IgA and IgG antibodies in SARS-CoV-2 vaccinated health workers by a homemade ELISA diagnostic test

Abstract The SARS-CoV-2 pandemic has accelerated vaccine development and testing, an important step in its eradication. Health workers were included among the first subjects to be vaccinated, therefore it was necessary to check their immunological status after the first and second dose of vaccine. Serum samples belonging to 24 health workers undergoing vaccination for SARS-CoV-2 (Pfizer-BioNTech) were analysed: for 2 of them it was also possible to obtain a serum sample prior to the first dose of vaccine (zero time); antibody dosing was performed in 18 out of 24 after the first and second vaccination dose; in the remaining 6 healthcare workers a serum sample was obtained only after the second dose. In each serum sample, IgA and IgG antibodies to "Spike Receptor Binding Domain" (RBD) and "Nucleocapsid" (N) proteins were searched by ELISA test. Except for the two subjects for whom it was possible to have a serum sample before vaccination, the others were collected on day 18 from the first dose of vaccine and on day 8 from the second dose. Serum samples collected after the first dose of vaccine showed reactivity to anti-RBD IgG in 11 out of 18 healthcare workers and to IgA in 2 subjects. After the second dose all 24 samples showed the presence of anti-S IgG, while 5 revealed a reactivity for anti-S IgA. Three samples showed reactivity towards anti-N IgG. The ELISA test has shown all its effectiveness in controlling post vaccine immunity and in discriminating natural immunity from vaccine induced immunity

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p < 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

Purinergic receptors in a pleural mesotelioma cell line.

We here demonstrated the expression of 4 (P2Y1,4,6,11) purinoceptors mRNAs coupled to Gq protein. The extracellular ATP, UTP, ADP and UDP caused a transient [Ca2+]i peak followed by a sustained lower phase. Removal of extracellular Ca2+ decreased the initial transient and abolished the plateau phase. Ca2+ signal was blocked by the inhibitor of PLCβ, U73122. Through experiments of fluorescence quenching, it was seen that nucleotides increased plasmamembrane Ca2+ permeability. These results indicate that [Ca2+]i increase was due to activation of P2Y receptors exclusively and that Ca2+ is released from intracellular stores and also enters from the extracellular liquid. We also investigated the effects of purinoceptors on cell proliferation. UTP, ATP and UDP had no significant effects, while ADP causes a drastic cell proliferation decrease in a dose- and time-dependent manner. In conclusion, these results indicate that the expressed purinergic receptors act via PLC activation and that ADP may have a therapeutic potential in MM

Adenosine diphosphate regulates MMP2 and MMP9 activity in malignant mesothelioma cells

Although an association between cancer progression and matrix metalloproteinase (MMP) 2 and MPP9 expression has been known, the expression, nuclear localization, and physiologically controlled activation of these two MMPs have not been investigated in malignant mesothelioma cells. We examined the expression and intracellular localization of MMP2/9 in ZL55 malignant mesothelioma cells, as well as their regulation by ADP. Using real-time PCR, we showed that activation of the P2Y1 receptor by ADP increased the expression of MMP2/9 mRNAs; MMP2/9 collected from conditioned media also showed an increase in activity; and ADP induced the nuclear localization of MMP2/9. The effects of ADP on transcription of the MMPs were due to activation of c-Src, Akt, and NF-kappa B, while ERK1/2 phosphorylation was needed for the increase in enzymatic activity and the regulation of nuclear import. We also showed that the nuclear localization of MMP2/9 induced by ADP causes the cleavage and inactivation of poly-ADP-ribose polymerase-1. These findings may help to elucidate the mechanisms regulating MMP2/9 activation in ZL55 human epithelioid mesothelioma cells, and perhaps other cells. Therapeutic approaches that promote ADP accumulation in a tumor environment may constitute an effective means to induce anticancer activity

PKC-δ/PKC-α activity balance regulates the lethal effects of cisplatin

Cisplatin is commonly employed in therapy of mesothelioma but its efficacy is limited and the mechanisms by which induces its effects are not clearly understood. PKCs can regulate cisplatin sensitivity. PKCs effects on cellular sensitivity/resistance depend on the pattern of active PKC isozymes as well as on cellular context. The present study was undertaken to determine if specific PKC isoforms regulate cisplatin-induced apoptosis in the human mesothelioma ZL55 cells. Cells were treated with cisplatin at various concentrations and for different incubation periods. Cytotoxicity assays and Western blottings of various proteins involved in apoptosis and survival were then performed. Exposure of ZL55 cells to cisplatin at concentrations ranging from 1 to 200 μM resulted in a dose-dependent inhibition of cell survival and the activation of the mitochondrial apoptotic pathway. Cisplatin activated full-length PKC-δ and generated a PKC-δ fragment. PKC-δ inhibition (by PKC-δ-siRNA) decreased ZL55 cell apoptosis. Full-length PKC-δ translocated to the nucleus and activated caspase-3 expression, whereas PKC-δ fragment preferentially localized to mitochondria. Cisplatin also provoked the generation of reactive oxygen species (ROS) by NADPH oxidase. ROS increment was responsible for the PKC-α activation that provoked EGFR transactivation and consequential phosphorylation of ERK1/2. The inhibition of this pathway at various level (PKC-α, EGFR or ERK1/2) increased cisplatin-induced cytotoxicity. The results suggest that PKC-δ is an essential part of the apoptotic program in mesothelioma cells, whereas PKC-α mediates a pro-survival response to cisplatin

