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Background and aimsInducible T-cell Co-Stimulator (ICOS) present on T-lymphocytes and its ligand ICOSL expressed by myeloid cells play multiple roles in regulating T-cell functions. However, recent evidence indicates that reverse signalling involving ICOSL is also important in directing the differentiation of monocyte-derived cells. In this study, we investigated the involvement of ICOS/ICOSL dyad in modulating macrophage functions during the evolution of metabolic dysfunction-associated steatohepatitis (MASH).ResultsIn animal models of MASH, ICOS was selectively up-regulated on CD8+ T-cells in parallel with an expansion of ICOSL-expressing macrophages. An increase in circulating soluble ICOSL was also evident in patients with MASH as compared to healthy individuals. ICOSL knockout (ICOSL-/-) mice receiving choline/methionine deficient (MCD) diet for 6 weeks had milder steatohepatitis than wild type mice. MASH improvement was confirmed in mice fed with cholesterol-enriched Western diet for 24 weeks in which ICOSL deficiency greatly reduced liver fibrosis along with the formation of crown-like macrophage aggregates producing the pro-fibrogenic mediators osteopontin (OPN) and galectin-3 (Gal-3). These effects associated with a selective shewing of F4-80+/CD11bhigh monocyte-derived macrophages (MoMFs) expressing the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) to CD11blow/F4-80+ cells positive for the Kupffer cell marker C-type lectin-like type 2 receptor (CLEC-2), thus indicating an increased MoMF maturation toward monocyte-derived Kupffer cells.ConclusionsThese results suggest that CD8+ T-cells interaction with monocyte-derived macrophages through ICOS/ICOSL critically supports a specific subset of TREM2+-expressing cells contributing to the evolution of steatohepatitis. The data also point ICOS/ICOSL dyad as a possible target for therapeutic interventions in MASH.

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.

Inducible T cell co-stimulator (ICOS), an immune checkpoint protein expressed on activated T cells and its unique ligand, ICOSL, which is expressed on antigen-presenting cells and non-hematopoietic cells, have been extensively investigated in the immune response. Recent findings showed that a soluble recombinant form of ICOS (ICOS-Fc) can act as an innovative immunomodulatory drug as both antagonist of ICOS and agonist of ICOSL, modulating cytokine release and cell migration to inflamed tissues. Although the ICOS-ICOSL pathway has been poorly investigated in the septic context, a few studies have reported that septic patients have reduced ICOS expression in whole blood and increased serum levels of osteopontin (OPN), that is another ligand of ICOSL. Thus, we investigated the pathological role of the ICOS-ICOSL axis in the context of sepsis and the potential protective effects of its immunomodulation by administering ICOS-Fc in a murine model of sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in five-month-old male wild-type (WT) C57BL/6, ICOS-/-, ICOSL-/- and OPN-/- mice. One hour after the surgical procedure, either CLP or Sham (control) mice were randomly assigned to receive once ICOS-Fc, F119SICOS-Fc, a mutated form uncapable to bind ICOSL, or vehicle intravenously. Organs and plasma were collected 24 h after surgery for analyses. When compared to Sham mice, WT mice that underwent CLP developed within 24 h a higher clinical severity score, a reduced body temperature, an increase in plasma cytokines (TNF-α, IL-1β, IL-6, IFN-γ and IL-10), liver injury (AST and ALT) and kidney (creatinine and urea) dysfunction. Administration of ICOS-Fc to WT CLP mice reduced all of these abnormalities caused by sepsis. Similar beneficial effects were not seen in CLP-mice treated with F119SICOS-Fc. Treatment of CLP-mice with ICOS-Fc also attenuated the sepsis-induced local activation of FAK, P38 MAPK and NLRP3 inflammasome. ICOS-Fc seemed to act at both sides of the ICOS-ICOSL interaction, as the protective effect was lost in septic knockout mice for the ICOS or ICOSL genes, whereas it was maintained in OPN knockout mice. Collectively, our data show the beneficial effects of pharmacological modulation of the ICOS-ICOSL pathway in counteracting the sepsis-induced inflammation and organ dysfunction.

