iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

An Overview of In Vitro Assays of 64Cu-, 68Ga-, 125I-, and 99mTc-Labelled Radiopharmaceuticals Using Radiometric Counters in the Era of Radiotheranostics.

Radionuclides are unstable isotopes that mainly emit alpha (α), beta (ß) or gamma (γ) radiation through radiation decay. Therefore, they are used in the biomedical field to label biomolecules or drugs for diagnostic imaging applications, such as positron emission tomography (PET) and/or single-photon emission computed tomography (SPECT). A growing field of research is the development of new radiopharmaceuticals for use in cancer treatments. Preclinical studies are the gold standard for translational research. Specifically, in vitro radiopharmaceutical studies are based on the use of radiopharmaceuticals directly on cells. To date, radiometric ß- and γ-counters are the only tools able to assess a preclinical in vitro assay with the aim of estimating uptake, retention, and release parameters, including time- and dose-dependent cytotoxicity and kinetic parameters. This review has been designed for researchers, such as biologists and biotechnologists, who would like to approach the radiobiology field and conduct in vitro assays for cellular radioactivity evaluations using radiometric counters. To demonstrate the importance of in vitro radiopharmaceutical assays using radiometric counters with a view to radiogenomics, many studies based on 64Cu-, 68Ga-, 125I-, and 99mTc-labeled radiopharmaceuticals have been revised and summarized in this manuscript.

Long-Term Effectiveness of BNT162b2 Pfizer-BioNTech mRNA-Based Vaccine on B Cell Compartment: Efficient Recall of SARS-CoV-2-Specific Memory B Cells.

At present, there is a lack of clinical evidence about the impact and long-term durability of the immune response induced by the third dose of mRNA vaccines. In this study, we followed up the B cell compartment behavior in a cohort of immunocompetent individuals three and six months after the third dose of vaccine. During this period, some subjects contracted the virus. In uninfected vaccinated subjects, we did not report any changes in serum spike-specific IgG levels, with a significant reduction in IgA. Instead, subjects recovered from natural infection showed a significant increase in both specific IgG and IgA. Moreover, we showed a time-related decrease in IgG neutralizing potential to all SARS-CoV-2 variants of concern (VOC) in uninfected compared to recovered subjects, who displayed an increased neutralizing ability, particularly against the omicron variant. Finally, we underlined the presence of a pool of SARS-CoV-2-specific B cells in both groups that are prone to respond to restimulation, as demonstrated by their ability to differentiate into plasma cells and to produce anti-SARS-CoV-2-specific immunoglobulins. These data lead us to assert the long-term effectiveness of the BNT162b2 vaccine in contrasting the severe form of the pathology and prevent COVID-19-associated hospitalization.

Immune Response after the Fourth Dose of SARS-CoV-2 mRNA Vaccine Compared to Natural Infection in Three Doses' Vaccinated Solid Organ Transplant Recipients.

Solid organ transplant recipients (SOTRs) show higher rates of COVID-19 breakthrough infection than the general population, and nowadays, vaccination is the key preventative strategy. Nonetheless, SOTRs show lower vaccine efficacy for the prevention of severe COVID-19. Moreover, the emergence of new SARS-CoV-2 variants of concern has highlighted the need to improve vaccine-induced immune responses by the administration of repeated booster doses. In this study, we analyzed the humoral and cellular responses in a cohort of 25 SOTRs, including 15 never-infected SOTRs who received the fourth dose of the mRNA vaccine and 10 SOTRs who contracted SARS-CoV-2 infection after the third dose. We analyzed the serum IgG and IgA levels through CLIA or ELISA, respectively, and the Spike-specific T cells by ELISpot assay. We report a significant increase in anti-Spike IgG and no differences in IgA secretion in both groups of patients before and after the booster dose or the natural infection. Still, we show higher IgA levels in recovered SOTRs compared to the fourth dose recipients. Conversely, we show the maintenance of a positive Spike-specific T-cell response in SOTRs who received the fourth dose, which, instead, was significantly increased in SOTRs who contracted the infection. Our results suggest that the booster, either through the fourth dose or natural infection, in vulnerable poor responder SOTRs, improves both humoral and cellular-specific immune responses against SARS-CoV-2.

Analysis of the Specific Immune Response after the Third Dose of mRNA COVID-19 Vaccines in Organ Transplant Recipients: Possible Spike-S1 Reactive IgA Signature in Protection from SARS-CoV-2 Infection.

