COVID-19 Associated Vasculitis Confirmed by the Tissues RT-PCR: A Case Series Report.

BACKGROUND: Several cases of skin and central nervous system vasculopathy associated with COVID-19 in children have been published, but the information is rather limited. Our study aimed to describe these cases of vasculitis associated with COVID-19 in children. METHODS: In the retrospective-prospective case series study we included information regarding four children with COVID-19-associated vasculitis. In every case, we had a morphological description and the etiology was confirmed via real-time polymerase chain reaction during a tissue biopsy. RESULTS: The most involved systems were skin (4/4), respiratory (3/4), cardiovascular (2/4), nervous (1/4), eye (1/4), kidney (1/4), and inner year (1/4). All patients had increased inflammatory markers and thrombotic parameters (D-dimer). No patient met the criteria for multisystem inflammatory syndrome in children. Two patients met polyarteritis nodosa criteria, one met Henoch-Schonlein purpura criteria, and one met unclassified vasculitis criteria. All patients were treated with systemic glucocorticosteroids (two-pulse therapy). Non-biologic DMARDs were prescribed in all cases; 1/4 patients (25%) was treated with intravenous immunoglobuline, and 3/4 (75%) were treated with biologics (etanercept, tocilizumab, and adalimumab). CONCLUSIONS: Vasculitis associated with COVID-19 could be a life-threatening condition; SARS-CoV-2 might be a new trigger or etiological agent for vasculitis and other immune-mediated diseases. Further research and collection of similar cases are required.

SARS-CoV-2 escape from cytotoxic T cells during long-term COVID-19.

Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties. While escape from humoral immunity certainly contributes to intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin's lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented the binding of known immunogenic SARS-CoV-2 HLA class I antigens. By experimentally assessing the effect of a subset of the escape mutations, we show that they resulted in a loss of as much as ~1% of effector CD8 T cell response. Our results indicate that CD8 T cell escape represents a major underappreciated contributor to SARS-CoV-2 evolution in humans.

Analysis of Expression Pattern of snoRNAs in Human Cells A549 Infected by Influenza A Virus.

Small nucleolar RNAs (snoRNAs) are a highly expressed class of non-coding RNAs known for their role in guiding post-transcriptional modifications of ribosomal RNAs and small nuclear RNAs. Emerging studies suggest that snoRNAs are also implicated in regulating other vital cellular processes, such as pre-mRNA splicing and 3'-processing of mRNAs, and in the development of cancer and viral infections. There is an emerging body of evidence for specific snoRNA's involvement in the optimal replication of RNA viruses. In order to investigate the expression pattern of snoRNAs during influenza A viral infection, we performed RNA sequencing analysis of the A549 human cell line infected by influenza virus A/Puerto Rico/8/1934 (H1N1). We identified 66 that were upregulated and 55 that were downregulated in response to influenza A virus infection. The increased expression of most C/D-box snoRNAs was associated with elevated levels of 5'- and 3'-short RNAs derived from this snoRNA. Analysis of the poly(A)+ RNA sequencing data indicated that most of the differentially expressed snoRNAs synthesis was not correlated with the corresponding host genes expression. Furthermore, influenza A viral infection led to an imbalance in the expression of genes responsible for C/D small nucleolar ribonucleoprotein particles' biogenesis. In summary, our results indicate that the expression pattern of snoRNAs in A549 cells is significantly altered during influenza A viral infection.

COVID-19 pandemic in Saint Petersburg, Russia: Combining population-based serological study and surveillance data.