ADP sensitizes ZL55 cells to the activity of cisplatin

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor in which cisplatin therapy is commonly used, although its effectiveness is limited. It follows that research efforts dedicated to identify promising combinations that can synergistically kill cancer cells are needed. Because we recently demonstrated that ADP inhibits the proliferation of ZL55 cells, an MPM-derived cell line obtained from bioptic samples of asbestos-exposed patients. Our objective in this study was to investigate the hypothesis that ADP also potentiates the cytotoxic activity of cisplatin. Results show that in ZL55 cells ADP enhanced (a) the cytotoxicity of cisplatin by 12-fold, (b) the restraint of cell clonogenic potential cisplatin-mediated, and (c) the number of apoptotic cells. Cisplatin, but not ADP, caused caspases activation; nevertheless, poly(ADP-ribose) polymerase-1 was not only cleaved in cisplatin-treated cells but also in cells treated with ADP alone. Furthermore, ADP, but not cisplatin, decreased mTOR and 6SK phosphorylations. Both ADP and cisplatin increased p53 protein, but ADP was also able to enhance p53 messenger RNA. P53 silencing resulted in a very large decrement of cell death induced by ADP or by cisplatin and reverted ADP effects on mTOR/S6K phosphorylation, suggesting that activated p53 may act as a negative regulator of mTOR. Consistently, the inhibition of mTOR by rapamycin also sensitized cells to cisplatin, and the effects of cisplatin plus rapamycin were identical to those obtained with cisplatin plus ADP. These findings suggest that the combination of ADP and cisplatin may be a promising strategy for the clinical treatment of cisplatin-resistant MPM

Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway

Extracellular nucleotides can regulate cell proliferation in both normal and tumorigenic tissues. Here, we studied how extracellular nucleotides regulate the proliferation of ZL55 cells, a mesothelioma-derived cell line obtained from bioptic samples of asbestos-exposed patients. ADP and 2-MeS-ADP inhibited ZL55 cell proliferation, whereas ATP, UTP, and UDP were inactive. The nucleotide potency profile and the blockade of the ADP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 suggest that P2Y1 receptor controls ZL55 cell proliferation. The activation of P2Y1 receptor by ADP leads to activation of intracellular transduction pathways involving [Ca(2+) ]i , PKC-δ/PKC-α, and MAPKs, ERK1/2 and JNK1/2. Cell treatment with ADP or 2-MeS-ADP also provokes the activation of p53, causing an accumulation of the G1 cyclin-dependent kinase inhibitors p21(WAF1) and p27(Kip) . Inhibition of ZL55 cell proliferation by ADP was completely reversed by inhibiting MEK1/2, or JNK1/2, or PKC-δ, and PKC-α. Through the inhibition of ADP-activated transductional kinases it was found that PKC-δ was responsible for JNK1/2 activation. JNK1/2 has a role in transcriptional up-regulation of p53, p21(WAF1/CIP1) , and p27(kip1) . Conversely, the ADP-activated PKC-α provoked ERK1/2 phosphorylation. ERK1/2 increased p53 stabilization, required to G1 arrest of ZL55 cells. Concluding, the importance of the study is twofold: first, results shed light on the mechanism of cell cycle inhibition by ADP; second, results suggest that extracellular ADP may inhibit mesothelioma progression

[Pt(O,O′-acac)(γ-acac)(DMS)] inhibits malignant pleural mesothelioma cells in vitro and in vivo

The aim of this study was to compare antitumor activity of [Pt(O,O’-acac)(γ-acac)(DMS)] in epithelioid and sarcomatoid type of MPM. Thus, we employed the human cell line of epithelioid derivation ZL55, and the sarcomatoid cell line ZL34 in vitro and SCID mice. In epithelioid cells, [Pt(O,O’-acac)(γ-acac)(DMS)] was approximately 12-fold more cytotoxic than cisplatin after 24 h of incubation (IC50 were 3.9±0.11 μM for [Pt(O,O’-acac)(γ-acac)(DMS)] and 46.8±0.6 μM for cisplatin, n=6). Similarly, in sarcomatoid cells cisplatin was significantly less cytotoxic than [Pt(O,O’-acac)(γ-acac)(DMS)] (IC50 48.7±1.7 μM and ND n=4, for [Pt(O,O’-acac)(γ-acac)(DMS) and cisplatin, respectively). In addition, we employed a preclinical model based on the subcutaneous injection of ZL55 and ZL34 malignant pleural mesotelioma cell lines in SCID mice. Remarkably, [Pt(O,O’-acac)(γ-acac)(DMS)] stands out for higher anticancer activity than cisplatin toward both the murine tumor models examined, inducing up to 50% inhibition of tumor growth. Mice inoculated with MPM cells showed a statistically significant reduction of tumor volume at every time point in the [Pt(O,O’-acac)(γ-acac)(DMS)] groups compared with both not treated and cisplatin-treated mice (p < 0.05). In summary, our findings show that [Pt(O,O’-acac)(γ-acac)(DMS)] seems to be more potent than cisplatin in MPM, thus providing a solid starting point for its validation as a suitable candidate for further pharmacological testing

Correction: Apoptosis by [Pt(O,O'-acac)(γ-acac)(DMS)] requires PKC-δ mediated p53 activation in malignant pleural mesothelioma.

[This corrects the article DOI: 10.1371/journal.pone.0181114.]

In Vitro and In Vivo Antitumor Activity of [Pt(O,O'-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O'-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC-siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing