IgG subclasses of FVIII inhibitors in an argentine cohort of severe hemophilia A patients: Analysis by flow cytometry.

INTRODUCTION: FVIII inhibitors consist of a polyclonal population of antibodies. Previous studies have demonstrated different distribution of IgG subclasses. IgG4 was associated to high level of FVIII inhibitors and failure of immune tolerance induction (ITI) treatment. This study monitored the relative distribution of IgG subclasses of anti-FVIII in patients with severe hemophilia A (SHA). METHODS: Anti-FVIII antibodies were measured employing an immunomethod, developed in our laboratory, that combines flow cytometry (FC) with microspheres coupled (FVIII-m) or not (Control-m) to FVIII. Seventy-five patients with SHA were studied, 17 without inhibitors (Group I); 58 with inhibitor history, 13 low responders: (LR: Group II), and 45 high responders (HR: Group III). Eight patients undergoing ITI were also included. RESULTS: We found anti-FVIII antibodies in 11 of 27 patients (40%) without inhibitors and in 45 of 48 with inhibitors at the moment of the study. IgG4 was predominant only in the Group III: P=0.02 in patients with low level of inhibitors and P=0.0001 with high titer of inhibitors. Longitudinal analysis performed on patients undergoing ITI showed a gradual decrease of IgG4 values that was associated to improvement of clinical parameters during treatment. CONCLUSION: We suggest the use of the FC method to supplement functional traditional assays and to help to improve the management of patients with SHA.

Hepatitis C virus long-term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1.

Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus-infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV-monoinfected and 25 HIV/HCV-coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010-2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed-genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38-15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC-associated variants persisted for 10 years in some subjects, emphasizing their role as long-term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV-infected patients.

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

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.

A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay.

In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme-linked immunosorbent assay (ELISA) that detects both inhibitory (I-Ab) and non-inhibitory (NI-Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I-Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII-m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control-m). Captured anti-FVIII antibodies were detected using anti-human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I-Ab, <0.5 BU mL(-1)); 13 PI (patients with I-Ab, 1.1-8200 BU mL(-1)). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII-m and control-m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI-Ab by FC, and two of them developed high levels of I-Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 µL of plasma or serum is required especially making it useful for paediatric patients.

Anti-polymorphonuclear neutrophil antibodies in patients with leukopenia or neutropenia.

INTRODUCTION: Immune humoral neutropenia (Np) could be the consequence of anti-polymorphonuclear neutrophil (PMN) antibodies, circulating immune complexes (CIC) and/or antibodies against myeloid precursors. Granulocyte immunofluorescence test (GIFT) and a leukoagglutination technique (LAGT) assays are recommended for its diagnosis. METHODS: Fifty adult patients with secondary Np were screened for anti-PMN. GIFT by flow cytometry from viable PMN and LAGT were employed. In addition, CIC levels, low expression of CD16(b) (CD16 (b)(low)), PMN phenotype and sera tumor necrosis factor-alpha (TNF-alpha) were also evaluated. RESULTS: Direct IgG-PMN binding (dir-GIFT) was positive in 16% of the patients. Antibodies against autologous PMN were detected in 32% of the samples by indirect (ind)-GIFT and demonstrated in 70% of the sera by both ind-GIFT and/or LAGT. Predominance of human neutrophil alloantigen (HNA)-1b and HNA-2 expression was confirmed. CD16(b)(low) was detected in 16% of the patient's PMN and TNF-alpha in 68% of sera patients. CONCLUSION: Our results suggest that diagnosis of immune Np in the laboratory may be improved by focusing on patient's PMN together with the assessment of cellular markers.

HCV recovery from peripheral blood mononuclear cell culture supernatants derived from HCV-HIV co-infected haemophilic patients with undetectable HCV viraemia.

Hepatitis C viraemia, in 38 human immunodeficiency virus positive (HIV+)/hepatitis C virus positive (HCV+) patients, was determined in haemophilic patients during the 4 years since initiation of highly active antiretroviral therapy (HAART). Six of 38 patients had persistently HCV-negative viraemia for more than 2 years. No correlation between HCV-negative viraemia and CD4+ T-cell counts, HIV viral load, age, type or severity of haemophilia could be established. Reduced levels of HIV viral load and the immune reconstitution that follows the initiation of HAART were not enough to explain the disappearance of HCV from plasma. Individuals who cleared plasma HCV had significantly higher CD8+ T-cell counts (P=0.0013) (mean +/- SE: 1153 +/- 117.8 cells microL(-1)) than those with HCV-positive viraemia (819.1 +/- 40.72 cells microL(-1)). Because HCV could maintain a low replication level in peripheral blood mononuclear cells (PBMC), we cultured PBMC of five of six patients with undetectable HCV viraemia. We found four of five HCV RNA-positive cultures. The presence of HCV RNA in our cultures proved that these cells may be an important viral reservoir that could contribute to HCV recurrence in plasma even after long periods of negative viraemia. In summary, our results indicate that in spite of prolonged HCV-negative plasma viraemia, HCV patients that are co-infected with HIV may harbour replication-competent HCV in their PBMC. Therefore, true clearance of HCV infection is difficult to achieve in these patients.

