Costruzione di chimere molecolari dell'enzima Integrasi di HIV con attività idrolizzante delle LTR virali.

La Sindrome da Immunodeficienza Acquisita (AIDS o SIDA) causata da HIV-1 (Virus dell'Immunodeficienza umana) è caratterizzata dalla graduale compromissione del sistema immunitario del soggetto colpito. Le attuali terapie farmacologiche, purtroppo, non riescono a eliminare l'infezione a causa della comparsa di continui ceppi resistenti ai farmaci, e inoltre questi trattamenti non sono in grado di eliminare i reservoir virali latenti e permettere l'eradicazione definitiva del virus dall’organismo. E' in questo ambito che si colloca il progetto a cui ho lavorato principalmente in questi anni, cioè la creazione di una strategia per eradicare il provirus di HIV integrato nel genoma della cellula ospite. L'Integrasi di HIV-1 è un enzima che media l'integrazione del cDNA virale nel genoma della cellula ospite. La nostra idea è stata, quindi, quella di associare all'attività di legame dell'IN stessa, un'attività catalitica. A tal fine abbiamo creato una proteina chimerica costituita da un dominio DNA-binding, dato dall'Integrasi, e da un dominio con attività nucleasica fornito dall'enzima FokI. La chimera ottenuta è stata sottoposta a mutagenesi random mediante UV, ed è stata oggetto di selezione in vivo, al fine di ottenere una chimera capace di riconoscere, specificamente le LTR di HIV-1, e idrolizzare i siti di inserzione. Questo lavoro porterà a definire pertanto se l'IN di HIV può essere riprogrammata a catalizzare una nuova funzione mediante la sostituzione dell'attività del proprio dominio catalitico con quello di FokI.

Multiple pathogen detection using nanoplasmonic sensing

Opportunistic diseases caused by Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) is an omnipresent global challenge. In order to manage these epidemics, we need to have low cost and easily deployable platforms at the point-of-care in high congestions regions like airports and public transit systems. In this dissertation we present our findings in using Localized Surface Plasmon Resonance (LSPR)-based detection of pathogens and other clinically relevant applications using microfluidic platforms at the point-of-care setting in resource constrained environment. The work presented here adopts the novel technique of LSPR to multiplex a lab-on-a-chip device capable of quantitatively detecting various types of intact viruses and its various subtypes, based on the principle of a change in wavelength occurring when metal nano-particle surface is modified with a specific surface chemistry allowing the binding of a desired pathogen to a specific antibody. We demonstrate the ability to detect and quantify subtype A, B, C, D, E, G and panel HIV with a specificity of down to 100 copies/mL using both whole blood sample and HIV-patient blood sample discarded from clinics. These results were compared against the gold standard Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). This microfluidic device has a total evaluation time for the assays of about 70 minutes, where 60 minutes is needed for the capture and 10 minutes for data acquisition and processing. This LOC platform eliminates the need for any sample preparation before processing. This platform is highly multiplexable as the same surface chemistry can be adapted to capture and detect several other pathogens like dengue virus, E. coli, M. Tuberculosis, etc.

Immune reconstitution inflammatory syndrome in patients starting antiretroviral therapy for HIV infection: a systematic review and meta-analysis

In patients with HIV-1 infection who are starting combination antiretroviral therapy (ART), the incidence of immune reconstitution inflammatory syndrome (IRIS) is not well defined. We did a meta-analysis to establish the incidence and lethality of the syndrome in patients with a range of previously diagnosed opportunistic infections, and examined the relation between occurrence and the degree of immunodeficiency. Systematic review identified 54 cohort studies of 13 103 patients starting ART, of whom 1699 developed IRIS. We calculated pooled cumulative incidences with 95% credibility intervals (CrI) by Bayesian methods and did a random-effects metaregression to analyse the relation between CD4 cell count and incidence of IRIS. In patients with previously diagnosed AIDS-defining illnesses, IRIS developed in 37.7% (95% CrI 26.6-49.4) of those with cytomegalovirus retinitis, 19.5% (6.7-44.8) of those with cryptococcal meningitis, 15.7% (9.7-24.5) of those with tuberculosis, 16.7% (2.3-50.7) of those with progressive multifocal leukoencephalopathy, and 6.4% (1.2-24.7) of those with Kaposi's sarcoma, and 12.2% (6.8-19.6) of those with herpes zoster. 16.1% (11.1-22.9) of unselected patients starting ART developed any type of IRIS. 4.5% (2.1-8.6) of patients with any type of IRIS died, 3.2% (0.7-9.2) of those with tuberculosis-associated IRIS died, and 20.8% (5.0-52.7) of those with cryptococcal meningitis died. Metaregression analyses showed that the risk of IRIS is associated with CD4 cell count at the start of ART, with a high risk in patients with fewer than 50 cells per microL. Occurrence of IRIS might therefore be reduced by initiation of ART before immunodeficiency becomes advanced.

