Protocol of comparison of the effects of single plasma exchange and double filtration plasmapheresis on peripheral lymphocyte phenotypes in patients with Chronic Inflammatory Demyelinating Polyradiculoneuropathy: a monocentric prospective study with single-case experimental design.

BACKGROUND: Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP), a rare disorder affecting young adults, causes gradual weakness of the limbs, areflexia and impaired sensory function. New CIDP phenotypes without pathogenic antibodies but with modified cell profiles have been described. Treatments include corticotherapy, intravenous immunoglobulins, and plasmapheresis but the latter's action mechanisms remain unclear. Plasmapheresis supposedly removes toxic agents like antibodies from plasma but it is uncertain whether it has an immune-modulating effect. Also, the refining mechanisms of the two main plasmapheresis techniques-single plasma exchange and double filtration plasmapheresis (DFPP) - are different and unclear. This study aims to compare the evolution of peripheral lymphocyte profiles in patients with CIDP according to their treatment (single centrifugation plasmapheresis or DFPP) to better grasp the action mechanisms of both techniques. METHOD: In this proof-of-concept, monocentric, prospective, Single-Case Experimental Design study, 5 patients are evaluated by alternating their treatment type (single plasma exchange or DFPP) for 6 courses of treatment after randomization to their first treatment type. Each course of treatment lasts 2-4 weeks. For single plasma exchange, 60 ml/kg plasma will be removed from the patient and replaced with albumin solutes, with a centrifugation method to avoid the immunological reaction caused by the membrane used with the filtration method. For DFPP, 60 ml/kg plasma will be removed from the patient with a plasma separator membrane, then processed via a fractionator membrane to remove molecules of a greater size than albumin before returning it to the patient. This technique requires no substitution solutes, only 20 g of albumin to replace what would normally be lost during a session. The primary outcome is the difference between the two plasmapheresis techniques in the variation of the TH1/TH17 ratio over the period D0H0-D0H3 and D0H0-D7. Secondary outcomes include the variation in lymphocyte subpopulations at each session and between therapeutic plasmapheresis techniques, the clinical evolution, tolerance and cost of treatments. DISCUSSION: Understanding the action mechanisms of single plasma exchange and DFPP will help us to offer the right treatment to each patient with CIPD according to efficacy, tolerance and cost. TRIAL REGISTRATION: ClinicalTrials.gov under the no. NCT04742374 and date of registration 10 December 2020.

Two-drug vs. three-drug combinations for HIV-1: Do we have enough data to make the switch?

Three-drug combination antiretroviral therapy (ART) became available in 1996, dramatically improving the prognosis of people living with HIV. The clinical benefits of ART are due to the sustained viral load suppression and CD4 T cell gains. Major drawbacks of the first ART regimens were adverse events, and high pill burden, which led to the reduction of drug adherence resulting in frequent treatment discontinuations and the development of drug resistance. Due to increased viral potency of new antiretroviral drugs consideration of a two-drug combination therapy repositioning occurred in an effort to reduce adverse events, drug-drug interactions and cost, while maintaining a sustained antiviral effect. Various combinations of two-drug regimens have been studied, and non-inferiority compared to a three-drug regimen has been shown only for some of them. In addition, a two-drug combination regimen may not be suitable for every patient, especially those who are pregnant, those with tuberculosis or coexisting HBV infection. Furthermore no information has been generated concerning the secondary transmission of HIV from patients who have undetectable plasma viral load on two-drug regimens. Additional studies of two-drug combinations are also necessary to evaluate the debated existence of low viral replication in tissues and on immune activation. While there is no urgent need to routinely switch patients to two-drug combination therapy, due to the availability of drug combinations without significant toxicities, dual regimens represent a suitable option that deserve long-term evaluation before being introduced to clinical practice.

Immune activation in the course of HIV-1 infection: Causes, phenotypes and persistence under therapy.

