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Objective: To report the clinicopathologic features and outcome of myositis in patients treated with immune checkpoint inhibitors (ICIs) (irMyositis)., Methods: We retrospectively analyzed patients diagnosed with irMyositis in tertiary centers in Paris, France, and Berlin, Germany, from January 2015 to July 2017. The main outcomes were clinical manifestations and muscle histology, which included major histocompatibility complex class I (MHC-I), C5b-9, CD3, CD4, CD8, CD20, CD68, programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L) 1, and programmed cell death 1 ligand 2 (PD-L2)., Results: Ten patients with metastatic cancer were included; median age was 73 (range 56-87) years. Median follow-up duration was 48 (range 16-88) weeks. Six patients developed myositis during nivolumab therapy, 1 patient during pembrolizumab, 1 patient during durvalumab, and 2 patients during combined nivolumab and ipilimumab. Median delay between ICI initiation and myositis onset was 25 (range 5-87) days. Clinical manifestations were dominated by acute or subacute myalgia (8 patients) and limb-girdle (7), axial (7), and oculomotor (7) weakness. Four patients had evidence of myocarditis. In all patients, creatine kinase levels were elevated (median 2,668, range 1,059-16,620 U/L), while anti-acetylcholine receptor and myositis-associated antibodies were negative. Electrodiagnostic studies showed myopathic process without decrement in all patients. Muscle biopsy constantly showed multifocal necrotic myofibers, sarcolemmal MHC-I, and endomysial inflammation, consisting mainly of CD68+ cells expressing PD-L1 and CD8+ cells expressing PD-1. ICI treatment was withdrawn in all patients; 9 patients received immunosuppressive therapy, which consistently led to marked clinical improvement., Conclusions: irMyositis presents with remarkably homogeneous and unique clinicopathologic features, expanding the nosologic spectrum of inflammatory myopathies in patients with cancer. ICI withdrawal and treatment with corticosteroids improve outcome., (© 2018 American Academy of Neurology.)

Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, β1i or β5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8 + T-cells and CD68  +  macrophages in IIMs expressed β1i or β5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during in vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate β1i or β5i. Selective inhibition or depletion of β5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of β1i or β5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, we investigated the cytokine profiles of 14 treatment-naive HIV-infected patients on the initiation of highly active antiretroviral therapy (HAART). At baseline, plasma levels of TNF-alpha and its mRNA in peripheral blood mononuclear cells (PBMC) were highest in the most severely immunocompromised patients (<200 CD4+ cells/mm3). After 12 months of HAART, the virus was undetectable in the plasma of all patients (<200 copies/ml), and median CD4 T cell counts had increased (+164 cells/mm3). We also observed a gradual decrease in the number of proviral DNA copies in PBMC and in immune activation, with lower levels of IFN-gamma mRNA in PBMC associated with weaker activation of CD8+ T cells and lower levels of plasma TNF-alpha. IL-2 mRNA levels in PBMC were found to increase in parallel. The decrease in TNF-alpha and IFN-gamma levels and the increase in IL-2 production appear to be correlated with the efficacy of HAART in naive immunocompromised HIV-infected individuals.

Haemophagocytic syndrome (HPS) and HIV infection are both associated with cytokine network dysregulation. We therefore analysed plasma levels and mRNA synthesis in peripheral blood mononuclear cells (PBMC) of cytokines, chemokines and chemokine receptors in one HIV-infected patient with HPS. We compared the results with those for eight HIV-infected patients with similar CD4+ T cell counts (207/mm3 versus controls: median 214/mm3) and plasma virus load (4.1 log copies/ml, versus controls: median 4.2 log copies/ml). The HPS patient had a lower viral DNA load in PBMC and higher plasma levels of interferon-gamma, IL-10, and macrophage inflammatory protein (MIP)-1beta. No difference in plasma tumour necrosis factor-alpha (TNF-alpha), IL-6 and MIP-1alpha concentration was observed between the HPS patient and control patients. No difference was observed in TNF-alpha, IL-1beta, IL-10, IL-4, MIP-1alpha, MIP-1beta, RANTES, CXCR-4, and CCR-5 mRNA levels in PBMC, but IL-6 levels were higher in the HPS patient. Our results emphasize the role of IL-10 in the control of immune hyperactivation that is observed in HPS.

