Long-term modifications of the peripheral immune repertoire after switching from sequestering disease-modifying treatments in multiple sclerosis.

BACKGROUND: Scarce data are available on the long-term immunological effects of multiple sclerosis (MS) disease-modifying treatments (DMTs). OBJECTIVES: This study aimed to investigate the long-term modifications of the peripheral immune repertoire on interruption of a sequestering DMT (natalizumab, fingolimod) and switch to another high-efficacy DMT. METHODS: Lymphocyte subpopulations were assessed, every 6 months up to 48 months, in patients switched from fingolimod or natalizumab to ocrelizumab, and in patients switched from fingolimod to natalizumab, compared to patients switched to ocrelizumab or natalizumab from a moderate-efficacy DMT and to naive patients. RESULTS: We included 389 MS patients (200 ocrelizumab and 189 natalizumab). After adjusting for baseline variables, patients switched from fingolimod to ocrelizumab showed lower CD3 + and CD4 + lymphocytes up to 48 months after switch (with lower percentage of naive CD4 +), and increased odds of total, CD3+, CD4+ lymphopenia. Patients switched from natalizumab to ocrelizumab showed higher CD3 + lymphocytes up to 36 months after switch, and higher CD4+, CD8+ lymphocytes up to 24 months. The frequency of infections was not influenced by previous treatment. CONCLUSIONS: A long-term persistence of the residual effects of the exposure to sequestering DMTs (fingolimod and less natalizumab) on the peripheral immune repertoire was observed after switching to another high-efficacy DMT.

Immunophenotype Anomalies Predict the Development of Autoimmune Cytopenia in 22q11.2 Deletion Syndrome.

BACKGROUND: Patients with 22q11.2 deletion syndrome (22q11.2DS) may develop severe thrombocytopenic purpura and hemolytic anemia. There are no reliable predictors for the development of hematologic autoimmunity (HA) in these patients. OBJECTIVE: To describe the peculiar B and T subpopulation defects in patients with 22q11DS who have developed HA and test if these defects precede the development of HA. METHODS: We performed a case-control multicenter study. Patients with HA were compared with a control population of 22q11.2DS without HA (non-HA). A complete immunological evaluation was performed at diagnosis and at the last follow-up including extensive T and B phenotypes. RESULTS: Immunophenotype at the last follow-up was available in 23 HA and 45 non-HA patients. HA patients had significantly decreased percentage of naïve CD4+ cells (26.8% vs 43.2%, P = .003) and recent thymic emigrants (48.6% vs 80.5%, P = .046); decreased class-switched B cells (2.0% vs 5.9%, P = .04) and increased naive B cells (83.5% vs 71.4%, P = .02); increased CD16+/56+ both in absolute number (312 vs 199, P = .009) and percentage (20.0% vs 13.0%, P = .03). Immunophenotype was performed in 36 patients (11 HA and 25 non-HA) at diagnosis. Odds ratio (OR) of immune cytopenia were estimated for both CD4 naïve ≤30% (OR 14.0, P = .002) and switched memory B cells ≤2% (OR 44.0, P = .01). The estimated survival curves reached statistical significance, respectively, P = .0001 and P = .002. CONCLUSIONS: Among patients with 22q11.2DS, those with HA have characteristic lymphocyte anomalies that appear considerably before HA onset. Systematic immunophenotyping of patients with 22q11.2DS at diagnosis is advisable for early identification of patients at risk for this severe complication.

Low synovial double negative T and γδ T cells predict longer free-disease survival in oligoarticular JIA.