Background: Several studies have indicated that anti-SARS-CoV-2 mRNA vaccinations are less effective in inducing robust immune responses among solid organ transplant recipients (SOTRs) compared with the immunocompetent. The third dose of vaccine in SOTRs showed promising results of immunogenicity, even though clinical studies have suggested that immunocompromised subjects are less likely to build a protective immune response against SARS-CoV-2 resulting in lower vaccine efficacy for the prevention of severe COVID-19. Methods: Serological IgG and IgA were analyzed through CLIA or ELISA, respectively, while Spike-specific T cells were detected by ELISpot assay after the second and third dose of vaccine in 43 SOTRs. Results: The third dose induced an improvement in antibody response against SARS-CoV-2. We also reported a strong correlation between specific humoral and cellular responses after the third dose, even though we did not see significant changes in the magnitude of the SARS-CoV-2-specific T cell response. SOTRs who contracted the SARS-CoV-2 infection after the third dose, despite eliciting a positive IgG response, failed to mount an anti-Spike-S1 IgA response, both after the third dose and after SARS-CoV-2 infection. Conclusions: We can conclude that serum IgA detection can be helpful, along with IgG detection, for the evaluation of vaccine efficacy, principally in fragile subjects at high risk of infection.

Specific Anti-SARS-CoV-2 Humoral and Cellular Immune Responses After Booster Dose of BNT162b2 Pfizer-BioNTech mRNA-Based Vaccine: Integrated Study of Adaptive Immune System Components.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is modifying human activity all over the world with significant health and economic burden. The advent of the SARS-CoV-2 pandemic prompted the scientific community to learn the virus dynamics concerning transmissibility, epidemiology, and usefulness of vaccines in fighting emerging health hazards. Pieces of evidence suggest that the first and second doses of mRNA vaccines induce a significant antibody response in vaccinated subjects or patients who recovered from SARS-CoV-2 infection, demonstrating the importance of the previously formed memory. The aim of this work has been to investigate the effects of BNT162b2 Pfizer-BioNTech mRNA-based vaccine booster dose in a cohort of 11 uninfected immunocompetent (ICs), evaluating the humoral and cellular responses, with more carefulness on memory B and T cells. Our findings underscore the potential benefit of the third dose of mRNA vaccine on the lifespan of memory B and T cells, suggesting that booster doses could increase protection against SARS-CoV-2 infection.

Erratum: Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

[This corrects the article DOI: 10.1016/j.omtm.2021.05.002.].

Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

Endotoxin content is a critical factor that affects the safety of biological pharmaceutical products. International pharmacopoeias describe several reference methods to determine endotoxin levels in advanced therapy medicinal product (ATMP) preparations. Administration of ATMPs must be done as rapidly as possible to ensure complete viability and potency of the cellular product. To evaluate the endotoxin content in the shortest time possible, we chose to validate an alternative method based on the use of the Charles River Portable Testing System (PTS) and FDA-approved cartridges, compliant with the requirements of the European Pharmacopoeia and providing results in <20 min. Here, we describe a unique and complete validation approach for instrument, personnel, and analytical method for assessment of endotoxins in ATMP matrices. The PTS system provides high sensitivity and fast quantitative results and uses less raw material and accessories compared with compendial methods. It is also less time consuming and less prone to operator variability. Our validation approach is suitable for a validated laboratory with trained personnel capable of conducting the ATMP release tests, and with very low intra-laboratory variability, and meets the criteria required for an alternative approach to endotoxin detection for in-process and product-release testing of ATMPs.

Successful Use of Heterologous CMV-Reactive T Lymphocyte to Treat Severe Refractory Cytomegalovirus (CMV) Infection in a Liver Transplanted Patient: Correlation of the Host Antiviral Immune Reconstitution with CMV Viral Load and CMV miRNome.

Cytomegalovirus (CMV) infection is the most significant viral infection in hosts with compromised immune systems as solid organ transplant patients. Despite significant progress being made in the prevention of CMV disease in these patients, further therapeutic strategies for CMV disease and for the CMV reactivation prevention are needed. Here, we describe the outcome of the infusion of in vitro expanded CMV-reactive T-cells, taken from a healthy CMV-seropositive donor, in a liver-transplanted recipient with a refractory recurrent CMV. In this particular case, adoptive transfer of allogenic CMV-reactive T-lymphocytes resulted in the clearance of CMV infection and resolution of the pathological manifestations of the patient. In the study we also investigated circulating miRNAs, both cellular and viral, as potential biomarkers during the course of CMV infection. The results indicate that the infusion of allogenic CMV-reactive T-cells can be an effective strategy to treat CMV infection recurrence when the generation of autologous virus specific T cell clones is not possible.

