Advancements in Immunotherapeutic Treatments for Hepatocellular Carcinoma: Potential of Combination Therapies.

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and a significant global health burden, with increasing incidence rates and limited treatment options. Immunotherapy has become a promising approach due to its ability to affect the immune microenvironment and promote antitumor responses. The immune microenvironment performs an essential role in both the progression and the development of HCC, with different characteristics based on specific immune cells and etiological factors. Immune checkpoint inhibitors, including programmed death-1/programmed death-ligand 1 inhibitors (pembrolizumab, nivolumab, and durvalumab) and cytotoxic T lymphocyte antigen-4 inhibitors (tremelimumab and ipilimumab), have the potential to treat advanced HCC and overcome adverse effects, such as liver failure and chemoresistance. Phase II and phase III clinical trials highlight the efficacy of pembrolizumab and nivolumab, respectively, in advanced HCC patients, as demonstrated by their positive effects on overall survival and progression-free survival. Tremelimumab has exhibited modest response rates, though it does possess antiviral activity. Thus, it is still being investigated in ongoing clinical trials. Combination therapies with multiple drugs have demonstrated potential benefits in terms of survival and tumor response rates, improving patient outcomes compared to monotherapy, especially for advanced-stage HCC. This review addresses the clinical trials of immunotherapies for early-, intermediate-, and advanced-stage HCC. Additionally, it highlights how combination therapy can significantly enhance overall survival, progression-free survival, and objective response rate in advanced-stage HCC, where treatment options are limited.

Cell Culture Adaptive Amino Acid Substitutions in FMDV Structural Proteins: A Key Mechanism for Altered Receptor Tropism.

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology.

Inhibition of NS2B-NS3 protease from all four serotypes of dengue virus by punicalagin, punicalin and ellagic acid identified from Punica granatum.

Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV.Communicated by Ramaswamy H. Sarma.

Correction: Evolution and consequences of individual responses during the COVID-19 outbreak.

[This corrects the article DOI: 10.1371/journal.pone.0273964.].

Surfactin-Conjugated Silver Nanoparticles as an Antibacterial and Antibiofilm Agent against Pseudomonas aeruginosa.

The emergence of antimicrobial resistance is an alarming global health concern and has stimulated the development of novel functional nanomaterials to combat multi-drug-resistant (MDR) bacteria. In this work, we demonstrate for the first time the synthesis and application of surfactin-coated silver nanoparticles as an efficient antibacterial and antibiofilm agent against the drug-resistant bacteria Pseudomonas aeruginosa for safe dermal applications. Our in vivo studies showed no significant superficial dermal irritation, edema, and erythema, while microscopic analysis revealed that surfactin-coated silver nanoparticles caused no pathological alterations at the applied concentrations. These results support the potential use of surfactin-coated silver nanoparticles against drug-resistant bacterial biofilm infections and in skin wound dressing applications.

Exome sequencing in a Romanian Bardet-Biedl syndrome cohort revealed an overabundance of causal BBS12 variants.

Bardet-Biedl syndrome (BBS), is an emblematic ciliopathy hallmarked by pleiotropy, phenotype variability, and extensive genetic heterogeneity. BBS is a rare (~1/140,000 to ~1/160,000 in Europe) autosomal recessive pediatric disorder characterized by retinal degeneration, truncal obesity, polydactyly, cognitive impairment, renal dysfunction, and hypogonadism. Twenty-eight genes involved in ciliary structure or function have been implicated in BBS, and explain the molecular basis for ~75%-80% of individuals. To investigate the mutational spectrum of BBS in Romania, we ascertained a cohort of 24 individuals in 23 families. Following informed consent, we performed proband exome sequencing (ES). We detected 17 different putative disease-causing single nucleotide variants or small insertion-deletions and two pathogenic exon disruptive copy number variants in known BBS genes in 17 pedigrees. The most frequently impacted genes were BBS12 (35%), followed by BBS4, BBS7, and BBS10 (9% each) and BBS1, BBS2, and BBS5 (4% each). Homozygous BBS12 p.Arg355* variants were present in seven pedigrees of both Eastern European and Romani origin. Our data show that although the diagnostic rate of BBS in Romania is likely consistent with other worldwide cohorts (74%), we observed a unique distribution of causal BBS genes, including overrepresentation of BBS12 due to a recurrent nonsense variant, that has implications for regional diagnostics.