BACKGROUND: The COVID-19 pandemic in Russia has already resulted in 500,000 excess deaths, with more than 5.6 million cases registered officially by July 2021. Surveillance based on case reporting has become the core pandemic monitoring method in the country and globally. However, population-based seroprevalence studies may provide an unbiased estimate of the actual disease spread and, in combination with multiple surveillance tools, help to define the pandemic course. This study summarises results from four consecutive serological surveys conducted between May 2020 and April 2021 at St. Petersburg, Russia and combines them with other SARS-CoV-2 surveillance data. METHODS: We conducted four serological surveys of two random samples (May-June, July-August, October-December 2020, and February-April 2021) from adults residing in St. Petersburg recruited with the random digit dialing (RDD), accompanied by a telephone interview to collect information on both individuals who accepted and declined the invitation for testing and account for non-response. We have used enzyme-linked immunosorbent assay CoronaPass total antibodies test (Genetico, Moscow, Russia) to report seroprevalence. We corrected the estimates for non-response using the bivariate probit model and also accounted the test performance characteristics, obtained from independent assay evaluation. In addition, we have summarised the official registered cases statistics, the number of hospitalised patients, the number of COVID-19 deaths, excess deaths, tests performed, data from the ongoing SARS-CoV-2 variants of concern (VOC) surveillance, the vaccination uptake, and St. Petersburg search and mobility trends. The infection fatality ratios (IFR) have been calculated using the Bayesian evidence synthesis model. FINDINGS: After calling 113,017 random mobile phones we have reached 14,118 individuals who responded to computer-assisted telephone interviewing (CATI) and 2,413 provided blood samples at least once through the seroprevalence study. The adjusted seroprevalence in May-June, 2020 was 9.7% (95%: 7.7-11.7), 13.3% (95% 9.9-16.6) in July-August, 2020, 22.9% (95%: 20.3-25.5) in October-December, 2021 and 43.9% (95%: 39.7-48.0) in February-April, 2021. History of any symptoms, history of COVID-19 tests, and non-smoking status were significant predictors for higher seroprevalence. Most individuals remained seropositive with a maximum 10 months follow-up. 92.7% (95% CI 87.9-95.7) of participants who have reported at least one vaccine dose were seropositive. Hospitalisation and COVID-19 death statistics and search terms trends reflected the pandemic course better than the official case count, especially during the spring 2020. SARS-CoV-2 circulation showed rather low genetic SARS-CoV-2 lineages diversity that increased in the spring 2021. Local VOC (AT.1) was spreading till April 2021, but B.1.617.2 substituted all other lineages by June 2021. The IFR based on the excess deaths was equal to 1.04 (95% CI 0.80-1.31) for the adult population and 0.86% (95% CI 0.66-1.08) for the entire population. CONCLUSION: Approximately one year after the COVID-19 pandemic about 45% of St. Petersburg, Russia residents contracted the SARS-CoV-2 infection. Combined with vaccination uptake of about 10% it was enough to slow the pandemic at the present level of all mitigation measures until the Delta VOC started to spread. Combination of several surveillance tools provides a comprehensive pandemic picture.

From Bedside to the Bench-A Call for Novel Approaches to Prognostic Evaluation and Treatment of Empyema.

Empyema, a severe complication of pneumonia, trauma, and surgery is characterized by fibrinopurulent effusions and loculations that can result in lung restriction and resistance to drainage. For decades, efforts have been focused on finding a universal treatment that could be applied to all patients with practice recommendations varying between intrapleural fibrinolytic therapy (IPFT) and surgical drainage. However, despite medical advances, the incidence of empyema has increased, suggesting a gap in our understanding of the pathophysiology of this disease and insufficient crosstalk between clinical practice and preclinical research, which slows the development of innovative, personalized therapies. The recent trend towards less invasive treatments in advanced stage empyema opens new opportunities for pharmacological interventions. Its remarkable efficacy in pediatric empyema makes IPFT the first line treatment. Unfortunately, treatment approaches used in pediatrics cannot be extrapolated to empyema in adults, where there is a high level of failure in IPFT when treating advanced stage disease. The risk of bleeding complications and lack of effective low dose IPFT for patients with contraindications to surgery (up to 30%) promote a debate regarding the choice of fibrinolysin, its dosage and schedule. These challenges, which together with a lack of point of care diagnostics to personalize treatment of empyema, contribute to high (up to 20%) mortality in empyema in adults and should be addressed preclinically using validated animal models. Modern preclinical studies are delivering innovative solutions for evaluation and treatment of empyema in clinical practice: low dose, targeted treatments, novel biomarkers to predict IPFT success or failure, novel delivery methods such as encapsulating fibrinolysin in echogenic liposomal carriers to increase the half-life of plasminogen activator. Translational research focused on understanding the pathophysiological mechanisms that control 1) the transition from acute to advanced-stage, chronic empyema, and 2) differences in outcomes of IPFT between pediatric and adult patients, will identify new molecular targets in empyema. We believe that seamless bidirectional communication between those working at the bedside and the bench would result in novel personalized approaches to improve pharmacological treatment outcomes, thus widening the window for use of IPFT in adult patients with advanced stage empyema.