BACKGROUND: Oligoarticular juvenile idiopathic arthritis (oJIA) is the most frequent form of chronic arthritis in children; the clinical course is extremely variable. In this study we have characterized by flow cytometry synovial B and T cells subsets in patients with oJIA in order to identify any parameters that could predict a more aggressive course of disease. METHODS: B and T cells from synovial fluid (SF) of 39 patients with oJIA were characterized by flow cytometry. In 22 patients SF was analysed at the onset of the disease (GroupA), in 17 SF was analysed at articular relapse (Group B). All patients in Group A were followed up for at least for 2 years after SF analysis: 13 patients relapsed during the follow-up period. RESULTS: Comparison of SF from Group A and Group B demonstrated an activated phenotype in relapsed patients, with higher Switched Memory B cells (58.53 vs 36.07% of CD19+, P-value 0.004) and lower Naïve B cells (8.53 vs 25.9 of CD19+, P-value 0.002) in Group B. Furthermore, patients from Group A who did not relapse showed lower percentages of synovial DNT (2.38 vs 1.50% of CD3 + TCRalpha/beta+, P-value 0.025) and γδ T cells (19.1 vs 15.0% of CD3+ cells, P-value 0.004) at the onset, if compared with other Group A patients. CONCLUSIONS: In oJIA relapse SF present an activated B phenotype. Patients at disease onset with DNTs <1.8% and/or γδ T cells <16% of CD3+ in synovial fluid have longer free-disease survival. © 2017 International Clinical Cytometry Society.

In vitro anti-neuroblastoma activity of saquinavir and its association with imatinib.

Neuroblastoma (NB) has a poor prognosis when in advanced stages, highlighting the need for new therapeutic options. The human immunodeficiency virus (HIV) protease inhibitor saquinavir is active in vitro against chronic myeloid leukaemia cells, in synergy with the tyrosine kinase inhibitor imatinib. Here, we evaluated the effects of saquinavir, alone or in association with imatinib, on cell proliferation (count of viable cells after trypan blue exclusion), apoptosis (Annexin V binding) and invasion (through a transwell membrane coated with Matrigel) in SJ-N-KP, IMR5, AF-8, SK-N-SH and SK-N-BE NB lines, all expressing c-kit and PDGF-R (determined by flow cytometry). Saquinavir showed a dose-dependent anti-proliferative and anti-invasive activity on NB lines, increased by the association with imatinib when the two drugs were utilized at clinically attainable concentrations. The same low saquinavir concentrations inhibited in NB cells the nuclear activation of NF-κB (Western immuno-blotting for nuclear NF-κB p50 and p65). Saquinavir at high concentrations also exerted a pro-apoptotic activity on NB lines, significantly increased by the association with imatinib. In conclusion, saquinavir and imatinib are both drugs utilized for long-term therapies, with good oral bioavailability and a well-known toxicity profile. The anti-NB activity of saquinavir and of its association with imatinib suggests a potential usefulness in the treatment of NB, particularly for remission maintenance.

In CD28-costimulated human naïve CD4+ T cells, I-κB kinase controls the expression of cell cycle regulatory proteins via interleukin-2-independent mechanisms.

Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.

Aberrantly glycosylated IgA1 induces mesangial cells to produce platelet-activating factor that mediates nephrin loss in cultured podocytes.

The reaction of mesangial cells with aberrantly glycosylated IgA1 has been implicated in the etiology of IgA nephropathy (IgAN). Tumor necrosis factor, which is assumed to mediate the interaction between mesangial cells and podocytes, also induces the expression of platelet-activating factor (PAF). In this study, we determined whether PAF affects the expression of nephrin (an adhesion molecule critical to glomerular permselectivity) and cytoskeletal F-actin organization in podocytes. We treated human mesangial cells with atypically glycosylated IgA1 either prepared in vitro or derived from the sera of patients with IgAN. We then prepared conditioned media from these cells and added them to cultured human podocytes in the presence of PAF receptor antagonists. Podocytes transfected to overexpress acetylhydrolase, the main catabolic enzyme of PAF, served as controls. Downregulation of nephrin expression and F-actin reorganization occurred when podocytes were cultured with mesangial cell-conditioned medium. Preincubation of podocytes with a PAF receptor antagonist prevented the loss and redistribution of nephrin. In control podocytes overexpressing acetylhydrolase, nephrin loss was abrogated. Our results suggest that atypically glycosylated IgA-induced PAF from mesangial cells is a mediator of podocyte changes, which, when more directly tested elsewhere, were found to be associated with proteinuria. Hence, it is possible that these in vitro findings may be relevant to the proteinuria of IgAN.

cAMP response element binding protein (CREB) overexpression CREB has been described as critical for leukemia progression.

CREB has been described as critical for leukemia progress. We investigated CREB expression in ALL and AML pediatric patients. CREB protein was significantly high (p<0.001) at diagnosis but not during remission. This study underlines the role of CREB in leukemia and suggests new insights into the transformation process.

Immunomodulatory effects of two HIV protease inhibitors, Saquinavir and Ritonavir, on lymphocytes from healthy seronegative individuals.

UNLABELLED: Immunological and clinical benefits of highly active antiretroviral therapy (HAART) do not always correlate with its efficacy in controlling HIV replication. This could be due to biological effects on the cell of retroviral agents. DESIGN: To this view we evaluated possible direct immunomodulatory effects of two HIV protease inhibitors, such as Saquinavir (SQ) and Ritonavir (RIT). METHODS: In particular we assessed the PHA- and anti-CD3-driven T cell proliferation, mixed lymphocyte reaction (MLR) and cytokine production on PBMCs from HIV-uninfected subjects incubated with increasing concentrations (2, 5, 10 and 20 microM) of SQ or RIT. RESULTS: Treatment of PBMCs with RIT resulted in a dose-dependent reduction of lymphoproliferative responses. Such an effect was also marked with SQ. MLR was significantly reduced in a concentration-dependent fashion after incubation with either drug. The percentages of stimulated PBMCs and mostly of CD4+ cells expressing TNF-alpha, IL-2 and IFN-gamma were also reduced by SQ or RIT. CONCLUSION: At therapeutic doses both SQ and RIT exhibit potent immunomodulatory activity, which may contribute to correct the HIV-driven cytokine dysregulation and account for some clinical and immunological benefits of therapy in patients with virologic failure. In view of autoreactive immunopathology occurring in AIDS, these direct biologic effects raise intriguing speculations on anti-HIV strategy.

The effects of saquinavir on imatinib-resistant chronic myelogenous leukemia cell lines.

We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity.

The HIV protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcriptase inhibitors, adversely affect human proteasome function.

BACKGROUND: In HIV-infected patients some clinical and immunological benefits of antiretroviral therapy, which frequently include a combination of HIV protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), cannot be solely explained by the drugs' action on viral enzymes. Proteasomes constitute the central protease of the ubiquitin ATP-dependent pathway involved in many cellular processes, as well as in HIV maturation and aggressiveness. OBJECTIVE: To explore whether the PIs nelfinavir and saquinavir and the RTIs abacavir, nevirapine, delavirdine, stavudine and didanosine affect proteasome function in vitro and in vivo. METHODS: Peptidase activity of purified human 26S and 20S proteasomes was assayed with and without the drugs at different concentrations. Intracellular proteasome proteolytic activity was evaluated by searching for ubiquitin-tagged proteins in HL60 cells incubated with and without the drugs. RESULTS: At therapeutic dosages, nelfinavir and saquinavir inhibited proteasome peptidase activity and caused intracellular accumulation of polyubiquitinated proteins, a hallmark of proteasome proteolytic inhibition in vivo; the RTIs failed to evoke either effect. CONCLUSION: Proteasomes are targeted by the two PIs but not the RTIs. Therefore, in HIV-infected patients the beneficial effect of a therapy including one of the two PIs should partly rely on inhibition of host proteasome function.

Imatinib mesylate (STI571) interference with growth of neuroectodermal tumour cell lines does not critically involve c-Kit inhibition.