Chronic inflammation contributes to the pathogenesis of many diseases, including apparently unrelated conditions such as metabolic disorders, cardiovascular diseases, neurodegenerative diseases, osteoporosis, and tumors, but the use of conventional anti-inflammatory drugs to treat these diseases is generally not very effective given their adverse effects. In addition, some alternative anti-inflammatory medications, such as many natural compounds, have scarce solubility and stability, which are associated with low bioavailability. Therefore, encapsulation within nanoparticles (NPs) may represent an effective strategy to enhance the pharmacological properties of these bioactive molecules, and poly lactic-co-glycolic acid (PLGA) NPs have been widely used because of their high biocompatibility and biodegradability and possibility to finely tune erosion time, hydrophilic/hydrophobic nature, and mechanical properties by acting on the polymer’s composition and preparation technique. Many studies have been focused on the use of PLGA-NPs to deliver immunosuppressive treatments for autoimmune and allergic diseases or to elicit protective immune responses, such as in vaccination and cancer immunotherapy. By contrast, this review is focused on the use of PLGA NPs in preclinical in vivo models of other diseases in which a key role is played by chronic inflammation or unbalance between the protective and reparative phases of inflammation, with a particular focus on intestinal bowel disease; cardiovascular, neurodegenerative, osteoarticular, and ocular diseases; and wound healing.

Monogenic autoimmune disorders represent an important tool to understand the mechanisms behind central and peripheral immune tolerance. Multiple factors, both genetic and environmental, are known to be involved in the alteration of the immune activation/immune tolerance homeostasis typical of these disorders, making it difficult to control the disease. The latest advances in genetic analysis have contributed to a better and more rapid diagnosis, although the management remains confined to the treatment of clinical manifestations, as there are limited studies on rare diseases. Recently, the correlation between microbiota composition and the onset of autoimmune disorders has been investigated, thus opening up new perspectives on the cure of monogenic autoimmune diseases. In this review, we will summarize the main genetic features of both organ-specific and systemic monogenic autoimmune diseases, reporting on the available literature data on microbiota alterations in these patients.

This work presents a computational model to study the degradation behavior of polyester-based three-dimensional (3D) functionalized scaffolds for bone regeneration. As a case study, we investigated the behavior of a 3D-printed scaffold presenting a functionalized surface with ICOS-Fc, a bioactive protein able to stimulate bone regeneration and healing, inhibiting osteoclast activity. The aim of the model was to optimize the scaffold design to control its degradation and thus the release of grafted protein over time and space. Two different scenarios were considered: (i) a scaffold without macroporosity presenting a functionalized external surface; and (ii) a scaffold presenting an internal functionalized macroporous architecture with open channels to locally deliver the degradation products.

The most important limitations of chemotherapeutic agents are severe side effects and the development of multi-drug resistance. Recently, the clinical successes achieved with immunotherapy have revolutionized the treatment of several advanced-stage malignancies, but most patients do not respond and many of them develop immune-related adverse events. Loading synergistic combinations of different anti-tumor drugs in nanocarriers may enhance their efficacy and reduce life-threatening toxicities. Thereafter, nanomedicines may synergize with pharmacological, immunological, and physical combined treatments, and should be increasingly integrated in multimodal combination therapy regimens. The goal of this manuscript is to provide better understanding and key considerations for developing new combined nanomedicines and nanotheranostics. We will clarify the potential of combined nanomedicine strategies that are designed to target different steps of the cancer growth as well as its microenvironment and immunity interactions. Moreover, we will describe relevant experiments in animal models and discuss issues raised by translation in the human setting.