Systemic immune activation is a striking consequence of HIV-1 infection. Even in virologically suppressed patients, some hyperactivity of the immune system and even of the endothelium and of the coagulation pathway may persist. Apart from immune deficiency, this chronic activation may contribute to various morbidities including atherothrombosis, neurocognitive disorders, liver steatosis and osteoporosis, which are currently main challenges. It is therefore of major importance to better understand the causes and the phenotypes of immune activation in the course of HIV-1 infection. In this review we will discuss the various causes of immune activation in HIV-1 infected organisms: the presence of the virus together with other microbes, eventually coming from the gut, CD4+ T cell lymphopenia, senescence and dysregulation of the immune system, and/or genetic factors. We will also describe the activation of the immune system: CD4+ and CD8+ T cells, B cells, NKT and NK cells, dendritic cells, monocytes and macrophages, and neutrophils of the inflammation cascade, as well as of the endothelium and the coagulation system. Finally, we will see that antiretroviral therapy reduces the hyperactivity of the immune and coagulation systems and the endothelial dysfunction, but often does not abolish it. A better knowledge of this phenomenon might help us to identify biomarkers predictive of non AIDS-linked comorbidities, and to define new strategies aiming at preventing their emergence.

Strategy for anti-aquaporin-4 auto-antibody identification and quantification using a new cell-based assay.

NMO-IgG is a specific biomarker of neuromyelitis optica (NMO) that targets the aquaporin-4 (AQP4) water channel protein. The current gold standard for NMO-IgG identification is indirect immunofluorescence (IIF). Our aim in this study was to develop a new quantitative cell-based assay (CBA) and to propose a rational strategy for anti-AQP4 Ab identification and quantification. We observed an excellent correlation between the CBA and IIF for NMO-IgG/anti-AQP4 detection. The CBA appeared more sensitive than IIF but on the other hand, IIF allows the simultaneous detection of various auto-Abs, underlining the complementarity between both methods. In conclusion, we propose to use IIF for the screening of patients at diagnosis in order to identify auto-Abs targeting the central nervous system. A highly sensitive, AQP4 specific and quantitative assay such as our CBA could be used thereafter to specifically identify the target of the Ab and to monitor its serum concentration under treatment.

[CCR5 antagonists and HIV-1 infection: Bases and consequences of this therapeutic approach]. / Antagonistes du récepteur CCR5 et infection par le VIH-1 : bases et conséquences de cette approche thérapeutique.

CCR5 molecule is a chemokine receptor with an important role in infectious diseases; not only is it the main coreceptor for HIV-1, but it has also been involved in the immune defense against various transmissible agents. CCR5 antagonists constitute a new class of antiretrovirals. Three molecules of this class have reached phases 2B and 3 of clinical development: aplaviroc (GlaxoSmithKine), vicriviroc (Schering-Plough) and maraviroc (Pfizer). The development of aplaviroc was stopped because of some cases of drug-induced hepatitis. In ACTG 5211 and Motivate trials, adding vicriviroc (in phase 3 trials) or maraviroc (now approved for clinical use) respectively to an optimized background regimen in treatment-experienced patients has resulted in a significant virologic benefit. The place of this new therapeutic class in strategies of initial, switch or rescue treatment needs further investigation, and its interest in immunological non-responders, in severe immunosuppressed patients or in subjects harbouring non-R5 HIV-1 strains, remains to be addressed. Major concerns about their use still remain, including long-term tolerability, the risk of inducing an R5 to X4 switch, particularly in compartments other than blood, and the risk of interfering with some immune responses.

[CCR5 and the natural history of HIV infection]. / L'impact du niveau d'expression du corécepteur CCR5 sur l'histoire naturelle de l'infection par VIH.