Perfluorochemical-facilitated plasminogen activator delivery to the airways: A novel treatment for inhalational smoke-induced acute lung injury.

BACKGROUND: Effective clinical management of airway clot and fibrinous cast formation of severe inhalational smoke-induced acute lung injury (ISALI) is lacking. Aerosolized delivery of tissue plasminogen activator (tPA) is confounded by airway bleeding; single-chain urokinase plasminogen activator (scuPA) moderated this adverse effect and supported transient improvement in gas exchange and lung mechanics. However, neither aerosolized plasminogen activator (PA) yielded durable improvements in physiologic responses or reduction in cast burden. Here, we hypothesized that perfluorochemical (PFC) liquids would facilitate PA distribution and sustain improvements in physiologic outcomes in ISALI. METHODS: Spontaneously breathing adult sheep (n = 36) received anesthesia and analgesia and were instrumented, exposed to cotton smoke inhalation, and supported by mechanical ventilation for 48 h. Groups (n = 6/group) were studied without supplemental treatment, or, starting 4 h post injury, they received intratracheal low volume (8 mL) PFC liquid alone or a dose range of tPA/PFC or scuPA/PFC suspensions (4 or 8 mg in 8 mL PFC) every 8 h. Outcomes were evaluated by sequential measurements of cardiopulmonary parameters, lung histomorphology, and biochemical analyses of bronchoalveolar lavage fluid. RESULTS: Dose-response and PA-type comparisons of outcomes demonstrated sustained superiority with low-volume PFC suspensions of scuPA over tPA or PFC alone, favoring the highest dose of scuPA/PFC suspension over lower doses, without airway bleeding. CONCLUSIONS: We propose that this improved profile over previously reported aerosolized delivery is likely related to improved dose distribution. Sustained salutary responses to scuPA/PFC suspension delivery in this translational model are encouraging and support the possibility that the observed outcomes could be of clinical importance.

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes.

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer-related signalling pathways. In this context, the deregulated expression of p62 - Sequestosome1 (p62/SQSTM1) - a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62-based medicine. In veterinary oncology the role of p62 in cancer-related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non-neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62-negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour-rejection antigen for an anti-cancer immunotherapy.

Formulation for a novel inhaled peptide therapeutic for idiopathic pulmonary fibrosis.

A caveolin-1 scaffolding domain, CSP7, is a newly developed peptide for the treatment of idiopathic pulmonary fibrosis. To develop a CSP7 formulation for further use we have obtained, characterized and compared a number of lyophilized formulations of CSP7 trifluoroacetate with DPBS and in combination with excipients (mannitol and lactose at molar ratios 1:5, 70 and 140). CSP7 trifluoroacetate was stable (>95%) in solution at 5 and 25 °C for up to 48 h and tolerated at least 5 freeze/thaw cycles. Lyophilized cakes of CSP7 trifluoroacetate with excipients were stable (>96%) for up to 4 weeks at room temperature (RT), and retained more than 98% of the CSP7 trifluoroacetate in the solution at 8 h after reconstitution at RT. The lyophilized CSP7 formulations were stable for up to 10 months at 5 °C protected from moisture. Exposure of the lyophilized cakes of CSP7 to 75% relative humidity (RH) resulted in an increase in the absorbed moisture, promoted crystallization of the excipients and induced reversible formation of CSP7 aggregates. Increased molar ratio of mannitol slightly affected formation of the aggregates. In contrast, lactose significantly decreased (up to 20 times) aggregate formation with apparent saturation at the molar ratio of 1:70. The possible mechanisms of stabilization of CSP7 trifluoroacetate in solid state by lactose include physical state of the bulking agent and the interactions between lactose and CSP7 trifluoroacetate (e.g. formation of a Schiff base with the N-terminal amino group of CSP7). Finally, CSP7 trifluoroacetate exhibited excellent stability during nebulization of formulations containing mannitol or lactose.

Precision-guided, Personalized Intrapleural Fibrinolytic Therapy for Empyema and Complicated Parapneumonic Pleural Effusions: The Case for the Fibrinolytic Potential.