Growth response to antiretroviral treatment in HIV-infected children: a cohort study from Lilongwe, Malawi

Objective  Malnutrition is common in HIV-infected children in Africa and an indication for antiretroviral treatment (ART). We examined anthropometric status and response to ART in children treated at a large public-sector clinic in Malawi. Methods  All children aged <15 years who started ART between January 2001 and December 2006 were included and followed until March 2008. Weight and height were measured at regular intervals from 1 year before to 2 years after the start of ART. Sex- and age-standardized z-scores were calculated for weight-for-age (WAZ) and height-for-age (HAZ). Predictors of growth were identified in multivariable mixed-effect models. Results  A total of 497 children started ART and were followed for 972 person-years. Median age (interquartile range; IQR) was 8 years (4–11 years). Most children were underweight (52% of children), stunted (69%), in advanced clinical stages (94% in WHO stages 3 or 4) and had severe immunodeficiency (77%). After starting ART, median (IQR) WAZ and HAZ increased from −2.1 (−2.7 to −1.3) and −2.6 (−3.6 to −1.8) to −1.4 (−2.1 to −0.8) and −1.8 (−2.4 to −1.1) at 24 months, respectively (P < 0.001). In multivariable models, baseline WAZ and HAZ scores were the most important determinants of growth trajectories on ART. Conclusions  Despite a sustained growth response to ART among children remaining on therapy, normal values were not reached. Interventions leading to earlier HIV diagnosis and initiation of treatment could improve growth response.

The complex clinical picture of side effects to biologicals

Biologicals are proteins used as drugs. Biologicals target clearly defined molecular structures, being part of established pathogenetic pathways. Therefore, their focused mode of action seems to render them superior to classic small molecular drugs regarding "off-target" adverse drug reactions (ADR). Nevertheless, the increasing use of biologicals for the treatment of different diseases has revealed partially unexpected adverse reactions. The often direct interaction of a biological with the immune system provides a clue to most side effects, which have consequently been subclassified, based on pathogenetic principles, into 5 subtypes named alpha, beta, gamma, delta, and epsilon, reflecting overstimulation (high cytokine values, type alpha), hypersensitivity (type beta), immune deviation (including immunodeficiency, type gamma), cross-reactivity (type delta), and nonimmune mediated side effects (type epsilon). This article presents typical clinical manifestations of these subtypes of ADR to biologicals, proposes general rules for treating them, and provides a scheme for a thorough allergological workup. This approach should help in future handling of these often very efficient drugs.

Frequency and determinants of unprotected sex among HIV-infected persons: the Swiss HIV cohort study

Access to antiretroviral therapy may have changed condom use behavior. In January 2008, recommendations on condom use for human immunodeficiency virus (HIV)-positive persons were published in Switzerland, which allowed for unprotected sex under well-defined circumstances ("Swiss statement"). We studied the frequency, changes over time, and determinants of unprotected sex among HIV-positive persons.

Is it safe to discontinue primary Pneumocystis jiroveci pneumonia prophylaxis in patients with virologically suppressed HIV infection and a CD4 cell count <200 cells/microL?

Current guidelines suggest that primary prophylaxis for Pneumocystis jiroveci pneumonia (PcP) can be safely stopped in human immunodeficiency virus (HIV)-infected patients who are receiving combined antiretroviral therapy (cART) and who have a CD4 cell count >200 cells/microL. There are few data regarding the incidence of PcP or safety of stopping prophylaxis in virologically suppressed patients with CD4 cell counts of 101-200 cells/microL.

Molecular epidemiology reveals long-term changes in HIV type 1 subtype B transmission in Switzerland

Sequence data from resistance testing offer unique opportunities to characterize the structure of human immunodeficiency virus (HIV) infection epidemics.

Incidence and risk factors for chronic elevation of alanine aminotransferase levels in HIV-infected persons without hepatitis b or c virus co-infection

Chronic liver disease in human immunodeficiency virus (HIV)-infected patients is mostly caused by hepatitis virus co-infection. Other reasons for chronic alanine aminotransferase (ALT) elevation are more difficult to diagnose.

Xenotransplantation of cryopreserved ovarian tissue from patients with ovarian tumors into SCID mice--no evidence of malignant cell contamination

This study examined the possible presence of malignant cells in ovarian cortex from patients with ovarian tumors after xenografting of the ovarian tissue into severe combined immunodeficiency mice. None of the mice presented symptoms of reintroduced malignancy nor did microscopic and immunohistochemical evaluation of the grafts raise any suspicion of residual malignant disease.