Reservoirs of HIV replication after successful combined antiretroviral treatment.

Sustained reduction of viral replication can be achieved in HIV infected patients after treatment with combinations of drugs (HAART) that inhibit the viral reverse transcriptase, and protease enzymes. However, replication competent virus can still be recovered from latently infected resting memory CD4+ T-cell lymphocytes. Moreover, "covert" virus replication has been demonstrated in patients who experienced reductions in plasma viremia to levels below the limit of detection of the most sensitive PCR assays. In most studies, preferential attention has been given to latent resting CD4+ T-lymphocytes as a source of HIV persistence. However, insufficient suppression of HIV replication could also lead to viral re-emergence after HAART interruption. In addition to CD4+ T- lymphocytes, other host cells such as long-lived resident macrophages or recently infected blood monocytes could also contribute to maintain persistent HIV replication after HAART. Establishing the origin of re-emerging HIV in patients under HAART upon treatment interruption is important to design optimal treatment schemes. Therapeutic strategies aimed at reducing the number of latently infected cells involve immune activation with IL-2, or other stimulatory factors, in the presence of antiretroviral drugs. Elimination of replication-competent virus would require intensification of HAART, or the use of antiretroviral drugs achieving an effective concentration at the site of HIV replication. In this review the mechanisms of HIV persistence and the methods that can be used to distinguish latent from covert HIV replication in different cell types will be discussed.

Immortalized Epstein-Barr virus-positive B-cell lines obtained by prolonged culture of peripheral blood mononuclear cells from human immunodeficiency virus type 1-positive patients.

OBJECTIVES: To study the factors that determine malignant B cell growth in human immunodeficiency virus type 1 (HIV-1)-infected patients. STUDY DESIGN: B-cell lines (lymphocyte cell lines [LCL]) were developed after nonstimulated culture of peripheral blood mononuclear cells (PBMC) from HIV-1-positive (HIV-1(+)) patients. Human immunodeficiency virus type 1 replication in culture, Epstein-Barr virus (EBV) latent oncogene expression, and cell-to-cell interaction were studied after nonstimulated culture of HIV-1(+) PBMC, analyzing their contribution to LCL appearance. METHODS: Nonstimulated PBMC cultures of HIV-1(+) PBMC and controls (N-PBMC) were established. Lymphocyte cell lines were characterized. Epstein-Barr virus latent membrane protein 1 (LMP-1) and Epstein-Barr nuclear antigen 2 were detected by polymerase chain reaction (PCR). Clonality of LCL was determined by light chain restriction (flow cytometry) and immunoglobulin H chain rearrangement (semi-nested PCR). Peripheral blood mononuclear cell phenotypes were studied at different intervals of culture. RESULTS: Lymphocyte cell lines were obtained in 73% of HIV-1(+) PBMC cultures, compared with 6% in N-PBMC. All LCL were EBV-positive (EBV(+)). B-cell lineage was established, and up to 12 different B-cell clones were expanded from the same individual. Occurrence of LCL was more frequent in cultures with HIV-1 replication, high LMP-1 expression in viable B cells, and high CD4:CD8 ratio. Human immunodeficiency virus type 1 replication persisted in 53% of the LCL. CONCLUSIONS: In vitro HIV-1 replication and persistence of viable EBV(+) lymphoblasts favor spontaneous in vitro outgrowth of LCL in HIV-1(+) patients.

Monocyte differentiation and HIV replication after prolonged culture of peripheral blood mononuclear cells from HIV-infected individuals.

Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.

[Human immunodeficiency virus (HIV) co-receptors expression in mononuclear leucocytes (ML) of HIV+ patients]. / Expresion de co-receptores para el virus de la inmunodeficiencia humana (VIH) luego del cultivo de leucocitos mononucleares (LM) de pacientes VIH+.

Variations of the expression of CXCR4 and CCR5 HIV co-receptors after non stimulated culture of peripheral blood mononuclear cells (PBMC) from HIV+ patients were studied. Expression of CCR5 on both CD4+ and CD8+ T lymphocytes was reduced after 7 days and remained low throughout the culture. CXCR4 levels remained stable in both lymphocyte subpopulations. No significant changes were observed in control HIV- PBMC cultures. In order to ascertain if the CCR5 changes were associated to in vitro HIV replication, 6 days pre-cultured HIV- PBMC were infected with HIV+ culture supernatants. After 3 days CCR5 expression was reduced both in CD4+ and in CD8+ T lymphocytes, while CXCR4 expression was not, coincident with initiation of HIV replication in culture. These results suggest that CCR5 down modulation in CD4+ or CD8+ T lymphocytes is a consequence of HIV replication.

Nonhepatosplenic gamma delta T-cell lymphoma with initial testicular compromise.

