Raúl Pérez Bianco

Analysis of factor VIII gene intron 1 inversion in Argentinian families with severe haemophilia A and a review of the literature.

Besides intron 22 factor VIII gene inversion (Inv22), intron 1 inversion (Inv1) has recently been reported as a further recurrent mutation that causes approximately 5% of severe haemophilia A (HA) cases. We analysed the presence of the Inv1 in a group of 64 severe HA-affected families from Argentina, and found only one positive case. This Inv1 patient has not developed a factor VIII inhibitor, and the screening for small mutations in the coding sequences of the factor VIII gene did not detect any additional defect in this case. The Inv1 genotyping was further applied to analyse the haemophilia carrier status of the probands sister. In addition, we studied the accuracy of the current polymerase chain reaction-based method to investigate the Inv1, and confirmed the absence of amplimer length polymorphisms associated to the Inv1-specific polymerase chain reaction amplifications in 101 X-chromosome haplotypes from unrelated Argentinian healthy males. In order to discuss Inv1 mutation frequency in severe HA and the risk of inhibitor formation, a review of the literature was included. Our data highlight the importance of analysis of the Inv1 in Inv22-negative severe HA cases. This will benefit both genetic counselling and the study of the relationship between genotype and inhibitor development.

Evidence of occult HCV genotypes in haemophilic individuals with unapparent HCV mixed infections

Summary.  Individuals with haemophilia who received non heat‐treated factor concentrates were likely to undergo multiple exposures to the hepatitis C virus (HCV). Therefore, HCV mixed‐genotype infections might be more frequent in these patients than in the general population. Their prevalence is extremely variable in similar groups of patients tested by different assays due to the fact that currently available genotyping techniques are not suitable to detect multiple HCV genotypes in a viral population. As an HCV viral reservoir, the peripheral blood mononuclear cell (PBMC) might harbor viral variants distinct from the genotypes detected in plasma. We investigated the presence of HCV genotypes in a group of chronically infected haemophilic patients in the PBMC compartment using a non‐stimulated cell culture system that allows the detection of the HCV genome in culture supernatants. We compared them to the HCV genotypes found in plasma samples. Cell culture experiments performed with PBMC demonstrated the presence of additional HCV genotypes that were undetected in the corresponding plasma samples with the same genotyping technique. Although mixed infections at HCV genotype level became evident in 5.6% of the patients (16/288), the culture methodology increased the number of HCV infections with multiple genotypes to 62.5% (10/16) (P < 0.0001). Once more, the role of mononuclear cells as HCV viral reservoirs is emphasized. Considering minor strains could influence the outcome of treatment, detection of covert HCV mixed‐genotype infections might be essential for choosing the adequate therapeutic regimen.

Structural aberrations of chromosomes 17 and 12 in chronic B-cell disorders.

Abstract: Objectives: Genomic aberrations can now be identified in approximately 80% of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients. In the present study, four new structural changes involving chromosomes 17 and 12 in CLL/SLL patients are described.

Decreased recovery of replication-competent HIV-1 from peripheral blood mononuclear cell-derived monocyte/macrophages of HIV-positive patients after 3 years on highly active antiretroviral therapy.

We studied the release of p24 antigen from peripheral blood mononuclear cell-derived monocyte/macrophages obtained from 13 HIV-positive patients receiving highly active antiretroviral therapy (HAART). Although HIV-infected monocyte/macrophages were detected in 80% of patients after 36 months of continuous treatment, additional exposure to HAART reduced the chance of detecting HIV-releasing monocyte/macrophages. Therefore, after more than 3 years of HAART, recently infected monocytes may play a less important role as a source of emerging HIV-1 upon HAART interruption.

Inhibition of normal natural killer cytotoxicity by sera from hemophilic patients

In this study we searched for circulating antibodies or other serum factors that could account for the natural killer (NK) defect observed in hemophiliacs (He) infected with the human immunodeficiency virus (HIV). We analyzed the effect of negative or positive sera for HIV from He on normal NK activity. We showed that sera from He interfered with normal NK cytotoxicity. The inhibitory activity was higher in HIV+ sera and increased as the HIV disease progressed. HIV- sera also inhibited NK function, although to a lesser extent than HIV+, and it was probably due to isoimmunization through replacement treatment with plasma-derived concentrates. For each individual, no direct correlation was found between NK inhibition (NK-INH) of sera and the NK activity of He peripheral blood mononuclear cells (PBMC). Furthermore, He serum was poorly inhibitory on autologous PBMC. Preincubation of allogenic effector or target cells with He sera revealed that the inhibitory effect was the result of the reaction with these cells. A positive correlation was found by comparing NK-INH of whole He sera with the serum levels of circulating immune complexes. When the NK-INH assay was performed using the same concentration of DEAE-purified IgG from N, HIV- or HIV+, we found that HIV+ AIDS IgG was more inhibitory than the others.

