell membrane. In order to establish if the expression of the HIV-1 variant (F12/HIV) could be per se sufficient to induce the homologous viral interference shown in the F12 cells, the whole F12/HIV provirus was cloned and transfected in He-La CD4+ cells. In F12/HIV expressing He-La CD4+ clones, both the viral proteins expressed and the HIV nonproducer phenotype remain unmodified compared to F12 cells. Furthermore, despite the full expression of CD4 HIV receptors, the life cycle of the superinfecting HIV could be either strongly inhibited or totally abolished, depending on the cell clone considered. The inhibition of the superinfecting HIV was also reproduced when an HIV infectious molecular clone was transfected in F12/HIV He-La CD4+ clones, thus indicating that a post-cDNA synthesis block may operate against the superinfecting HIV. These data demonstrate that HIV susceptibility could be abrogated in cells expressing the F12/HIV genome, even in absence of any CD4 down-regulation.

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.

Background The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. Results Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 μg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. Conclusion We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.

A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G140E, V153L and E177G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4+ cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G177E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E140G and L153V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.

F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef -defective (Δ nef ) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in- trans expression of wt nef . Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in- cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.

A Hut-78 celi clone (F12) harboring a non-producer human immunodeficiency virus (HIV-1) variant shows complete resistance to HIV-1 or HIV-2 superinfection. The F12-HIV provirus produces an altered HIV-1 protein pattern and cannot generate even immature viral particles. We demonstrated that HeLa CD4+ cells transfected with the F12-HIV genome resist HIV superinfection through a CD4-independent mechanism. As F12-HIV appears to be a useful system to induce anti-HIV intracellular immunization, we constructed various retroviral vectors containing the F12-HIV genome, modified by elimination of the F12 3’ LTR and part of its nef gene, inserted “antisense” with respect to the Moloney murine leukemia virus 5’ LTR. Here we show that recombinant retroviral particles carrying the N2/F12-HIV nef-(as) construct can stably transduce both CEMss human cells and primary human peripheral blood lymphocytes, inducing the expression of the F12-HIV genome. These results could open the way to an anti-AIDS gene therapy strategy based on F12-HIV-induced intracellular immunization.

Recent findings have shown that the expression of the seven trans-membrane G-protein–coupled CXCR4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4 molecule. In the human system, the role of CXCR4 in HIV infection has been determined through env-mediated cell fusion assays and confirmed by blocking viral entry in CD4+/CXCR4+ cells by SDF-1 pretreatment. We observed that the human megakaryoblastic CD4+ UT-7 cell line fails to express CXCR4 RNA and is fully resistant to HIV entry. Transfection of an expression vector containing the CXCR4 c-DNA rendered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named UT-7/fus) induces the formation of polynucleated cells through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2–infected UT-7/fus cells. These findings further strengthen the role of CXCR4 as a molecule necessary for the replication of T-lymphotropic HIV-1 and HIV-2 isolates and provide a useful model to study the functional role of CD4 coreceptors in HIV infection.