The interest elicited by the large microbial population colonizing the human gut has ancient origins and has gone through a long evolution during history. However, it is only in the last decades that the introduction of high-throughput technologies has allowed to broaden this research field and to disentangle the numerous implications that gut microbiota has in health and disease. This comprehensive ecosystem, constituted mainly by bacteria but also by fungi, parasites, and viruses, is proven to be involved in several physiological and pathological processes that transcend the intestinal homeostasis and are deeply intertwined with apparently unrelated body systems, such as the immune and the nervous ones. In this regard, a novel speculation is the relationship between the intestinal microbial flora and the pathogenesis of some neurological and neurodevelopmental disorders, including the clinical entities defined under the umbrella term of autism spectrum disorders. The bidirectional interplay has led researchers to coin the term gut-brain-immune system axis, subverting the theory of the brain as an immune-privileged site and underscoring the importance of this reciprocal influence already from fetal life and especially during the pre- and post-natal neurodevelopmental process. This revolutionary theory has also unveiled the possibility to modify the gut microbiota as a way to treat and even to prevent different kinds of pathologies. In this sense, some attempts have been made, ranging from probiotic administration to fecal microbiota transplantation, with promising results that need further elaboration. This state-of-art report will describe the main aspects regarding the human gut microbiome and its specific role in the pathogenesis of autism and its related disorders, with a final discussion on the therapeutic and preventive strategies aiming at creating a healthy intestinal microbial environment, as well as their safety and ethical implications.

The liver capacity to recover from acute liver injury is a critical factor in the development of acute liver failure (ALF) caused by viral infections, ischemia/reperfusion or drug toxicity. Liver healing requires the switching of pro-inflammatory monocyte-derived macrophages(MoMFs) to a reparative phenotype. However, the mechanisms involved are still incompletely characterized. In this study we investigated the contribution of T-lymphocyte/macrophage interaction through the co-stimulatory molecule Inducible T-cell co-stimulator (ICOS; CD278) and its ligand (ICOSL; CD275) in modulating liver repair. The role of ICOS/ICOSL dyad was investigated during the recovery from acute liver damage induced by a single dose of carbon tetrachloride (CCl4). Flow cytometry of non-parenchymal liver cells obtained from CCl4-treated wild-type mice revealed that the recovery from acute liver injury associated with a specific up-regulation of ICOS in CD8+ T-lymphocytes and with an increase in ICOSL expression involving CD11bhigh/F4-80+ hepatic MoMFs. Although ICOS deficiency did not influence the severity of liver damage and the evolution of inflammation, CCl4-treated ICOS knockout (ICOS-/-) mice showed delayed clearance of liver necrosis and increased mortality. These animals were also characterized by a significant reduction of hepatic reparative MoMFs due to an increased rate of cell apoptosis. An impaired liver healing and loss of reparative MoMFs was similarly evident in ICOSL-deficient mice or following CD8+ T-cells ablation in wild-type mice. The loss of reparative MoMFs was prevented by supplementing CCl4-treated ICOS-/- mice with recombinant ICOS (ICOS-Fc) which also stimulated full recovery from liver injury. These data demonstrated that CD8+ T-lymphocytes play a key role in supporting the survival of reparative MoMFs during liver healing trough ICOS/ICOSL-mediated signaling. These observations open the possibility of targeting ICOS/ICOSL dyad as a novel tool for promoting efficient healing following acute liver injury.

Vertebral compression fractures are typical of osteoporosis and their treatment can require the injection of a cement through a minimally invasive procedure to restore vertebral body height. This study reports the development of an injectable calcium sulphate-based composite cement able to stimulate bone regeneration while inhibiting osteoclast bone resorption. To this aim, different types of strontium-containing mesoporous glass particles (Sr-MBG) were added to calcium sulphate powder to impart a pro-osteogenic effect, and the influence of their size and textural features on the cement properties was investigated. Anti-osteoclastogenic properties were conferred by incorporating into poly(lactic-co-glycolic)acid (PLGA) nanoparticles, a recombinant protein able to inhibit osteoclast activity (i.e., ICOS-Fc). Radiopaque zirconia nanoparticles (ZrO2) were also added to the formulation to visualize the cement injection under fluoroscopy. The measured cement setting times were suitable for the clinical practice, and static mechanical testing determined a compressive strength of ca. 8 MPa, comparable to that of human vertebral bodies. In vitro release experiments indicated a sustained release of ICOS-Fc and Sr2+ ions up to 28 days. Overall, the developed cement is promising for the treatment of vertebral compression fractures and has the potential to stimulate bone regeneration while releasing a biomolecule able to limit bone resorption.