The human immunodeficiency virus type 1 (HIV-1) uses, in addition to the CD4 molecule, a chemokine receptor as a receptor to infect T lymphocytes. Most viral strains use the chemokine receptor CCR5 as a coreceptor. The density of CCR5 molecules on CD4+ T cells varies widely among individuals, but is constant over time for a given individual. Infected subjects with high CCR5 expression present high viral load, progress rapidly, respond poorly to antiretroviral therapies, and have high viral rebond after treatment interruption. This is due to the fact that in cells expressing high surface CCR5 densities, the binding of the virus to its coreceptor triggers strong activation signals, that transit through Gai proteins, and facilitate the reverse transcription of the viral RNA. Thus, CCR5 is not only a dock for HIV-1 but also a choke preparing the target cell to replicate the virus. This model could explain intercellular variabilities in infectibility by differences in the capacity of the virus to activate the cell it infects. Moreover, this model opens new therapeutic opportunities targeting the pathways activated by the virus for his advantage.

Jacalin, a lectin with anti-HIV-1 properties, and HIV-1 gp120 envelope protein interact with distinct regions of the CD4 molecule.

Jacalin is a multimeric plant lectin able to interact with the lymphocyte cell-surface molecule CD4, a known receptor for the human immunodeficiency virus type 1 (HIV-1). Moreover, jacalin is able to block HIV-1 infection of CD4+ lymphoblastoid cells. Here we studied whether jacalin prevents HIV-1 gp120-CD4 interactions. We found (i) that jacalin did not inhibit HIV-1 Lai-induced syncytium formation that requires gp120-CD4 interactions; (ii) that jacalin prevented neither rgp120 binding to cell-surface CD4 nor sCD4 binding to viral envelope proteins expressed at the surface of HIV-1-infected lymphoblastoid cells; (iii) that jacalin did not compete for binding to CD4 with anti-CD4 mAb specific for the CDR2- or CDR3-like regions of the D1 domain of CD4; (iv) that jacalin did not bind a recombinant soluble molecule containing the D1/D2 domains of CD4; and, (iv) that jacalin binding to CD4 is inhibited by sugars known to interact with the lectinic-site of jacalin. These data have implications for the understanding of the mechanism by which jacalin blocks HIV-1 infection of CD4+ cells.

CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression.

OBJECTIVE AND DESIGN: We have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression. METHODS: Using a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Delta 32 allele) was also determined. RESULTS: CCR5 density was stable over time on non-activated, HLA-DR(-)CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomatic and non-treated, we observed a correlation between CCR5 density on HLA-DR(-)CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Delta 32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002). CONCLUSION: These results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

Serum procalcitonin and C-reactive protein concentrations to distinguish mildly infected from non-infected diabetic foot ulcers: a pilot study.

AIMS/HYPOTHESIS: Infection of diabetic foot ulcers is common; at early stages it is difficult to differentiate between non-infected ulcers (or those colonised with normal flora) and ulcers infected with virulent bacteria that lead to deterioration. This pilot study aimed to assess the diagnostic accuracy of inflammatory markers as an aid to making this distinction. METHODS: We included 93 diabetic patients who had an episode of foot ulcer and had not received antibiotics during the 6 months preceding the study. Ulcers were classified as infected or uninfected, according to the Infectious Diseases Society of America-International Working Group on the Diabetic Foot classification. Diabetic patients without ulcers (n=102) served as controls. C-reactive protein (CRP), orosomucoid, haptoglobin and procalcitonin were measured together with white blood cell and neutrophil counts. The diagnostic performance of each marker, in combination (using logistic regression) or alone, was assessed. RESULTS: As a single marker, CRP was the most informative for differentiating grade 1 from grade 2 ulcers (sensitivity 0.727, specificity 1.000, positive predictive value 1.000, negative predictive value 0.793) with an optimal cut-off value of 17 mg/l. In contrast, white blood cell and neutrophil counts were not predictive. The most relevant combination derived from the logistic regression was the association of CRP and procalcitonin (AUC 0.947), which resulted in a significantly more effective determination of ulcer grades, as shown by comparing receiver operating characteristic curves. CONCLUSIONS/INTERPRETATION: Measurement of only two inflammatory markers, CRP and procalcitonin, might be of value for distinguishing between infected and non-infected foot ulcers in subgroups of diabetic patients, to help ensure the appropriate allocation of antibiotic treatment. Nevertheless, external validation of the diagnostic value of procalcitonin and CRP in diabetic foot ulcers is needed before routine use can be recommended.