This study evaluates the consequences of antiretroviral treatment of the acute simian immunodeficiency virus (SIV) primary infection on virus load and cytokine responses. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate (SIVmac251). Animals were pretreated with 10.8 mg/kg/day of dideoxyinosine (ddI) from 4 days before inoculation, and treatment was continued for 28 days. Proinflammatory (IL6, IL1 beta and TNF alpha) and antiinflammatory (IL10) cytokine and lymphokine (IL2, IL4 and IFN gamma) polymerase chain reaction (PCR) ratios were monitored in unmanipulated peripheral blood mononuclear cells (PBMCs) during acute infection by using a semiquantitative reverse transcription (RT)-PCR method. PBMC-associated virus loads were dramatically reduced compared to those of placebo-treated macaques. Nevertheless, a transient rise in IL6, IL1 beta, TNF alpha and IL10 mRNA expression was observed in PBMCs. IL2, IL4 and IFN gamma mRNAs were either undetectable or weakly detectable throughout the study, with no major changes. Despite a dramatic reduction in the acute viral loads in ddI-treated monkeys, early cytokine mRNA profiles were comparable to those of untreated SIVmac251-infected monkeys. Contrary to what was previously evidenced during primary infection with an attenuated SIV clone, no increase in IL2 and IL4 mRNA was detected in PBMCs of the ddI-treated monkeys, although these monkeys exhibited virus loads similar to those evidenced in macaques infected by attenuated SIV. These data indicate that differential lymphokine expression patterns found in pathogenic and Nef-truncated SIV-infected monkeys may not be strictly dependent on virus load levels.

Various methods are currently used to monitor cytokine mRNA expression levels in clinical samples. In instances where sample size is limited (e.g. biopsies), amplification procedures such as reverse transcription followed by polymerase chain reaction amplification appear to be the only ones that are currently useful. This paper provides a review of such techniques for the detection and quantitation of cytokine message in clinical samples, and compares semiquantitative and competitive techniques in terms of sensitivity, reproducibility, feasibility and costs.

Problem: Although the overall anti-infectious and anti-parasitic immunity of parous women appears normal, several aspects of maternal cell-mediated and humoral immunity are altered during pregnancy. This has been suggested to occur via preferential local and systemic secretion of Th-2 type cytokines, which down-regulate or prevent secretion/action of Th-1 type cytokines, in animals as well as in humans., Method: To evaluate the influence of gestation on the maternal immune system, we have measured, in pregnant women, the mRNAs coding for several cytokines (TNF alpha, IFN gamma, GM-CSF, IL-1 beta, IL-2, IL-4, IL-6, and IL-10) in peripheral blood mononuclear cells, by use of semi-quantitative RT-PCR., Results: Our results show significant modulations of IFN gamma, IL-1 beta, IL-4, and IL-6 genes expression especially during the third trimester and near parturition., Conclusion: Cytokine expression is thus finely tuned in peripheral blood during pregnancy, in a previously unexpected complex pattern, related to gestational stage.

Inflammation is a frequent result of radiation damage, especially after therapeutic irradiation. Monocytes and macrophages play an important role in inflammatory cytokine secretion. However, knowledge of monocyte/ macrophage involvement in the pathogenesis of inflammation is still incomplete. In this study, we investigated the inflammatory cytokine regulation after in vitro gamma-irradiation of monocytes/macrophages. Semiquantitative RT-PCR after irradiation revealed no significant induction of interleukin 1 beta or tumour necrosis factor-alpha mRNA expression. When induction was observed, for only two donors out of seven, only interleukin 6 gene expression was affected by irradiation, with a 5-14-fold increase in level according to the donor, 2h after irradiation compared with sham-irradiated cells. For only one of seven donors tested, monocytes/macrophages responded to 10-Gy gamma-rays by releasing inflammatory cytokines.