HIV infection and natural killer cytotoxicity in hemophilic patients

In this study we analyzed the ability of peripheral blood mononuclear cells (PBMC) from hemophilic patients (He) with negative or positive serology for the human immunodeficiency virus (HIV), to increase natural killer (NK) cytotoxicity upon stimulation with physiological and non physiological agents. Purified interleukin-2 (IL-2), the interferon (IFN)-inducer polyinosinic polycytidylic acid (PIC), recombinant alpha- and gamma-IFN and the protein kinase activator phorbol myristate acetate (PMA) were used as stimulatory agents. The NK functional response was correlated with the presence of PBMC bearing phenotypic markers of activated cells (IL-2 receptor, IL-2R) and of different NK cell maturation stages. Our results demonstrate that NK effector cells with slight lytic activity (Leu 7+ CD16-) predominated in HIV+ He patients. On the other hand the occurrence of IL-2R positive cells was similarly high in both HIV+ and HIV- individuals and was probably more related to chronic replacement treatment with Factor VIII or Factor IX concentrates than to HIV infection. The ability to respond to physiological NK regulators such as IL-2 and IFNs, or to the IFN-inducer PIC was impaired in HIV+ He, especially in HIV+ LAS individuals, suggesting that the inability of these cells to increase NK cell activity after appropriate induction was due to an intrinsic defect. Since phosphoinositide turnover and subsequent protein kinase C activation are thought to be part of the physiological mechanism of NK cytotoxicity, we studied the effect of PMA on PBMC from each group of patients. The ability to respond to PMA was lost only in PBMC from HIV+ LAS patients, indicating that impairment of the NK lytic mechanism progresses as the disease gets worse.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe haemophilia A patients have reduced numbers of peripheral memory B cells.

Summary.  The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII‐specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti‐FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti‐FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non‐switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti‐FVIII response and to monitor the success of ITI.

Mixed Genotypes in Hepatitis C Virus Infection

Before the existence of commercial clotting factor concentrates, bleeding was the number one cause of death in persons with hemophilia and the only alternative treatment was cryoprecipitate. During the 1970s human freeze-dried (lyophilized) FVIII and FIX became available. The life of individuals with hemophilia was revolutionized because patients were able to treat themselves conveniently at home, as soon as spontaneous bleeds occurred. The commercial blood-derived products had a tremendous positive impact on physical, psychological and social lives. Unfortunately, they also carried an increased risk of bloodborne viral infections, largely due to their preparation from pools of plasma collected from thousands of donors. Consequently, the use of clotting factor concentrates resulted in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) epidemics in this population (Lee C, 1995, 2009; Eyster, 2008; Ragni et al., 2010). As reported in different cohorts around the world, many patients with hemophilia became infected with HIV between 1982 and 1985. In some hemophilic populations, subsequent testing of stored frozen plasma samples revealed that the first infections with HIV occurred in 1978-79, that the bulk of patients were infected in 1981-82, and that there were very few new infections by the end of 1984 (Eyster, 2008; Goedert et al., 1985). The HCV epidemic was a much longer one, occurring between 1961 and 1985. The first patients became infected from the first large pool plasma-derived FIX concentrates and the epidemic ended with the dry heating of concentrates in 1985 (Lee C, 2009). Particularly in Argentina, commercial factor concentrates were not accessible until 1975. As a result of the economic situation of the country, the non-availability of more expensive products leaded to a lower rate of HIV-infected people that reached 17% of our hemophilic population. Heat-inactivated factor concentrates were not available until November 1985. Virtually, all hemophiliacs who received clotting factor concentrates prior to implementation of viral inactivation techniques became infected with hepatitis C virus at the time of the first infusion (Morfini et al, 1994; Lee C et al, 2002; Ragni et al, 2010). Prevalence rates of HCV infection up to 100% were reported in hemophilia patients treated with concentrates before 1985 (Yee et al., 2000; Lee C, 2009; Manucci, 2008; Arnold et al., 2006). Even though the introduction of heat-treated factor concentrates progressively decreased HCV transmission, the true risk ended when new regulations in blood donor screening together with the implementation of second and third generation immunoassays for the detection of antibodies against HCV was introduced in 1991 in Europe, in 1992 in the US and 1993 in