A therapeutic role of STI571 (imatinib mesylate) has been anticipated in patients with c-Kit positive neuroectodermal tumors. We examined the efficacy of STI571 to inhibit expansion of c-Kit positive neuroectodermal tumor cell lines in vitro and in a mouse model inoculated with ES (Ewing sarcoma) derived tumor cells, and investigated the molecular mechanism of STI571 action. Eleven tumor lines of ES, PNET (primitive neuroectodermal tumors) and NB (neuroblastoma) were assayed in the presence of 1, 5, 10, 15, 20 or 30 micro M STI571 for 24, 48, 72 h and 7 days. The mechanism of STI571 action was investigated using a microphysiometer cytosensor that determines cellular metabolic rates in the presence of STI571. c-Kit and global protein phosphorylation was assayed by immunoprecipitation and a direct enzyme-linked immunoadsorbent assay after 72 h of 10 micro M STI571. Apoptosis was investigated by propidium iodide (PI), Annexin V staining and by enzymatic activity of caspase-3. Moreover, apoptotic gene expression was investigated using microarray technology. In nude mice, tumor volume and histology were analyzed in STI571 treated and untreated mice, and apoptotic gene expression analysis was performed on tumor masses. A decrease in cell proliferation and increase of cell apoptosis was caused by STI571 in a concentration- and time-dependent manner. Cytosensor microphysiometer and immunoprecipitation experiments demonstrated a time- and concentration-dependent decrease of cellular metabolic activity and global protein dephosphorylation after STI571 exposure. The inhibition by STI571 appeared at least to some extent independent of c-Kit inhibition since cells remained sensitive to SCF stimulation. Tumor volume was significantly reduced in STI571-treated mice compared to tumors from control inoculated non-treated mice. The apoptosis pathway in response to STI571 appeared not to be dependent on caspase activation, while gene expression profiles suggested accumulation of reactive oxygen species (ROS) resulting in cell death after exposure to STI571. The results point to the potential relevance of STI157 for neuroectodermal tumor therapy in view of its inhibitory effect on tumor cell growth, in spite of the observation that the inhibition of the c-Kit signaling pathway is not critically involved.

The human 26S proteasome is a target of antiretroviral agents.

BACKGROUND: Proteasomes constitute the degradative machinery of the ubiquitin/adenosine triphosphate-dependent proteolytic pathway, which is involved in many cell functions, including immune response and apoptosis, and in HIV maturation and infectivity. OBJECTIVE: To examine whether proteasomes are targeted by antiretroviral agents. METHODS: Chymotrypsin-like, trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of purified human 26S and 20S proteasomes, the latter depleted or enriched in 11S regulator, were assayed after incubation with indinavir, lamivudine and zidovudine at 1-80 microM alone and in combination. To assess the drug effects on cellular functions regulated by proteasomes, the accumulation of ubiquitin-tagged proteins, the processing of the nuclear factor kappa B precursor p105, and the degradation of the inhibitor of nuclear factor kappa B, isoform alpha (IkappaBalpha) were evaluated by Western immunoblotting in Jurkat cells after incubation for 6 h with the drugs above. RESULTS: Trypsin-like and mostly chymotrypsin-like activities of purified 26S proteasome were inhibited by each drug from 10 to 80 microM, more by double combinations and mostly by the triple combination. The peptidyl-glutamyl-peptide hydrolysing activity of the 26S proteasome and the three peptidase activities of the 20S proteasome, depleted or enriched in 11S regulator, were unaffected. The accumulation of ubiquitin-tagged proteins, reduced IkappaBalpha degradation and p105 processing were appreciable in intact cells with the triple drug combination. CONCLUSION: The human 26S proteasome is a target of antiretroviral agents. This suggests that the antiviral action and some clinical and immunological benefits of combined antiretroviral therapy rely not only on its known effects on viral enzymes, but also on host cell components.