The expression of cytotoxic mediators is altered in mononuclear cells of patients with melanoma and increased by interferon-alpha treatment.

BACKGROUND: The role of cytotoxic cells in the control of cancer is now well established. OBJECTIVES: To evaluate the expression of perforin and granzyme A in cytotoxic cells of patients with melanoma and to look for a link between this expression and natural tumour progression; to check if interferon (IFN)-alpha administration increased expression of cytotoxic mediators; and to evaluate if this increase was correlated with the antitumoral effect of IFN-alpha. METHODS: To determine in patients with melanoma the expression of the cytotoxic mediators perforin and granzyme A in peripheral blood natural killer (NK) and T cells, we used flow cytometry before and after IFN-alpha administration. RESULTS: Compared with healthy volunteers, we observed in 82 patients a low percentage of NK cells harbouring perforin [75% (95% confidence interval (CI) 70-79) vs. 92% (95% CI 89-95), P < 0.001] and granzyme A [48% (95% CI 41-55) vs. 73% (95% CI 66-81), P < 0.001]. By contrast, a high percentage of T cells, and particularly of CD56+ T cells, expressed perforin [56% (95% CI 41-71) vs. 28% (95% CI 18-38), P < 0.001], whereas a low percentage of CD56+ T cells expressed granzyme A [30% (95% CI 24-36) vs. 54% (95% CI 43-65), P < 0.001]. In untreated patients, the percentage of CD56+ T cells expressing granzyme A was higher in progressors than in nonprogressors [49% (95% CI 39-58) vs. 16% (95% CI 0-33), P = 0.003]. We followed cytotoxic mediator expression in 17 patients treated with IFN-alpha. IFN-alpha administration increased granzyme A expression in NK cells [44% (95% CI 27-61) and 65% (95% CI 54-76) before and after treatment, respectively, P = 0.010], rather than perforin expression, whereas expression of both perforin [46% (95% CI 30-62), and 58% (95% CI 44-73), P = 0.112] and especially granzyme A [27% (95% CI 14-40) vs. 45% (95% CI 26-64), P = 0.016] was increased in CD56+ T cells after IFN-alpha administration. Yet, this effect was not correlated with the clinical response to IFN-alpha. CONCLUSIONS: Thus, the expression of cytotoxic mediators is altered in cytotoxic cells of patients with melanoma, and increased under IFN-alpha administration.

Anti-HLA antigen class I heavy chain monoclonal antibodies inhibit human immunodeficiency virus production by peripheral blood mononuclear cells.

We have examined the capacity of monoclonal antibodies (mAb) specific for HLA class I heavy chain to interfere with the human immunodeficiency virus (HIV) replicative cycle in human T cells. Among six anti-HLA class I heavy chain-specific mAb assayed, two mAb, RL4-24-6 and W6/32, were able to delay HIV1 and HIV2 cytopathic effect on MT4 cells, a human T cell leukemia virus type I (HTLVI) immortalized T cell line, mAb RL4-24-6, chosen for further studies, also inhibited HIV1 production by peripheral blood mononuclear cells (PBMC), and this inhibition was dose dependent. However, no effect was observed when mAb treatment was performed with either the CEM or Jurkat T cell lines. Our investigation of how RL4-24-6 interferes with the HIV replicative cycle revealed that: (a) incubation of PBMC with RL4-24-6 prior to HIV exposure did not change the susceptibility of these cells to HIV infection, (b) syncytia formation between CD4+ MT4 cells and HIV chronically infected PBMC was not affected by RL4-24-6 and (c) treatment of freshly infected PBMC with RL4-24-6, however, inhibited viral production. These data, together with those we previously reported using anti-beta 2-microglobulin (beta 2m) mAb, suggest that anti-HLA class I/beta 2m complex mAb can modify an early step of the HIV replicative cycle without affecting the viral entry.

Anti-HIV effects of HIV vectors.

We have recently developed an HIV-1 packaging cell line, psi 422, as an improved tool for anti-HIV gene therapy. After stable transfection with an HIV-1 or HIV-2 vector, psi 422 has been shown to synthesize virions able to transduce CD4+ T cells and macrophages. We now report that HIV vectors per se, in the absence of antiviral genes, inhibit HIV infection of transduced cells. This antiviral effect was shown to be due, at least in part, to a TAR and RRE decoy effect. These data highlight further advantages of HIV-derived gene delivery systems for HIV therapy, in addition to CD4 cell targeting and the ability to transduce nondividing cells.

An antibody that binds the immunoglobulin CDR3-like region of the CD4 molecule inhibits provirus transcription in HIV-infected T cells.

We used the polymerase chain reaction (PCR) to study which step(s) of the human immunodeficiency virus type 1 (HIV-1) life cycle may be blocked following treatment of HIV-exposed CEM cells with 13B8-2, a monoclonal antibody (mAb) specific for the immunoglobulin (Ig) CDR3-like region of the CD4 molecule and able to inhibit the productive infection of CEM cells by HIV-1. The presence of viral RNA was investigated and found in 13B8-2 mAb-treated CEM cells 30 min after viral exposure; the full-length viral DNA was found at 24 h post-infection. We also found integrated forms of viral DNA at 24 h post-infection. However, the integrated provirus was transcriptionally inactive in 13B8-2 mAb-treated cells, as demonstrated by the absence of spliced HIV-1 mRNA. The lack of HIV transcription under 13B8-2 mAb treatment was confirmed by chloramphenicol acetyltransferase (CAT) assay. We conclude that the inhibition of viral gene transcription accounts for the lack of progeny virions in culture supernatants of cells treated with this anti-CD4 mAb. We also demonstrate that 13B8-2 blocks viral production from chronically infected cells and restores CD4 cell-surface expression on CEM cells containing an integrated provirus(es). We found this effect to be reversible. Moreover, we demonstrate that 13B8-2 mAb treatment is efficient on different HIV-1 and HIV-2 virus isolates. These results may have major implications for the treatment of AIDS.

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.

Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line.

By transfecting fibroblast cells with an HIV-1-MN molecular clone with a deletion of the major packaging sequence, we have developed a stable HIV-1 packaging cell line, psi 422, psi 422 cells form syncytia with CD4-positive cells, correctly express HIV-1 structural proteins, and produce a large amount of mature particles with normal reverse transcriptase activity. Yet these particles, in which RNA was not detected by reverse transcriptase-PCR, are not infectious. When stably transfected with an HIV-1-based retroviral vector, the psi 422 cell line produces virions capable of transducing CD4-positive cells with high efficiency (up to 10(5) cells per ml). The availability of this stable noninfectious HIV-1 packaging cell line capable of generating high-titer HIV-1 vectors represents a new step toward the use of an HIV-1 delivery system in gene therapy.

Transduction of human macrophages using a stable HIV-1/HIV-2-derived gene delivery system.

We have previously established a stable HIV-1 packaging cell line, psi 422, which yielded high titers of an HIV-1 vector capable of efficiently transducing CD4+ cells. In order to increase the safety of this gene delivery system, we have now replaced the HIV-1 vector with an HIV-2 vector to abolish any risk of homologous recombination between the packaging cells and the vector. The HIV-2 vector was also modified by insertion of a cis-acting constitutive transport element which confers Rev independence. The supernatant of psi 422 cells stably transfected with this new vector was capable of transducing CD4+ cells with a titer of 10(4) transducing units per milliliter. This result shows that cross-packaging of HIV-2 vectors with the HIV-1 packaging cells is quite efficient. Using this new stable HIV-1/HIV-2 gene delivery system, we were able to transduce human monocyte-derived primary macrophages, which are refractory to murine retrovirus-mediated transduction. The availability of a stable HIV-based gene delivery system for macrophages, a key target cell in HIV infection; is an important advance in gene therapy for AIDS.