We report here a case of nonhepatosplenic gammadelta T-cell lymphoma with undescribed initial localization in testis, without hepatosplenomegaly or adenopathies, and subsequent development in the maxillary sinus. The maxillar mass biopsy revealed a T-cell infiltration, and its immunologic characterization by flow cytometry showed a gammadelta T-cell phenotype (CD45+, CD3+, CD2+, TCR gammadelta+), without expression of CD7, CD5, CD1a, TdT, CD4, CD8, TCR alphabeta, or NK antigens (CD16, CD56, and CD57). Clonal gamma-chain gene rearrangement by polymerase chain reaction (PCR) was detected in testicular and maxillar biopsies. Epstein-Barr virus type 1 (EBV) sequences were detected by molecular biology in the biopsy material, suggesting that this oncogenic virus may play a role in the genesis of the clonal expansion of gammadelta T-cells. The patient was initially treated with standard chemotherapeutic protocols, with poor response and aggressive course.

[Apoptosis and human immunodeficiency virus infection (HIV]. / Apoptosis e infección por el virus de la inmunodeficiencia humana (HIV).

Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.

Apoptosis e infección por el virus de la inmunodeficiencia humana (HIV) / [Apoptosis and human immunodeficiency virus infection (HIV]

Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.

Protein kinase C regulates NO-cGMP pathway in muscarinic receptor activation by HIV+-IgA.

In this work we demostrate that IgA purified from HIV infected patients recognizes a band with a molecular weight corresponding to radiolabelled ileal muscarinic acethylcholine receptors (mAChR) by immunoblotting. HIV+-IgA triggers the signals that are the consequence of mAChR stimulation in the intestine, regulating protein kinase C (PKC) and nitric oxide synthase (NOS) activity as well as 3',5'-cyclic guanosine monophosphate (cGMP) formation. On the one hand PKC activation by HIV+-IgA induces NOS inhibition and as a consequence, low amounts of NO that could improve local immunosuppression in the intestine; on the other hand HIV+-IgA stimulates cGMP production which could potentiate ileal motility and loss of water/electrolytes involved in intestinal damage in AIDS.

Tyrosine kinase regulatory action on ileal muscarinic effects of IFN-gamma.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has a large number of immunologic and nonimmunologic functions. We have described that IFN-gamma could activate muscarinic cholinergic receptors (mAchR) of rat intestine, stimulating ileal motility. We also observed that mAchR activation induced inhibition of cAMP levels and stimulation of cGMP formation. The objectives of our work were to clarify the signal transduction pathways involved in regulation of ileal motility through mAchR activation by IFN-gamma. Our results demonstrate that this cytokine produces an ileal cholinergic response through tyrosine kinase activity. The activation of tyrosine kinase mediates ileal contractility, phosphoinositide hydrolysis by phospholipase C, nitric oxide synthase via protein kinase C, and cGMP synthesis. The increment in ileal motility is probably due to hyperproduction of prostaglandin E2 (PGE2) by ileal tissue. This prostanoid is an important mediator because it stimulates ileal motility. We conclude that IFN-gamma not only immunomodulates the gut microenvironment but also exerts a local nonimmunologic regulation on intestinal motility.

CD8 expression in a case of chronic lymphocytic leukemia with trisomy 12.

We report a case of B-cell chronic lymphocytic leukemia (B-CLL) with aberrant expression of the T-cell-associated antigen CD8, as revealed by two-color flow-cytometric analysis. DNA studies showed immunoglobulin heavy-chain gene rearrangement, but not of gamma-chain T-cell receptor, confirming the B-cell origin of the neoplastic cells. Ploidy analysis showed a tetraploid population and high S-phase fraction. B-CLL cells also carried trisomy 12, detected by fluorescence in situ hybridization. The identification of more cases with the same features would be necessary to establish the prognosis of this subtype of B-CLL.

IgA from HIV+ haemophilic patients triggers intracellular signals coupled to the cholinergic system of the intestine.

IgA was obtained from HIV-infected haemophilic patients and the intracellular signals triggered by its reaction with isolated rat intestinal strips were studied. HIV+ IgA stained intestinal microvilli with a granular immunofluorescence pattern and bound to the muscarinic acetylcholine receptor (mAChR), displacing the specific muscarinic cholinergic antagonist QNB in a non-competitive manner. It triggered the signals that are the consequence of mAChR stimulation in the intestine. Thus, it decreased cAMP synthesis and increased guanosine 3':5'-cyclic monophosphate (cGMP) formation and phosphoinositide (PI) turnover of the intestine. In addition, it stimulated prostaglandin E2(PGE2) synthesis by intestinal strips. Through its effect on PGE2 synthesis, HIV+ IgA could have a dual action. On the one hand, it could enhance immunosuppression at a local level, favouring pathogen growth and subsequent intestinal dysfunction. On the other hand, PGE2 could directly increase intestinal motility and electrolyte/fluid loss. Both effects could be involved in intestinal damage in AIDS.