Cigarette smoke inhibits the NLRP3 inflammasome and leads to caspase-1 activation via the TLR4-TRIF-caspase-8 axis in human macrophages.

The NLRP3 inflammasome is formed by the sensor NLRP3, the adaptor ASC, and pro-caspase-1. Assembly and activation of the inflammasome trigger caspase-1-dependent cleavage of pro-IL-1ß and pro-IL-18 into their secreted forms. Cigarette smoke is a risk factor for chronic inflammatory diseases and is associated with macrophage dysfunction. The impact of cigarette smoke on NLRP3-dependent responses in macrophages is largely unknown. Herein, we investigated the effects of cigarette smoke extract (CSE) on the NLRP3 inflammasome in human monocyte-derived macrophages (MDMs) and THP-1 cells stimulated with lipopolysaccharide (LPS) and LPS plus the NLRP3 inflammasome activator ATP. We found that CSE inhibited the release of IL-1ß and IL-18 as well as the expression of NLRP3 acting mainly at the transcriptional level. Interestingly, we found that CSE increased the caspase-1 activity via an NLRP3-independent and TLR4-TRIF-caspase-8-dependent pathway. Activation of caspase-1 by CSE led to a reduction of the basal glycolytic flux and impaired glycolytic burst in response to LPS. Overall, our findings unveil novel pathways leading to immune-metabolic alterations in human macrophages exposed to cigarette smoke. These mechanisms may contribute to macrophage dysfunction and increased risk of infection in smokers.

Molecular and cellular interplay in virus-induced tumors in solid organ recipients.

Patients following solid organ transplantation show a higher risk of developing cancer compared to the general population. Elevated risk is likely due to the interplay of a combination of factors, such as chronic inflammation, coexisting medical conditions, immunosuppressive regimen and persistent infection with oncogenic viruses. In addition, the tumor microenvironment plays a pivotal role in cancer progression, by driving recruitment and in situ differentiation of anti-inflammatory cells of the adaptive and innate immune system such as regulatory T cells, Th17, Dendritic Cells, Myeloid Derived Suppressor Cells, Type 2 Macrophages. Here we discuss the molecular role and the contribution to oncogenesis of Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) and Hepatitis C virus (HCV) in immunocompromised patients and describe how these viruses may contribute to oncogenesis both directly and indirectly.

Global profiling of viral and cellular non-coding RNAs in Epstein-Barr virus-induced lymphoblastoid cell lines and released exosome cargos.

The human EBV-transformed lymphoblastoid cell line (LCL), obtained by infecting peripheral blood monocular cells with Epstein-Barr Virus, has been extensively used for human genetic, pharmacogenomic, and immunologic studies. Recently, the role of exosomes has also been indicated as crucial in the crosstalk between EBV and the host microenvironment. Because the role that the LCL and LCL exosomal cargo might play in maintaining persistent infection, and since little is known regarding the non-coding RNAs of LCL, the aim of our work was the comprehensive characterization of this class of RNA, cellular and viral miRNAs, and cellular lncRNAs, in LCL compared with PBMC derived from the same donors. In this study, we have demonstrated, for the first time, that all the viral miRNAs expressed by LCL are also packaged in the exosomes, and we found that two miRNAs, ebv-miR-BART3 and ebv-miR-BHRF1-1, are more abundant in the exosomes, suggesting a microvescicular viral microRNA transfer. In addition, lncRNA profiling revealed that LCLs were enriched in lncRNA H19 and H19 antisense, and released these through exosomes, suggesting a leading role in the regulation of the tumor microenvironment.

Expression levels and clinical-pathological correlations of HER2/neu in primary and metastatic human breast cancer.

In this retrospective study we assessed the expression of the HER2/neu oncogene product in a series of 574 consecutive breast cancer cases, all recruited at the Maurizio Ascoli Cancer Center of Civico Hospital, in Palermo, between January 1998 and June 2003. The HER2/neu expression was evaluated using immunohistochemistry and scored from 0 to +3 as per FDA recommendations. The HER2/neu expression levels were related to the clinical-pathological features of the disease, including tumor size, nodal and menopausal status, estrogen and progesterone receptors, and hormonal or chemotherapeutic treatment. In 108 patients with a follow-up period of 3 years or more, the HER2/neu expression was also related to their survival characteristics. A significant correlation (P = 0.011) between HER2/neu +3 and estrogen receptor-negative cases was observed in the 487 M0 patients. In addition, HER2/neu +3 cases were associated with a positive nodal status (57.4%), although this association was not quite significant (P = 0.06). More importantly, follow-up data revealed that, in the 91 M0 patients with an average follow-up period of 37 months, the percentage of HER2/neu +3 patients who relapsed was remarkably greater (54.8%) than that observed for the HER2/neu +1/0 cases when combined (34.2%). Furthermore, the disease-free interval (DFI) was 47 months in the HER2/neu +1/0 group, while it dropped to 45 months in c-HER2/neu +3 cases. Although the limited number of cases does not allow us to draw any definitive conclusions, our data suggest that high expression levels of HER2/neu +3 are associated with an early relapse and a shorter disease-free interval in M0 breast cancer patients.

