Combination Of Protease Inhibitors Research Articles

Abstract B054: Protease activity as a biomarker and potential target among pancreatic cancer patients

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

A Combination of Novel HIV-1 Protease Inhibitor and Cytochrome P450 (CYP) Enzyme Inhibitor to Explore the Future Prospective of Antiviral Agents: Evotaz.

Viruses belong to the class of micro-organisms that are well known for causing infections in the human body. Antiviral medications are given out to prevent the spread of disease-causing viruses. When the viruses are actively reproducing, these agents have their greatest impact. It is particularly challenging to develop virus-specific medications since viruses share the majority of the metabolic functions of the host cell. In the continuous search for better antiviral agents, the United States Food and Drug Administration (USFDA) approved a new drug named Evotaz on January 29, 2015 for the treatment of human immunodeficiency virus (HIV). Evotaz is a combined once-daily fixed drug, containing Atazanavir, an HIV protease inhibitor, and cobicistat, an inhibitor of the human liver cytochrome P450 (CYP) enzyme. The medication is created such that it can kill viruses by concurrently inhibiting protease and CYP enzymes. The medicine is still being studied for a number of criteria, but its usefulness in children under the age of 12 is currently unknown. The preclinical and clinical characteristics of Evotaz, as well as its safety and efficacy profiles and a comparison of the novel drug with antiviral medications presently available in the market, are the main topics of this review paper.

Elimination of hepatitis C virus in a prison: An 18-year experience

IntroductionDespite the decrease of hepatitis C in Spanish prisons in the last years, it still remains a reservoir for infection. The aim of this work is to analyze the characteristics of these patients and the response to antiviral treatment over the last 18 years. MethodsRetrospective observational study in inmates of Araba penitentiary center diagnosed with HCV infection between 2002 and 2020.A descriptive analysis of patient characteristics and the response to the three antiviral treatment modalities was performed: peg-interferon and ribavirin, peg-interferon, ribavirin and a first-generation protease inhibitor and different combinations of direct-acting antivirals. ResultsA total of 248 antiviral treatments were prescribed. Treatment response rate up to 2015 was 65% and 93,7% after that year. Interferon non-responders were the main cause of non-response to treatment in periods 1 and 2 (40%–50%). Conversely, in period 3 viral breakthrough (67%) was the main culprit. ConclusionAfter 18 years, active hepatitis C infection in prison inmates has resolved with treatment according to clinical criteria. Therefore, the stay in prison may represent an opportunity to reduce the reservoir of the disease in the community, together with continued health care for those released from prison.

Eliminación del virus de la hepatitis C en un centro penitenciario: una experiencia de 18 años

IntroductionDespite the decrease of hepatitis C in Spanish prisons in the last years, it still remains a reservoir for infection. The aim of this work is to analyze the characteristics of these patients and the response to antiviral treatment over the last 18 years. MethodsRetrospective observational study in inmates of Araba penitentiary center diagnosed with HCV infection between 2002 and 2020.A descriptive analysis of patient characteristics and the response to the 3 antiviral treatment modalities was performed: peg-interferon and ribavirin, peg-interferon, ribavirin and a first-generation protease inhibitor and different combinations of direct-acting antivirals. ResultsA total of 248 antiviral treatments were prescribed. Treatment response rate up to 2015 was 65% and 93,7% after that year. Interferon non-responders were the main cause of non-response to treatment in periods 1 and 2 (40-50%). Conversely, in period 3 viral breakthrough (67%) was the main culprit. ConclusionAfter 18 years, active hepatitis C infection in prison inmates has resolved with treatment according to clinical criteria. Therefore, the stay in prison may represent an opportunity to reduce the reservoir of the disease in the community, together with continued health care for those released from prison.

Lopinavir-ritonavir for SARS-CoV-2 infection: a systematic review

Abstract Background SARS-CoV-2 is a new virus that first emerged in Wuhan, China in December 2019. The virus quickly spread throughout the countries, transforming the initial outbreak into a global pandemic. Lopinavir-ritonavir is an antiretroviral protease inhibitor combination drug that was proposed for the treatment of COVID-19 in the early phases of the pandemic. We conducted a systematic literature search on clinical trials assessing the efficacy and safety of lopinavir-ritonavir in patients hospitalized for COVID-19. 10 studies satisfying the inclusion criteria were analyzed and discussed in the current review. Four studies were randomized controlled trials, while the remaining had an observational design. The reported results are mixed, with some of the studies demonstrating the benefits of the combination drug, while others show little or no effect. These contradictory reports may be explained by the differences in study design, study population, the onset of treatment, and severity of disease, rendering this topic a subject for further debate.

Lunasin protease inhibitor concentrate decreases pro-inflammatory cytokines and improves histopathological markers in dextran sodium sulfate-induced ulcerative colitis

Lunasin protease inhibitor concentrate (LPIC) is a novel combination of soy bioactive peptide lunasin, Kunitz and Bowman-Birk protease inhibitors. The reported anti-inflammatory and anticancer properties of each one of them suggest LPIC as a promising candidate for the treatment of inflammatory-related diseases. Our objective was to assess the in vivo anti-inflammatory properties of LPIC. First, an in vitro test was performed in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophages by measuring the production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) as inflammatory markers. For the in vivo model, ulcerative colitis (UC) was induced in mice via oral administration of dextran sodium sulfate (DSS). LPIC treatment was performed via daily intraperitoneal injection of 50 mg/kg body weight. Body weight, visible blood in stool and stool consistency were scored daily as macroscopic indicators of disease progression. Occult blood was evaluated by the presence of hemoglobin in stool every third day. Colon length, caecum weight, colonic myeloperoxidase activity (MPO), presence of pro-inflammatory cytokines in blood and colon, changes in the architecture, and expression of inducible nitric oxide synthase (iNOS) in colonic tissue were evaluated. In vitro, LPIC induced production of NO and maintained cytokine levels in comparison to activated untreated macrophages. In vivo, LPIC increased colonic bleeding and did not improve macroscopic markers of the disease, but reduced colonic IL-1β and IL-6, decreased systemic circulation of TNF-α, attenuated neutrophils infiltration and iNOS expression in colonic tissue, and diminished the damage in colonic architecture. Our results suggest that combinations of peptides in LPIC may counteract the anti-inflammatory properties in vitro; while in vivo, LPIC can significantly reduce the histopathological damage, hence is a possible therapeutic strategy to attenuate UC.

Hospitalization and Emergency Department Encounters for COVID-19 After Paxlovid Treatment - California, December 2021-May 2022.

Nirmatrelvir/ritonavir (Paxlovid) is a combination protease inhibitor that blocks replication of SARS-CoV-2 (the virus that causes COVID-19) and has been shown to reduce the risk for hospitalization and death among patients with mild to moderate COVID-19 who are at risk for progression to severe disease* (1). In December 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for early treatment with Paxlovid among persons with mild to moderate cases of COVID-19 who are at high risk for progression to severe disease (2). FDA and a small number of published case reports have documented recurrence of COVID-19 symptoms or a positive viral test result (COVID-19 rebound) 2-8 days after recovery or a negative SARS-CoV-2 test result among patients treated with Paxlovid (3-7); however, large-scale studies investigating severe illness after Paxlovid treatment are limited. This study used electronic health record (EHR) data from a large integrated health care system in California (Kaiser Permanente Southern California [KPSC]) to describe hospital admissions and emergency department (ED) encounters related to SARS-CoV-2 infections during the 5-15 days after pharmacy dispensation of a 5-day treatment course of Paxlovid. Among 5,287 persons aged ≥12 years who received Paxlovid during December 31, 2021-May 26, 2022, 73% had received ≥3 doses of COVID-19 vaccine†, and 8% were unvaccinated. During the 5-15 days after Paxlovid treatment was dispensed, six hospitalizations and 39 ED encounters considered to be related to SARS-CoV-2 infection were identified, representing <1% of all patients to whom Paxlovid treatment was dispensed during the study period. Among these 45 persons, 21 (47%) were aged ≥65 years, and 35 (78%) had at least one underlying medical condition§ (8). This study found that hospitalization or ED encounters for COVID-19 during the 5-15 days after Paxlovid treatment was dispensed for mild to moderate COVID-19 illness were rarely identified. When administered as an early-stage treatment, Paxlovid might prevent COVID-19-related hospitalization among persons with mild to moderate cases of COVID-19 who are at risk for progression to severe disease.

Real-Life Early Anthropometric, Lipid and Liver Changes after Direct-Acting Antiviral Therapy in PLWHIV with HCV Co-Infection.

Treatment with interferon-free direct-acting antivirals (DAA) has become the gold standard in chronic hepatitis C virus (HCV) infection. Nevertheless, little research about the metabolic impact of achieving sustained virological response (SVR) is available in HCV/HIV co-infected patients. This research aimed to evaluate early anthropometric, lipid and liver parameters changes after achieving SVR 12 weeks after treatment (SVR12). A real-life retrospective descriptive before-after study assessed 128 DAA treatment episodes from 2015 to 2019 in HCV/HIV co-infected patients. Anthropometric parameters (weight, body mass index), lipid profile, genotype (GT) and viral load, liver data (basics laboratory necroinflammatory parameters and transient elastography (TE)) were collected before treatment with DAA (baseline), and when SVR12 was achieved. Significant increases (p < 0.01) were found in the early lipid profile, measured by LDLc (84.6 ± 35.0 vs. 108.6 ± 35.1 mg/dL) and total cholesterol (161.3 ± 41.0 vs. 183.3 ± 41.6 mg/dL). Significant changes (p < 0.05) were found in liver parameters, measured by ALT (58.2 ± 34.0 vs. 22.0 ± 16.0 U/L), bilirubin (0.8 ± 0.6 vs. 0.6 ± 0.5 mg/dL), albumin (4.2 ± 0.4 vs. 4.3 ± 0.3 g/dL) and liver stiffness (LS) (13.7 ± 13.3 vs. 11.8 ± 12.1 kPa). The main conclusions were that the use of DAA has an early negative impact on lipid metabolism. Achieving SVR12 against HCV leads to an early improvement in liver function and LS in HCV/HIV co-infected patients without interference with antiretroviral treatment (ART) and DAA. Short-term close lipid monitoring may be necessary when combining protease inhibitors. HCV-GT-3/HIV co-infected patients might require further close monitoring for residual fibrosis. These findings can be relevant for actual clinical practice.

Evaluation of the Prognostic Significance of Cereblon Protein Expression in Multiple Myeloma.

Considerable progress has been made in the treatment of multiple myeloma (MM) patients with the development of various new agents that increased survival rates over the past fifteen years. Cereblon (CRBN) plays an important role in mediating the antitumor effects of immunomodulatory drugs (IMiDs) among these new agents. The aim of our study is to investigate immunohistochemically (IHC) cereblon protein expression status in MM. Immunohistochemically, CRBN expression and its relationship with various prognostic factors were evaluated in bone marrow biopsies of 96 patients with MM in a single centre. Cytoplasmic and nuclear CRBN expression was detected in all neoplastic cells. While a complete or partial response to treatment was obtained in 45 patients, the disease was stable in 13 and progressive in 17 patients. Survival was longer in those treated with IMiD-containing regimens (p = 0.044). Both the survival rate (p = 0.013) and the survival time were significantly increased (p = 0.023) in those who received the treatment protocol containing protease inhibitors. A significant relationship was found between the treatment protocol and treatment response in the chi-squared analysis (p = 0.008). Although the longest survival time - though not statistically significant - was detected in the group treated with protease inhibitors (log rank, p = 0.217). The survival analysis revealed the presence of a relationship between IgG and IgA positivity and survival. In this study, the survival time of the patients who received treatment regimens containing protease inhibitors and IMiD was longer, independent of the presence of strong nuclear CRBN expression. The survival rate was significantly higher in those who used IMiD and protease inhibitors in combination. Since the survival rate was found to be increased in IgG positive cases and we thought that evaluation of immunoglobulin tissue expression in MM cases can provide prognostic prediction.

Peripheral neuropathy in people living with human immunodeficiency virus and acquired immunodeficiency syndrome on antiretroviral therapy

Background: Peripheral neuropathy is a common neurological complication in people with human immunodeficiency virus (HIV) infection. HIV-associated sensory neuropathy (HIV-SN) is defined as the presence of neuropathic symptoms and at least an abnormal perception of vibrations of a 128 Hz tuning fork on the great toe or abnormal ankle reflexes or both. Brief peripheral neuropathy screening (BPNS) tool is employed in identifying HIV-SN based on a directed symptom questionnaire and limited clinical examination. The present study was conducted to determine the prevalence and drug regimens related to peripheral neuropathy in people living with HIV and Acquired immunodeficiency syndrome (AIDS) on antiretroviral therapy (ART) so as to help improve the care for those on ART. The objective was to study the prevalence of peripheral neuropathy in people living with HIV and AIDS on ART using AIDS clinical trials group validated BPNS tools.Methods: Cross-sectional study on 198 HIV-seropositive cases aged above 18 years on ART attending centre of excellence (CoE) ART centre, RIMS Imphal. The presence of peripheral neuropathy was examined by using a BPNS among the participants.Results: Peripheral neuropathy was found in 46 (23.2%) out of 198 participants using BPNS. In this study a positive association between the duration of treatment with ART and use of protease inhibitor regimes with the development of peripheral neuropathy was significant.Conclusions: Peripheral neuropathy in patient with HIV and AIDS on ART had significant association with duration of treatment with ART and use of protease inhibitor combination in the ART regime.

Robust analysis of angiotensin peptides in human plasma: Column switching-parallel LC/ESI-SRM/MS without adsorption or enzymatic decomposition

Angiotensin (Ang) peptides are the main effectors of the renin-angiotensin system (RAS) regulating diverse physiological conditions and are involved in renal and vascular diseases. Currently, quantitative analyses of Ang peptides in human plasma mainly rely on radioimmunoassay-based methods whose reported levels are quite divergent. Analyses are further complicated by the potential of Ang peptides to bind to solid surfaces, to be enzymatically decomposed during sample preparation, and to undergo post-translational modifications. A column switching-parallel LC/ESI-SRM/MS method has been developed for seven Ang peptides (Ang I, Ang II, Ang III, Ang IV, Ang 1–9, Ang 1–7, and Ang A) in human plasma. Aqueous acetonitrile (5%) containing 50 mM arginine (Arg) as a dissolving solution and a combination of protease inhibitors with formic acid were used to prevent adsorption and enzymatic degradation, respectively. Plasma samples were simply deproteinized with acetonitrile followed by clean-up with an on-line trap column via column-switching. Stable isotope dilution with [13C5,15N1-Val]-Ang peptides as internal standards was employed for quantitative analysis. The current methodology has been successfully applied to determine the plasma levels of Ang peptides in healthy participants, suggesting future applicability to studies of various diseases related to RAS.

Role of Iron Chelation and Protease Inhibition of Natural Products on COVID-19 Infection.

Although the epidemic caused by SARS-CoV-2 callings for international attention to develop new effective therapeutics, no specific protocol is yet available, leaving patients to rely on general and supportive therapies. A range of respiratory diseases, including pulmonary fibrosis, have been associated with higher iron levels that may promote the course of viral infection. Recent studies have demonstrated that some natural components could act as the first barrier against viral injury by affecting iron metabolism. Moreover, a few recent studies have proposed the combination of protease inhibitors for therapeutic use against SARS-CoV-2 infection, highlighting the role of viral protease in virus infectivity. In this regard, this review focuses on the analysis, through literature and docking studies, of a number of natural products able to counteract SARS-CoV-2 infection, acting both as iron chelators and protease inhibitors.

A novel method for isolation and culture of primary swine gastric epithelial cells

BackgroundCulturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.ResultsMechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.ConclusionsA new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

Anticytokine Therapies in Severe Coronavirus Disease 2019 Should Be Informed by Detailed Inflammatory Profiling and Specific Therapeutic Targets.

To the Editor: We read the article by Buckley et al (1), recently published in Critical Care Explorations, on the role of anticytokine therapies for severe coronavirus disease 2019 (COVID-19) with interest. Although we agree that anticytokine therapies may lead to improved outcomes in COVID-19 patients, we contend that current evidence from critically ill COVID-19 patients, which was recently published in this journal (2,3), warrants against empiric targeting of interleukin 6 (IL-6) and suggests tumor necrosis factor (TNF) and serine proteases as more specific targets for anticytokine interventions. As the authors suggest, COVID-19 is associated with an elevation in IL-6 levels (4,5). However, the median values of IL-6 in severe COVID-19 are 10- to 40-fold lower than those in historic cohorts of acute respiratory distress syndrome (ARDS) (6). In keeping with the authors comments regarding the pleotropic nature of inflammatory mediators, we contend that empiric interventions targeting isolated inflammatory mediators, such as IL-6, may not be effective and could be potentially harmful. Instead, we should focus on detailed examination of the inflammatory profile of COVID-19 patients and its comparison with non-COVID-19 controls, assess the temporal evolution of inflammatory markers, and establish the link between different inflammatory profiles and clinical outcomes. In our recent prospective study published in this journal, we used machine learning to demonstrate that top six analytes distinguishing COVID-19 positive ICU patients from COVID-19 negative ICU patients were TNF, granzyme B, heat shock protein 70 (HSP-70), IL-18, interferon-gamma-inducible protein 10, and elastase 2 (2). These preliminary data suggest that IL-6 may not be an appropriate target, especially considering that median levels of IL-6 in severe COVID-19 are lower than those in historic non-COVID ARDS cohorts (6). Furthermore, in contrast to previous studies from sepsis and ARDS cohorts, COVID-19 patients in our study had persistently elevated levels of circulating TNF (2), suggesting altered upstream regulation of inflammation and highlighting the importance of assessing temporal trajectories of inflammatory markers. Equally intriguing was the role of serine proteases, including granzyme B and elastase 2. Granzyme B is located in lymphocytes (7) and would accumulate in lung early, whereas subsequent neutrophilic invasion would bring elastase 2 (8). Both serine proteases would potentially digest resident tissues and contribute to lung remodeling. Thus, a combination of TNF and serine protease inhibitors seems more relevant as potential therapeutic options than therapies targeting IL-6 response. Linkage with clinical outcomes is also critical. In our first study in this journal (2), we demonstrate that HSP-70 measured on ICU day 1 accurately predicted mortality, whereas in our second study in this journal, we showed that proteome profiling in COVID-19 patients identified six proteins that predict mortality on the first day of an ICU admission with 100% accuracy (3). Although these are preliminary findings, they warrant further investigation as potential markers of disease severity and clinical outcomes both for prognostication purposes and as a means to stratify patients in future anticytokine clinical trials. We applaud the authors for their comprehensive and well-written review on potential anticytokine therapies to improve outcomes of COVID-19. Although assessment of proposed empiric anticytokine therapies for clinical effectiveness in ongoing clinical trials is important, we want to highlight that detailed profiling of the inflammatory response in COVID-19 patients recently published in this journal is essential for identifying therapeutic targets that are specific to COVID-19 disease. Future clinical trials should establish whether inhibition of TNF and serine proteases (granzyme B and/or elastase 2) results in better clinical outcomes than anti-IL-6 treatments. ACKNOWLEDGMENT We thank the Lawson COVID19 Study Team that contributed to cited work. Marat Slessarev, MD, MSc, Lawson Health Research Institute, London, ON, Canada, and Department of Medicine, Western University, London, ON, Canada; Douglas D. Fraser, MD, PhD, Lawson Health Research Institute, London, ON, Canada, and Department of Pediatrics, Western University, London, ON, Canada

Acute Kidney Injury Associated With Lopinavir/Ritonavir Combined Therapy in Patients With COVID-19

Lopinavir and low-dose ritonavir (LPV/RTV) are associated in a fixed-dose combination protease inhibitor therapy used in patients with HIV and AIDS. The recent outbreak of severe acute respiratory syndrome coronavirus 2 infections causing coronavirus disease 2019 (COVID-19) has rekindled the interest in LPV/RTV after preclinical studies.1 Although no benefit was observed with LPV/RTV treatment beyond standard care,2 other randomized controlled trials, such as DisCoVeRy (NCT04315948), are currently enrolling.

Continuous and discrete modeling of HIV-1 decline on therapy.

Mathematical models have shed light on the dynamics of HIV- 1 infection in vivo. In this paper, we generalize continuous mathematical models of drug therapy for HIV-1 by Perelson et al. (Science 271:1582-1586, 1996) and Perelson and Nelson (SIAM Rev 41:3-44, 1999) on time scales, i.e., a nonempty closed subset of real numbers in order to derive new discrete models that predict the total concentration of plasma virus as a function of time. One of our main goals is to compare discrete mathematical models with the continuous model in Perelson et al. (1996) where HIV infected patients were given protease inhibitors and sampled frequently thereafter. For the comparison, we use experimental data collected in Perelson et al. (1996) and estimate the parameters such as the virion clearance rate and the rate of loss of infected cells by fitting the total concentration of plasma virus to this data set. Our results show that discrete systems describe the best fit. In the previous models of this study, the efficacy of protease inhibitor is assumed to be perfect. Motivated by Perelson and Nelson (1999), we end the paper with a mathematical model of imperfect protease inhibitor and reverse transcriptase (RT) inhibitor combination therapy of HIV-1 infection on time scales with its stability analysis.

Combination of Antifungal Drugs and Protease Inhibitors Prevent Candida albicans Biofilm Formation and Disrupt Mature Biofilms.

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.

Lung Surfactant for Pulmonary Barrier Restoration in Patients With COVID-19 Pneumonia.

Corona virus SARS-CoV-2 has already spread around the whole world and is currently, with no vaccine available yet, unstoppable. As per today, COVID-19 affects more than 3,000,000 confirmed patients globally. First line medications are antiviral drugs and multiple urgent clinical trials are under way. However, a recent clinical trial testing the HIV protease inhibitor combination lopinavir and ritonavir showed no significant antiviral activity against SARS-CoV-2 in patients with severe disease (1). As long as we do not have specific antiviral therapies against SARS-CoV-2, we need to provide supportive symptomatic therapies to prevent pulmonary failure, the most common cause of COVID-19 mortality.

A Computational Approach for the Prediction of Treatment History and the Effectiveness or Failure of Antiretroviral Therapy.

Human Immunodeficiency Virus Type 1 (HIV-1) infection is associated with high mortality if no therapy is provided. Currently, the treatment of an HIV-1 positive patient requires that several drugs should be taken simultaneously. The resistance of the virus to an antiretroviral drug may lead to treatment failure. Our approach focuses on predicting the exposure of a particular viral variant to an antiretroviral drug or drug combination. It also aims at the prediction of drug treatment success or failure. We utilized nucleotide sequences of HIV-1 encoding protease and reverse transcriptase to perform such types of prediction. The PASS (Prediction of Activity Spectra for Substances) algorithm based on the naive Bayesian classifier was used to make a prediction. We calculated the probability of whether a sequence belonged (P1) or did not belong (P0) to the class associated with exposure of the viral sequence to the set of drugs that can be associated with resistance to the set of drugs. The accuracy calculated as the average Area Under the ROC (Receiver Operating Characteristic) Curve (AUC/ROC) for classifying exposure of the sequence to the HIV-1 protease inhibitors was 0.81 (±0.07), and for HIV-1 reverse transcriptase, it was 0.83 (±0.07). To predict cases of treatment effectiveness or failure, we used P1 and P0 values, obtained in PASS, along with the binary vector constructed based on short nucleotide descriptors and the applied random forest classifier. Average AUC/ROC prediction accuracy for the prediction of treatment effectiveness or failure for the combinations of HIV-1 protease inhibitors was 0.82 (±0.06) and of HIV-1 reverse transcriptase was 0.76 (±0.09).

Extraction of total protein from shoots of Cereus morphological variants (Cactaceae) for proteomic analysis

Since there is a hypothesis that qualitative and/or quantitative differences of specific proteins may be associated with morphological variants in cacti of the Cereus genus (phenotypes erect, tortuosus and monstruosus), in current study we tested three different methods for protein extraction from shoots of the phenotypic variants to obtain protein fractions for further proteomic analysis. The TCA/acetone method for protein extraction revealed a larger number of well-defined bands in SDS-PAGE system than the methods with phenol. The quantification of protein extracted by TCA/acetone ranged between 0.488 (tortuosus) and 2.92 µg·mL-1 (monstruosus). Although the use of phenol is the most appropriate procedure for protein extraction from recalcitrant tissues, results have shown that extraction buffer containing two antioxidant agents (EDTA and β-mercaptoethanol) and PMSF to prevent protein degradation was efficient to avoid proteolysis and lower protein yield, than using TCA/acetone precipitation for protein extraction from shoots of Cereus sp. The use of extraction buffer with appropriate combinations of antioxidant agents, phenol-complexing agents, and protease inhibitors may be an efficient alternative for proteins extraction from succulent and recalcitrant tissues (such as cactus plants) using a simple protein extraction method.