High-grade melanoma remains a major life-threatening illness despite the improvement in therapeutic control that has been achieved by means of targeted therapies and immunotherapies in recent years. This work presents a preclinical-level test of a multi-pronged approach that includes the loading of immunotherapeutic (ICOS-Fc), targeted (sorafenib), and chemotherapeutic (temozolomide) agents within Intralipid®, which is a biocompatible nanoemulsion with a long history of safe clinical use for total parenteral nutrition. This drug combination has been shown to inhibit tumor growth and angiogenesis with the involvement of the immune system, and a key role is played by ICOS-Fc. The inhibition of tumor growth in subcutaneous melanoma mouse models has been achieved using sub-therapeutic drug doses, which is most likely the result of the nanoemulsion’s targeting properties. If translated to the human setting, this approach should therefore allow therapeutic efficacy to be achieved without increasing the risk of toxic effects.

Conventional therapies for immune-mediated diseases, including autoimmune disorders, transplant reactions, and allergies, have undergone a radical evolution in the last few decades; however, they are still not specific enough to avoid widespread immunosuppression. The idea that vaccine usage could be extended beyond its traditional immunogenic function by encompassing the ability of vaccines to induce antigen-specific tolerance may revolutionize preventive and therapeutic strategies in several clinical fields that deal with immune-mediated disorders. This approach has been supported by improved data relating to the several mechanisms involved in controlling unwanted immune responses and allowing peripheral tolerance. Given these premises, several approaches have been developed to induce peripheral tolerance against the antigens that are involved in the pathological immune response, including allergens, autoantigens, and alloantigens. Technological innovations, such as nucleic acid manipulation and the advent of micro- and nanoparticles, have further supported these novel preventive and therapeutic approaches. This review focuses on the main strategies used in the development of tolerogenic vaccines, including the technological issues used in their design and the role of “inverse adjuvants”. Even though most studies are still limited to the preclinical field, the enthusiasm generated by their results has prompted some initial clinical trials, and they show great promise for the future management of immune-mediated pathological conditions.

Recently, we demonstrated that inducible T-cell costimulator (ICOS) shares its unique ligand (ICOSL) with osteopontin (OPN), and OPN/ICOSL binding promotes tumor metastasis and angiogenesis in the 4T1 breast cancer model. Literature showed that OPN promotes melanoma metastasis by suppressing T-cell activation and recruiting myeloid suppressor cells (MDSC). On the opposite, ICOS/ICOSL interaction usually sustains an antitumor response. Here, we engineered murine B16F10 melanoma cells, by transfecting or silencing ICOSL. In vitro data showed that loss of ICOSL favors anchorage-independent growth and induces more metastases in vivo, compared to ICOSL expressing cells. To dissect individual roles of the three molecules, we compared data from C57BL/6 with those from OPN-KO, ICOS-KO, and ICOSL-KO mice, missing one partner at a time. We found that OPN produced by the tumor microenvironment (TME) favors the metastasis by interacting with stromal ICOSL. This activity is dominantly inhibited by ICOS expressed on TME by promoting Treg expansion. Importantly, we also show that OPN and ICOSL highly interact in human melanoma metastases compared to primary tumors. Interfering with this binding may be explored in immunotherapy either for nonresponding or patients resistant to conventional therapies.