Complicated pleural effusions and empyema with loculation and failed drainage are common clinical problems. In adults, intrapleural fibrinolytic therapy is commonly used with variable results and therapy remains empiric. Despite the intrapleural use of various plasminogen activators; fibrinolysins, for about sixty years, there is no clear consensus about which agent is most effective. Emerging evidence demonstrates that intrapleural administration of plasminogen activators is subject to rapid inhibition by plasminogen activator inhibitor-1 and that processing of fibrinolysins is importantly influenced by other factors including the levels and quality of pleural fluid DNA. Current therapy for loculation that accompanies pleural infections also includes surgery, which is invasive and for which patient selection can be problematic. Most of the clinical literature published to date has used flat dosing of intrapleural fibrinolytic therapy in all subjects but little is known about how that strategy influences the processing of the administered fibrinolysin or how this influences outcomes. We developed a new test of pleural fluids ex vivo, which is called the Fibrinolytic Potential or FP, in which a dose of a fibrinolysin is added to pleural fluids ex vivo after which the fibrinolytic activity is measured and normalized to baseline levels. Testing in preclinical and clinical empyema fluids reveals a wide range of responses, indicating that individual patients will likely respond differently to flat dosing of fibrinolysins. The test remains under development but is envisioned as a guide for dosing of these agents, representing a novel candidate approach to personalization of intrapleural fibrinolytic therapy.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models.

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP.

Multiple Bacteriophage Selection Strategies for Improved Affinity of a Peptide Targeting ERBB2.

Due to the heterogeneity of ERBB2-expression between tumors and over the course of treatment, a non-invasive molecular imaging agent is needed to accurately detect overall ERBB2 status. Peptides are a highly advantageous platform for molecular imaging, since they have excellent tumor penetration and rapid pharmacokinetics. One limitation of peptides however, is their traditionally low target affinity, and consequently, tumor uptake. The peptide KCCYSL was previously selected from a bacteriophage (phage) display library to bind ERBB2 and did so with moderate affinity of 295 nM. In order to enhance tumor uptake and clinical utility of the peptide, a novel phage microlibrary was created by flanking the parent sequence with random amino acids, followed by reselection using parallel strategies for high affinity and specific ERBB2 binding in an attempt to affinity maturate the peptide. One limitation of traditional phage display selections is difficulty in releasing the highest affinity phages from the target by incubation of acidic buffer. In an attempt to recover high affinity second-generation peptides from the ERBB2 microlibrary, two elution strategies, sonication and target elution, were undertaken. Sonication resulted in an approximately 50-fold enhancement in recovered phage per round of selection in comparison to target elution. Despite the differences in elution efficiency, the affinities of phage-displayed peptides selected from either strategy were relatively similar. Although both selections yielded peptides with significantly improved affinity in comparison to KCCYSL, the improvements were modest, most likely because the parental peptide binding cannot be improved by additional amino acids.

The time course of resolution of adhesions during fibrinolytic therapy in tetracycline-induced pleural injury in rabbits.

The time required for the effective clearance of pleural adhesions/organization after intrapleural fibrinolytic therapy (IPFT) is unknown. Chest ultrasonography and computed tomography (CT) were used to assess the efficacy of IPFT in a rabbit model of tetracycline-induced pleural injury, treated with single-chain (sc) urokinase plasminogen activators (scuPAs) or tissue PAs (sctPA). IPFT with sctPA (0.145 mg/kg; n = 10) and scuPA (0.5 mg/kg; n = 12) was monitored by serial ultrasonography alone (n = 12) or alongside CT scanning (n = 10). IPFT efficacy was assessed with gross lung injury scores (GLIS) and ultrasonography scores (USS). Pleural fluids withdrawn at 0-240 min and 24 h after IPFT were assayed for PA and fibrinolytic activities, α-macroglobulin/fibrinolysin complexes, and active PA inhibitor 1 (PAI-1). scuPA and sctPA generated comparable steady-state fibrinolytic activities by 20 min. PA activity in the scuPA group decreased slower than the sctPA group (kobs = 0.016 and 0.042 min(-1)). Significant amounts of bioactive uPA/α-macroglobulin (but not tPA; P < 0.05) complexes accumulated at 0-40 min after IPFT. Despite the differences in intrapleural processing, IPFT with either fibrinolysin was effective (GLIS ≤ 10) in animals imaged with ultrasonography only. USS correlated well with postmortem GLIS (r(2) = 0.85) and confirmed relatively slow intrapleural fibrinolysis after IPFT, which coincided with effective clearance of adhesions/organization at 4-8 h. CT scanning was associated with less effective (GLIS > 10) IPFT and higher levels of active PAI-1 at 24 h following therapy. We concluded that intrapleural fibrinolysis in tetracycline-induced pleural injury in rabbits is relatively slow (4-8 h). In CT-scanned animals, elevated PAI-1 activity (possibly radiation induced) reduced the efficacy of IPFT, buttressing the major impact of active PAI-1 on IPFT outcomes.

