Francesco Silvestris

Cytokine Overproduction, T-Cell Activation, and Defective T-Regulatory Functions Promote Nephritis in Systemic Lupus Erythematosus

Lupus nephritis (LN) occurs in more than one-third of patients with systemic lupus erythematosus. Its pathogenesis is mostly attributable to the glomerular deposition of immune complexes and overproduction of T helper- (Th-) 1 cytokines. In this context, the high glomerular expression of IL-12 and IL-18 exerts a major pathogenetic role. These cytokines are locally produced by both macrophages and dendritic cells (DCs) which attract other inflammatory cells leading to maintenance of the kidney inflammation. However, other populations including T-cells and B-cells are integral for the development and worsening of renal damage. T-cells include many pathogenetic subsets, and the activation of Th-17 in keeping with defective T-regulatory (Treg) cell function regards as further event contributing to the glomerular damage. These populations also activate B-cells to produce nephritogenic auto-antibodies. Thus, LN includes a complex pathogenetic mechanism that involves different players and the evaluation of their activity may provide an effective tool for monitoring the onset of the disease.

PTHrP Produced by Myeloma Plasma Cells Regulates Their Survival and Pro‐Osteoclast Activity For Bone Disease Progression

To promote their survival and progression in the skeleton, osteotropic malignancies of breast, lung, and prostate produce parathyroid hormone–related protein (PTHrP), which induces hypercalcemia. PTHrP serum elevations have also been described in multiple myeloma (MM), although their role is not well defined. When we investigated MM cells from patients and cell lines, we found that PTHrP and its receptor (PTH‐R1) are highly expressed, and that PTHrP is secreted both as a full‐length molecule and as small subunits. Among these subunits, the mid‐region, including the nuclear localization sequence (NLS), exerted a proliferative effect because it was accumulated in nuclei of MM cells surviving in starvation conditions. This was confirmed by increased transcription of several genes enrolled in proliferation and apoptosis control. PTHrP was also found to stimulate PTH‐R1 in MM cells. PTH‐R1s selective activation by the full‐length PTHrP molecule or the NH2‐terminal fragment resulted in a significant increase of intracellular Ca2+ influx, cyclic adenosine monophosphate (cAMP) content, and expression of receptor activator of NF‐κB ligand (RANKL) and monocyte chemoattractant protein‐1 (MCP‐1). Our data definitely clarify the role of PTHrP in MM. The PTHrP peptide is functionally secreted by malignant plasma cells and contributes to MM tumor biology and progression, both by intracrine maintenance of cell proliferation in stress conditions and by autocrine or paracrine stimulation of PTH‐R1, which in turn reinforces the production of osteoclastogenic factors.

New insights into the molecular pathogenesis of langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare proliferative disorder characterized by an accumulation of cells sharing the major phenotypic features of cutaneous Langerhans cells. Given its variable clinical evolution, ranging from self-limiting lesions to multisystemic forms with a poor prognosis, in the last decades it has been debated whether LCH might not have a neoplastic rather than an inflammatory nature. However, although the fundamental events underlying the pathogenesis of LCH are still elusive, recent advances have strikingly improved our understanding of the disease. In particular, the identification of multiple interplays between LCH cells and their tumor microenvironment, along with the recognition of the lesional cytokine storm as a key determinant of LCH progression, has substantiated new opportunities for devising targeted therapeutic approaches. Strikingly, the detection of the rapidly accelerated fibrosarcoma isoform B(V600E) gain-of-function mutation as a genetic alteration recurring in more than 50% of patients has fueled the paradoxical picture of LCH as a tumor of the antigen-presenting cells that can evade rejection by the immune system. Thus, new evidence regarding the ontogeny of LCH cells, as well as a better understanding of the putative immune system frustrating strategy in LCH, may help to define the precise pathogenesis.

NETs: organ-related epigenetic derangements and potential clinical applications

High-throughput next-generation sequencing methods have recently provided a detailed picture of the genetic landscape of neuroendocrine tumors (NETs), revealing recurrent mutations of chromatin-remodeling genes and little-to-no pathogenetic role for oncogenes commonly mutated in cancer. Concurrently, multiple epigenetic modifications have been described across the whole spectrum of NETs, and their putative function as tumorigenic drivers has been envisaged. As result, it is still unclear whether or not NETs are epigenetically-driven, rather than genetically-induced malignancies. Although the NET epigenome profiling has led to the identification of molecularly-distinct tumor subsets, validation studies in larger cohorts of patients are needed to translate the use of NET epitypes in clinical practice. In the precision medicine era, recognition of subpopulations of patients more likely to respond to therapeutic agents is critical, and future studies testing epigenetic biomarkers are therefore awaited. Restoration of the aberrant chromatin remodeling machinery is an attractive approach for future treatment of cancer and in several hematological malignancies a few epigenetic agents have been already approved. Although clinical outcomes of epigenetic therapies in NETs have been disappointing so far, further clinical trials are required to investigate the efficacy of these drugs. In this context, given the immune-stimulating effects of epidrugs, combination therapies with immune checkpoint inhibitors should be tested. In this review, we provide an overview of the epigenetic changes in both hereditary and sporadic NETs of the gastroenteropancreatic and bronchial tract, focusing on their diagnostic, prognostic and therapeutic implications.