This study investigated the effects of free paclitaxel (PTX) and PTX-loaded in pyromellitic nanosponges (PTX-PNS) in reducing in vitro and in vivo melanoma cell growth and invasivity, and in inhibiting angiogenesis. To test the response of cells to the two PTX formulations, the cell viability was evaluated by MTT assay in seven continuous cell lines, in primary melanoma cells, both in 2D and 3D cultures, and in human umbilical vein endothelial cells (HUVECs) after exposure to different concentrations of PTX or PTX-PNS. Cell motility was assessed by a scratch assay or Boyden chamber assay, evaluating cell migration in presence or absence of diverse concentrations of PTX or PTX-PNS. The effect of PTX and PTX-PNS on angiogenesis was evaluated as endothelial tube formation assay, a test able to estimate the formation of three-dimensional vessels in vitro. To assess the anticancer effect of PTX and PTX-PNS in in vivo experiments, the two drug formulations were tested in a melanoma mouse model obtained by B16-BL6 cell implantation in C57/BL6 mice. Results obtained were as follows: 1) MTT analysis revealed that cell proliferation was more affected by PTX-PNS than by PTX in all tested cell lines, in both 2D and 3D cultures; 2) the analysis of the cell migration showed that PTX-PNS acted at very lower concentrations than PTX; 3) tube formation assay showed that PTX-PNS were more effective in inhibiting tube formation than free PTX; and 4) in vivo experiments demonstrated that tumor weights, volumes, and growth were significantly reduced by PTX-PNS treatment with respect to PTX; the angiogenesis and the cell proliferation, detected in the tumor samples with CD31 and Ki-67 antibodies, respectively, indicated that, in the PTX-PNS-treated tumors, the tube formation was inhibited, and a low amount of proliferating cells was present. Taken together, our data demonstrated that our new PTX nanoformulation can respond to some important issues related to PTX treatment, lowering the anti-tumor effective doses and increasing the effectiveness in inhibiting melanoma growth in vivo.

Anaplastic carcinoma of the thyroid (ATC) is a lethal human malignant cancer with median survival of 6 months. To date, no treatment has substantially changed its course, which makes urgent need for the development of novel drugs or novel formulations for drug delivery. Nanomedicine has enormous potential to improve the accuracy of cancer therapy by enhancing availability and stability, decreasing effective doses and reducing side effects of drugs. Camptothecin (CPT) is an inhibitor of DNA topoisomerase-I with several anticancer properties but has poor solubility and a high degradation rate. Previously, we reported that CPT encapsulated in β-cyclodextrin-nanosponges (CN-CPT) increased solubility, was protected from degradation and inhibited the growth of prostate tumor cells both in vitro and in vivo. The aim of this study was to extend that work by assessing the CN-CPT effectiveness on ATC both in vitro and in vivo. Results showed that CN-CPT significantly inhibited viability, clonogenic capacity and cell-cycle progression of ATC cell lines showing a faster and enhanced effect compared to free CPT. Moreover, CN-CPT inhibited tumor cell adhesion to vascular endothelial cells, migration, secretion of pro-angiogenic factors (IL-8 and VEGF-α), expression of β-PIX, belonging to the Rho family activators, and phosphorylation of the Erk1/2 MAPK. Finally, CN-CPT significantly inhibited the growth, the metastatization and the vascularization of orthotopic ATC xenografts in SCID/beige mice without apparent toxic effects in vivo. This work extends the previous insight showing that β-cyclodextrin-nanosponges are a promising tool for the treatment of ATC.

Osteoporotic bone fractures represent a critical clinical issue and require personalized and specific treatments in order to stimulate compromised bone tissue regeneration. In this clinical context, the development of smart nano-biomaterials able to synergistically combine chemical and biological cues to exert specific therapeutic effects (i.e., pro-osteogenic, anti-clastogenic) can allow the design of effective medical solutions. With this aim, in this work, strontium-containing mesoporous bioactive glasses (MBGs) were bio-functionalized with ICOS-Fc, a molecule able to reversibly inhibit osteoclast activity by binding the respective ligand (ICOS-L) and to induce a decrease of bone resorption activity. N2 adsorption analysis and FT-IR spectroscopy were used to assess the successful grafting of ICOS-Fc on the surface of Sr-containing MBGs, which were also proved to retain the peculiar ability to release osteogenic strontium ions and an excellent bioactivity after functionalization. An ELISA-like assay allowed to confirm that grafted ICOS-Fc molecules were able to bind ICOS-L (the ICOS binding ligand) and to investigate the stability of the amide binding to hydrolysis in aqueous environment up to 21 days. In analogy to the free form of the molecule, the inhibitory effect of grafted ICOS-Fc on cell migratory activity was demonstrated by using ICOSL positive cell lines and the ability to inhibit osteoclast differentiation and function was confirmed by monitoring the differentiation of monocyte-derived osteoclasts (MDOCs), which revealed a strong inhibitory effect, also proven by the downregulation of osteoclast differentiation genes. The obtained results showed that the combination of ICOS-Fc with the intrinsic properties of Sr-containing MBGs represents a very promising approach to design personalized solutions for patients affected by compromised bone remodeling (i.e., osteoporosis fractures).

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α4β1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

Osteopontin (OPN) regulates the immune response at multiple levels. Physiologically, it regulates the host response to infections by driving T helper (Th) polarization and acting on both innate and adaptive immunity; pathologically, it contributes to the development of immune-mediated and inflammatory diseases. In some cases, the mechanisms of these effects have been described, but many aspects of the OPN function remain elusive. This is in part ascribable to the fact that OPN is a complex molecule with several posttranslational modifications and it may act as either an immobilized protein of the extracellular matrix or a soluble cytokine or an intracytoplasmic molecule by binding to a wide variety of molecules including crystals of calcium phosphate, several cell surface receptors, and intracytoplasmic molecules. This review describes the OPN structure, isoforms, and functions and its role in regulating the crosstalk between innate and adaptive immunity in autoimmune diseases.

Solid lipid nanoparticles (SLNs) comprise a versatile drug delivery system that has been developed for the treatment of a variety of diseases. The present study will investigate the feasibility of entrapping an active doxorubicin prodrug (a squalenoyl-derivative) in SLNs. The doxorubicin derivative-loaded SLNs are spherically shaped, have a mean diameter of 300–400 nm and show 85% w/w drug entrapment efficiency. The effects on cell growth of loaded SLNs, free doxorubicin and the prodrug have been examined using cytotoxicity and colony-forming assays in both human ovarian cancer line A2780 wild-type and doxorubicin-resistant cells. Further assessments as to the treatment’s ability to induce cell death by apoptosis have been carried out by analyzing annexin-V staining and the activation of caspase-3. The in vitro data demonstrate that the delivery of the squalenoyl-doxorubicin derivative by SLNs increases its cytotoxic activity, as well as its apoptosis effect. This effect was particularly evident in doxorubicin-resistant cells.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development.

Background Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome and its variant Dianzani’s autoimmune lymphoproliferative disease. Analysis of the lymphocyte transcriptome from a patient with this latter condition detected striking over-expression of osteopontin and tissue inhibitor of metalloproteinases-1. Since previous work on osteopontin had detected increased serum levels in these patients, associated with variations of its gene, the aim of this work was to extend the analysis to tissue inhibitor of metalloproteinases-1.Design and Methods Tissue inhibitor of metalloproteinases-1 levels were evaluated in sera and culture supernatants from patients and controls by enzyme-linked immunosorbent assay. Activation- and Fas-induced cell death were induced, in vitro, using anti-CD3 and anti-Fas antibodies, respectively.Results Tissue inhibitor of metalloproteinases-1 levels were higher in sera from 32 patients (11 with autoimmune lymphoproliferative syndrome and 21 with Dianzani’s autoimmune lymphoproliferative disease) than in 50 healthy controls (P