Tuberculosis in HIV programmes in lower-income countries: practices and risk factors

BACKGROUND: Tuberculosis (TB) is a common diagnosis in human immunodeficiency virus (HIV) infected patients on antiretroviral treatment (ART). OBJECTIVE: To describe TB-related practices in ART programmes in lower-income countries and identify risk factors for TB in the first year of ART. METHODS: Programme characteristics were assessed using standardised electronic questionnaire. Patient data from 2003 to 2008 were analysed and incidence rate ratios (IRRs) calculated using Poisson regression models. RESULTS: Fifteen ART programmes in 12 countries in Africa, South America and Asia were included. Chest X-ray, sputum microscopy and culture were available free of charge in respectively 13 (86.7%), 14 (93.3%) and eight (53.3%) programmes. Eight sites (53.3%) used directly observed treatment and five (33.3%) routinely administered isoniazid preventive treatment (IPT). A total of 19 413 patients aged ≥16 years contributed 13 227 person-years of follow-up; 1081 new TB events were diagnosed. Risk factors included CD4 cell count (>350 cells/μl vs. <25 cells/μl, adjusted IRR 0.46, 95%CI 0.33–0.64, P < 0.0001), sex (women vs. men, adjusted IRR 0.77, 95%CI 0.68–0.88, P = 0.0001) and use of IPT (IRR 0.24, 95%CI 0.19–0.31, P < 0.0001). CONCLUSIONS: Diagnostic capacity and practices vary widely across ART programmes. IPT prevented TB, but was used in few programmes. More efforts are needed to reduce the burden of TB in HIV co-infected patients in lower income countries.

Antiretroviral therapy for prevention of HIV transmission in HIV-discordant couples

Antiretroviral drugs have been shown to reduce risk of mother-to-child transmission of human immunodeficiency virus (HIV) and are also widely used for post-exposure prophylaxis for parenteral and sexual exposures. Observational data, ecological studies and models suggest that sexual transmission may be lower in couples in which one partner is infected with HIV and the other is not and the infected partner is on antiretroviral therapy (ART).

Antiretroviral therapy for prevention of HIV transmission in HIV-discordant couples

Antiretroviral drugs have been shown to reduce risk of mother-to-child transmission of human immunodeficiency virus (HIV) and are also widely used for post-exposure prophylaxis for parenteral and sexual exposures. Observational data, ecological studies and models suggest that sexual transmission may be lower in couples in which one partner is infected with HIV and the other is not and the infected partner is on antiretroviral therapy (ART).

Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut

Using a systems biology approach, we discovered and dissected a three-way interaction between the immune system, the intestinal epithelium and the microbiota. We found that, in the absence of B cells, or of IgA, and in the presence of the microbiota, the intestinal epithelium launches its own protective mechanisms, upregulating interferon-inducible immune response pathways and simultaneously repressing Gata4-related metabolic functions. This shift in intestinal function leads to lipid malabsorption and decreased deposition of body fat. Network analysis revealed the presence of two interconnected epithelial-cell gene networks, one governing lipid metabolism and another regulating immunity, that were inversely expressed. Gene expression patterns in gut biopsies from individuals with common variable immunodeficiency or with HIV infection and intestinal malabsorption were very similar to those of the B cell-deficient mice, providing a possible explanation for a longstanding enigmatic association between immunodeficiency and defective lipid absorption in humans.

Tight junctions in brain barriers during central nervous system inflammation

Homeostasis within the central nervous system (CNS) is a prerequisite to elicit proper neuronal function. The CNS is tightly sealed from the changeable milieu of the blood stream by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). Whereas the BBB is established by specialized endothelial cells of CNS microvessels, the BCSFB is formed by the epithelial cells of the choroid plexus. Both constitute physical barriers by a complex network of tight junctions (TJs) between adjacent cells. During many CNS inflammatory disorders, such as multiple sclerosis, human immunodeficiency virus infection, or Alzheimer's disease, production of pro-inflammatory cytokines, matrix metalloproteases, and reactive oxygen species are responsible for alterations of CNS barriers. Barrier dysfunction can contribute to neurological disorders in a passive way by vascular leakage of blood-borne molecules into the CNS and in an active way by guiding the migration of inflammatory cells into the CNS. Both ways may directly be linked to alterations in molecular composition, function, and dynamics of the TJ proteins. This review summarizes current knowledge on the cellular and molecular aspects of the functional and dysfunctional TJ complexes at the BBB and the BCSFB, with a particular emphasis on CNS inflammation and the role of reactive oxygen species.