Using two independent PCR-based quantification techniques, we have determined the levels of IL1 beta, IL2, IL4, IL6, IL10, IFN gamma and TNF alpha mRNA in multiple sclerosis patients at the moment of diagnosis of the disease and prior to any immunosuppressive treatment. These patients exhibit markedly reduced IL2 and IL10 mRNA expression accompanied by decreased levels of TNF alpha mRNA. Our results add to the evidence that IL10 plays a role in multiple sclerosis and suggest that decreased production of this interleukin allows the proliferation of autoreactive T cells at the onset of the disease.

We used semiquantitative RT-PCR to monitor the expression of mRNA encoding cytokines (IL-1 beta, IL-6, TNF-alpha, and IL-10) and IFN-gamma in fresh isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs), of four cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus (SIVmac251). To investigate the effects of the viral load on the expression of the cytokines, two monkeys received 30 mg kg-1 day-1 of didanosine (ddI). The two nontreated monkeys became infected and seroconverted, whereas the ddI-treated monkeys were completely protected as demonstrated by all criteria of diagnosis of SIV infection. Concomitant with the peak of viral replication (2 weeks after the experimental inoculation), high levels of IL-6 mRNA were produced in PBMCs, LNMCs, and BALMCs of the two placebotreated infected monkeys. Overexpression of TNF-alpha and IL-10 mRNAs was sometimes observed in LNMCs and BALMCs. A progressive overexpression of IFN-gamma mRNA, starting 2 weeks after experimental inoculation, was observed in BALMCs from infected animals. Concurrently, a marked increase in the CD8+ lymphocyte percentage in the BAL fluids was detected by FACS analysis. Thus, our results emphasize the importance of a comparative study of the expression of cytokines in different tissues. They suggest the interactions of monocyte/macrophage monokine production with viral replication, as well as the role of IFN-gamma in the development of lung cellular immunity to SIV infection.

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.

To investigate the spectrum of cytokines expressed by peripheral blood mononuclear cells (PBMC) from cynomolgus macaques (Macaca fascicularis), we used a semi-quantitative RT-PCR to determine levels of mRNA coding for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. The PCR products were labelled and quantified using a new fluorescent tag TOTO-1 (thiazole orange dimer) and an automated fluorescence-based electrophoretic instrument. Using this assay, the base line levels of cytokine mRNA expression in unmanipulated PBMCs (ex vivo) from 10 healthy monkeys were compared with the mRNA levels for the same cytokines in PBMC samples from two pre-immunized monkeys following culture with previously defined optimal concentrations of purified protein derivative (PPD), tetanus toxoid (TT) and the mitogen concanavalin A (con-A). While transcripts or IL-2, IL-4 and IFN-gamma were either low or not detected in unmanipulated PBMCs, varying levels of IL-1 beta, IL-5, IL-10, and TNF-alpha were readily detected in the same samples. With the exception of IL-10, the mitogen con-A induced the highest levels of cytokine expression, followed by levels induced by culture with TT. The levels of cytokine expression induced by PPD however, were not significantly elevated, despite the fact that the cells showed marked proliferative responses. This assay is a simple and convenient method for evaluating the levels of cytokine expression in small PBMC samples and will allow for the concurrent evaluation of immune profiles with functional immune analyses.

SUMMARY In this study, we investigated the cytokine profiles of 14 treatment-naive HIV-infected patients on the initiation of highly active antiretroviral therapy (HAART). At baseline, plasma levels of TNF-α and its mRNA in peripheral blood mononuclear cells (PBMC) were highest in the most severely immunocompromised patients (<200 CD4+ cells/mm3 ). After 12 months of HAART, the virus was undetectable in the plasma of all patients (<200 copies/ml), and median CD4 T cell counts had increased (+164 cells/mm3 ). We also observed a gradual decrease in the number of proviral DNA copies in PBMC and in immune activation, with lower levels of IFN-γ mRNA in PBMC associated with weaker activation of CD8+ T cells and lower levels of plasma TNF-α . IL-2 mRNA levels in PBMC were found to increase in parallel. The decrease in TNF-α and IFN-γ levels and the increase in IL-2 production appear to be correlated with the efficacy of HAART in naive immunocompromised HIV-infected individuals. [ABSTRACT FROM AUTHOR]