LFA-1 expression on CD4+CD45RO+ peripheral blood T-lymphocytes in RR MS: effects induced by rIFNβ-1a

We investigate the in vivo and in vitro effects of short-term treatment with recombinant Interferon beta-1a (rIFNbeta-1a) on CD4(+)CD45RO(+) activated/memory peripheral blood T-lymphocytes (PBTLs) expressing Leukocyte Function Antigen-1 (LFA-1; CD11a/CD18) in relapsing-remitting (RR) Multiple Sclerosis (MS) patients. Blood samples were obtained from 10 RR MS patients before and after 2, 4 and 6 months of rIFNbeta-1a (Avonex) treatment. For each sample, the percentage of CD4(+)CD45RO(+)CD11a(+) (CD11a(dim) and CD11a(bright)) T-cells was evaluated in in vivo PBTLs and in untreated or rIFNbeta-1a (1000 U/ml) or recombinant soluble Intercellular Adhesion Molecule-1 (ICAM-1, the ligand for LFA-1) (400 ng/ml) treated cultured PBTLs by triple fluorescence flow-cytometry (FACS analysis). Soluble ICAM-1 (sICAM-1) serum levels were evaluated by ELISA. In vivo, the percentage of CD4(+)CD45RO(+), CD4(+)CD45RO(+)CD11a(+), CD4(+)CD45RO(+)CD11a(dim) PBTLs increased after 4 and 6 months of rIFNbeta-1a treatment compared to pretreatment and 2 months of treatment (p<0.05). The CD11a expression per se did not change during the time course. Soluble ICAM-1 (sICAM-1) serum levels also increased (p<0.05) after 4 and 6 months of treatment. When T-cells, obtained from the blood of the same patients before and during in vivo treatment, were cultured either untreated or treated with rIFNbeta-1a, they showed an increase in the percentage of CD4(+)CD45RO(+) T-cells expressing CD11a(bright) (p<0.05). The addition of recombinant sICAM-1 to untreated cultures decreased the percentage of CD4(+)CD45RO(+) T-cells expressing CD11a. This last finding seems to support an indirect effect in vivo of rIFNbeta-1a via sICAM-1 on this T-cell subset, since the ICAM-1 soluble form, induced in vivo in serum by rIFNbeta-1a but lacking in in vitro conditions, keeps the percentage of CD11a(+) unchanged within CD4(+)CD45RO(+) T-cells and induces their expression of CD11a(dim), probably preventing T-cells from transmigrating.

Serum exosomes as predictors of clinical response to ipilimumab in metastatic melanoma

ABSTRACT Immunotherapy is effective in metastatic melanoma (MM) but most studies failed in discovering a biomarker predictive of clinical response. Exosomes (Exo) from melanoma cells are detectable in sera of MM patients similarly to those produced by immune cells that control the tumor progression. Here, we investigated by flow-cytometry the levels of Exo from both T-cells and dendritic cells (DCs) in 59 patients with MM treated with IPI and the relative expression of PD-1, CD28 and ICOS as well as CD80 and CD86. We found a significant increment of PD-1 and CD28 expression in patients achieving a clinical response reflected by improvement of both PFS and OS. Furthermore, MM patients receiving IPI who showed extended PFS underwent increased expression of CD80 and CD86 on DC-derived Exo at the end of treatment. These results suggest a possible association of both PD-1 and CD28 up-regulation on immune cell-derived Exo in patients with better clinical response to IPI.

pIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo

BackgroundMesenchymal/stromal stem cells (MSCs) are favorably regarded in anti-cancer cytotherapies for their spontaneous chemotaxis toward inflammatory and tumor environments associated with an intrinsic cytotoxicity against tumor cells. Placenta-derived or TRAIL-engineered adipose MSCs have been shown to exert anti-tumor activity in both in-vitro and in-vivo models of multiple myeloma (MM) while TRAIL-transduced umbilical cord (UC)-MSCs appear efficient inducers of apoptosis in a few solid tumors. However, apoptosis is not selective for cancer cells since specific TRAIL receptors are also expressed by a number of normal cells. To overcome this drawback, we propose to transduce UC-MSCs with a bicistronic vector including the TRAIL sequence under the control of IL-6 promoter (pIL6) whose transcriptional activation is promoted by the MM milieu.MethodsUC-MSCs were transduced with a bicistronic retroviral vector (pMIGR1) encoding for green fluorescent protein (GFP) and modified to include the pIL6 sequence upstream of the full-length human TRAIL cDNA. TRAIL expression after stimulation with U-266 cell conditioned medium, or IL-1α/IL-1β, was evaluated by flow cytometry, confocal microscopy, real-time PCR, western blot analysis, and ELISA. Apoptosis in MM cells was assayed by Annexin V staining and by caspase-8 activation. The cytotoxic effect of pIL6-TRAIL+-GFP+-UC-MSCs on MM growth was evaluated in SCID mice by bioluminescence and ex vivo by caspase-3 activation and X-ray imaging. Statistical analyses were performed by Student’s t test, ANOVA, and logrank test for survival curves.ResultspIL6-TRAIL+-GFP+-UC-MSCs significantly expressed TRAIL after stimulation by either conditioned medium or by IL-1α/IL-1β, and induced apoptosis in U-266 cells. Moreover, when systemically injected in SCID mice intratibially xenografted with U-266, those cells underwent within MM tibia lesions and significantly reduced the tumor burden by specific induction of apoptosis in MM cells as revealed by caspase-3 activation.ConclusionsOur tumor microenvironment-sensitive model of anti-MM cytotherapy is regulated by the axis pIL6/IL-1α/IL-1β and appears suitable for further preclinical investigation not only in myeloma bone disease in which UC-MSCs would even participate to bone healing as described, but also in other osteotropic tumors whose milieu is enriched of cytokines triggering the pIL6.

Uric Acid, Metabolic Syndrome and Atherosclerosis: The Chicken or the Egg, Which Comes First?

BACKGROUND A great debate in literature exists nowadays on the role of uric acid as a marker of cardiovascular and metabolic organ damage or a risk factor for cardiovascular and metabolic disease. METHODS The study aimed to determine the relationship among serum uric acid and metabolic syndrome and atherosclerosis, by means of carotid intima media-thickness, in a cohort of 811 otherwise healthy overweight/obese subjects, without overt atherosclerosis not using any kind of drug. RESULTS Uric acid levels were positively related to male gender, waist circumference, BMI, systolic and diastolic pressure levels, fasting insulin, fasting glucose, HOMA-IR, triglycerides, total cholesterol, LDL cholesterol, the presence of metabolic syndrome and the number of the components of metabolic syndrome and negatively related to HDL cholesterol levels. No correlation was found between uric acid and carotid intima media thickness. At the multiple regression analysis, only waist circumference and triglycerides (positively) and HDL-cholesterol (negatively) maintained an independent association with uric acid as dependent variable, while age, female gender and uric acid showed a significant independent association with metabolic syndrome as dependent variable. Moreover, the analysis of the odd ratios showed that the risk of developing metabolic syndrome was consistent with uric acid levels ranging from 3 mg/dl to 8 mg/dl. CONCLUSION The presence of metabolic syndrome does not seem to provide hyperuricemia. By contrast, higher serum uric acid level may predict the risk of metabolic syndrome. Moreover, our results suggest that uric acid cannot be considered a risk factor for early atherosclerosis, at least when assessed using carotid ultrasound.

Immune system and melanoma biology: a balance between immunosurveillance and immune escape

Melanoma is one of the most immunogenic tumors and its relationship with host immune system is currently under investigation. Many immunomodulatory mechanisms, favoring melanomagenesis and progression, have been described to interfere with the disablement of melanoma recognition and attack by immune cells resulting in immune resistance and immunosuppression. This knowledge produced therapeutic advantages, such as immunotherapy, aiming to overcome the immune evasion. Here, we review the current advances in cancer immunoediting and focus on melanoma immunology, which involves a dynamic interplay between melanoma and immune system, as well as on effects of “targeted therapies” on tumor microenvironment for combination strategies.

Characterization of a Rare Nonpathogenic Methylenetetrahydrofolatereductase (MTHFR) Gene Mutation p.Lys215del in a Southern Italian family

To the Editor: We read with interest the article by Froese et al. [2016] reporting an exhaustively updated review on methylenetetrahydrofolatereductase (MTHFR, MIM# 607093) gene mutations. Among the 109 mutations from 192 patients described as causing severe MTHFR deficiency, the authors suggest that an inframe codon 215 deletion with consequent loss of a lysine at the protein level (c.643 645delAAG; p.Lys215del) can cause hyperhomocysteinemia, methylation alterations, and related diseases. This mutation was previously detected by the same investigators in a subject from 76 selected patients with clinical and biochemical evidence of MTHFR deficiency. However, in addition to the p.Lys215del, this patient carried a missense change (p.His127Tyr) on a FADbinding residue and the heterozygous c.677C>T (C677T) polymorphism [Burda et al., 2015], and showed brain atrophy and hydrocephalus by magnetic resonance imaging [Huemer et al., 2016]. The deletion was deposited as “Pathogenic” in the clinical variant database ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/;