Regulatory role of small nucleolar RNAs in human diseases.

Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2'-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury.

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1(-/-) mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1(-/-) mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1(-/-) mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1(-/-) mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector.

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors - B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.

Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids.

Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2(ß) = 40 ± 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 ± 70 nM of αM/uPA formed in vivo [second order association rate (kass) = 580 ± 60 M(-1)·s(-1)]. αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of αM/uPA; and 3) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.

Intrapleural adenoviral delivery of human plasminogen activator inhibitor-1 exacerbates tetracycline-induced pleural injury in rabbits.

Elevated concentrations of plasminogen activator inhibitor-1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, ß-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40-80 and 200-400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10-15 nM in control animals to 20-40 nM in hPAI-1-overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment.

Novel aspects of urokinase function in the injured lung: role of α2-macroglobulin.

The level of active urokinase (uPA) is decreased in lung fluids of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) whereas α(2)-macroglobulin (α(2)-M), a plasma proteinase inhibitor, is a major component of these fluids. Since there have been reports describing the ability of α(2)-M to form complexes with uPA in vitro, we hypothesized that α(2)-M may interact with uPA in the lung to modulate its biological activity. Pulmonary edema fluids and lung tissues from patients with ALI/ARDS were evaluated for the presence of uPA associated with α(2)-M. Complexes between α(2)-M and uPA were detected in alveolar edema fluids as well as in lungs of patients with ALI/ARDS where they were located mainly in close proximity to epithelial cells. While uPA bound to α(2)-M retains its amidolytic activity towards low-molecular-weight substrates, it is not inhibited by its main physiological inhibitor, plasminogen activator inhibitor 1. We also investigated the functional consequences of formation of complexes between uPA and α(2)-M in vitro. We found that when α(2)-M:uPA complexes were added to cultures of human bronchial epithelial cells (BEAS-2B), activation of nuclear factor-κB as well as production of interleukin-6 and -8 was substantially suppressed compared with the addition of uPA alone. Our findings indicate for the first time that the function of uPA in patients with ALI/ARDS may be modulated by α(2)-M and that the effects may include the regulation of the fibrinolytic and signaling activities of uPA.

The urokinase receptor supports tumorigenesis of human malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is a lethal neoplasm for which current therapy is unsatisfactory. The urokinase plasminogen activator receptor (uPAR) is associated with increased virulence of many solid neoplasms, but its role in the pathogenesis of MPM is currently unclear. We found that REN human pleural MPM cells expressed 4- to 10-fold more uPAR than MS-1 or M9K MPM cells or MeT5A human pleural mesothelial cells. In a new orthotopic murine model of MPM, we found that the kinetics of REN cell tumorigenesis is accelerated versus MS-1 or M9K cells, and that REN instillates generated larger tumors expressing increased uPAR, were more invasive, and caused earlier mortality. While REN, MS-1, and M9K tumors were all associated with prominent extravascular fibrin deposition, excised REN tumor homogenates were characterized by markedly increased uPAR at both the mRNA and protein levels. REN cells exhibited increased thymidine incorporation, which was attenuated in uPAR-silenced cells (P < 0.01). REN cells traversed three-dimensional fibrin gels while MS-1, M9K, and MeT5A cells did not. uPAR siRNA or uPAR blocking antibodies decreased REN cell migration and invasion, while uPA and fetal bovine serum augmented the effects. Transfection of relatively low uPAR expressing MS-1 cells with uPAR cDNA increased proliferation and migration in vitro and tumor formation in vivo. These observations link overexpression of uPAR to the pathogenesis of MPM, demonstrate that this receptor contributes to accelerated tumor growth in part through interactions with uPA, and suggest that uPAR may be a promising target for therapeutic intervention.