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells.

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.

Increased estrogen formation and estrogen to androgen ratio in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVE: It has been proposed that physiologic levels of estrogens stimulate immune responses whereas androgens suppress inflammatory reactions. Thus, prevalence of synovial androgens relative to estrogens would be favorable in rheumatoid arthritis (RA). We investigated synovial fluid (SF) concentrations of several estrogens and androgens and conversion products of the sex steroid precursor dehydroepiandrosterone (DHEA) in supernatants of mixed synoviocytes. METHODS: SF steroid concentrations were measured by high performance liquid chromotography and mass spectrometry in 12 patients with RA and 8 subjects with traumatic knee injury (noninflammatory controls). Conversion of DHEA to downstream hormones was measured by thin-layer chromatography and phosphorimaging detection in 3 patients with RA and 3 patients with osteoarthritis (OA). RESULTS: Overall, SF concentration of free estrogens tended to be higher in RA patients versus controls (p < 0.06). Molar ratio of free SF estrogens/free SF androgens was elevated in RA compared to controls (1.17 +/- 0.32 vs 0.29 +/- 0.08, without unit; p = 0.017). The free SF concentration of the precursor androstenedione was significantly higher in RA patients than in controls (104.6 +/- 32.6 vs 30.4 +/- 0.4 ng/ml; p = 0.011), and SF estrone the aromatase conversion product of androstenedione was also elevated in RA compared to controls (13.6 +/- 2.6 vs 6.6 +/- 0.8 ng/ml; p = 0.035). The biologically active estrogen derivatives, 16a-hydroxyestrone and 4-hydroxyestradiol, were both higher in RA compared to controls (p = 0.085 and p = 0.044, respectively). In mixed RA synoviocytes, DHEA conversion yielded high local levels of 17beta-estradiol (708 pmol/l = 0.193 ng/ml) compared to testosterone (88 pmol/l = 0.026 ng/ml). CONCLUSION: SF levels of estrogens relative to androgens are significantly elevated, while those of androgens are markedly reduced, in patients with RA compared to controls. This imbalance is most probably due to increased aromatase activity. Thus, an available steroid precursor, such as DHEA, may be rapidly converted to proinflammatory estrogens in the synovial tissue, which may in turn stimulate the inflammatory process in patients with RA.

Tissue content of hydroxyestrogens in relation to survival of breast cancer patients.

PURPOSE: The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long-term follow-up of patients. EXPERIMENTAL DESIGN: Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues. RESULTS: Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha-hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008). CONCLUSIONS: Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients.

Altered androgen metabolism eventually leads hepatocellular carcinoma to an impaired hormone responsiveness.

Sex steroid hormones are thought, among several other risk factors, to play a role in liver malignancies. For example, from epidemiological studies in hepatocellular carcinoma (HCC), a clear disadvantage for male sex is evident. In addition, elevated levels of serum testosterone (T) and increased T to Estradiol (E(2)) ratio have been reported to predict an increased risk of HCC for male cirrhotic patients. On the other hand, palliative treatment of liver cancer patients with anti-hormones has been widely used in the past. However, the molecular mechanism(s) underlying sex steroid action on either normal or transformed liver cells, have not yet been fully clarified, nor endocrine discriminants have been satisfactorily assessed for an adequate characterization of liver cancer tissues. In this paper, we report studies on hormonal status of human liver tissues and cells, especially focusing on androgens, to better define endocrine end-points of interest for HCC. A consistent evidence from ex vivo or in vitro systems strongly suggests that high affinity binding sites of androgens are expressed at sufficient concentrations to induce a biological response in either normal or phenotipically transformed hepatocytes; in the latter, however, high heterogeneity and/or more scattering concentrations were encountered. Further, experimental data seem to suggest that lack of response to androgens may be due to a rapid metabolic conversion of steroids by neoplastic tissues and cells. Cancer hepatocytes privilege in fact 5beta more than 5alpha metabolic pathway of androgens. This may eventually lead biologically active androgens to be transformed into less active derivatives, as it occurs for T which is massively converted (>90% at 6 h) thus hindering the whole mechanism of action of androgens.