Evolution and consequences of individual responses during the COVID-19 outbreak.

In a long-lasting major disease outbreak such as that of COVID-19, the challenge for public health authorities is to keep people motivated and keen on following safety guidelines. In this study, a compartmental model with a heterogeneous transmission rate (based on awareness) is utilized to hypothesize about the public adoption of preventive guidelines. Three subsequent outbreaks in South Korea, Pakistan, and Japan were analyzed as case studies. The transmission, behavior change, and behavioral change ease rates of the disease were measured in these countries. The parameters were estimated using the maximum likelihood method with an additional identifiability analysis performed to determine the uniqueness of the estimated parameters for quantitatively comparing them during the first three waves of COVID-19. The mathematical analysis and simulation results show that individual responses had a significant effect on the outbreak. Individuals declining to follow the public health guidelines in Korea and Japan between the second and third waves contributed to making the third peak the highest of the three peaks. In Pakistan, however, individual responses to following public health guidelines were maintained between the second and third waves, resulting in the third peak being lower than the first, rather than being associated with the highest transmission rate. Thus, maintaining a high level of awareness is critical for containing the spread. Improvised public health campaigns are recommended to sustain individual attention and maintain a high level of awareness.

Biotransformation and toxicity evaluation of functionalized manganese doped iron oxide nanoparticles.

Safe inorganic nanomaterials are tremendously used for diagnosis and therapies. However, essential processing in the microbiological environment changed the physical properties and in situ degradability, which is evaluated meticulously. In this research article, bare, Polyethylene glycol, and citrate coated manganese doped iron oxide nanoparticles are synthesized through the coprecipitation route. Structural, magnetic, optical, and morphological analyses are performed through different characterization tools. X-ray diffraction confirmed the formation of single-phase FeMnO3 with a crystallite size of 48.91 nm. Vibrating sample magnetometer analysis confirmed the formation of soft ferromagnetic behavior of bare and coated nanoparticles (NPs). Scanning electron microscopy and transmission electron microscopy confirmed the formation of spherical shaped nanoparticles. Single-dose in vivo acute toxicity testing is performed through the intraperitoneal route of administration on groups of healthy albino rats. Elevated enzyme levels of kidney and liver are observed at day 1 but a transient decrease is observed at later stages. Through optical follow-up, degradation effects are studied by adding prepared NPs in lysosomal like medium. Finally, metabolization of degraded products based on manganese/iron ions is studied by adding apoferritin into a lysosome like solution. These studies showed partial storage of manganese ions from NPs, while no substantial transfer is observed in the case of manganese salt.

Impact of glucocorticoids on systemic sirtuin 1 expression and activity in rats with adjuvant-induced arthritis.

The class III histone deacetylase sirtuin 1 (SIRT1) plays a pivotal role in numerous biological and physiological functions, including inflammation. An association between SIRT1 and proinflammatory cytokines might exist. In addition to their important role in inflammation associated with rheumatoid arthritis (RA), proinflammatory cytokines mediate the development of systemic effects. Here, we evaluated systemic SIRT1 expression and enzymatic activity, in peripheral blood mononuclear cells (PBMCs) and in liver isolated from rats with adjuvant-induced arthritis (AIA), treated or not with low or high doses of glucocorticoids (GCs). We also measured the production of tumour necrosis factor alpha (TNF) and interleukin-1 beta (IL-1 beta) in PBMCs and liver. We found that SIRT1 expression and activity increased in PBMCs of AIA rats compared to healthy controls and decreased under GC treatment. Similarly, we observed an increased SIRT1 activity in the liver of AIA rats compared to healthy controls which decreased under high doses of GCs. We also found an increase in IL-1 beta and TNF levels in the liver of AIA rats compared to healthy controls, which decreased under high doses of GC. We did not observe a significant correlation between SIRT1 activity and proinflammatory cytokine production in PBMC or liver. In contrast, a strong positive correlation was found between the liver levels of TNF and IL-1 beta (rho=0.9503, p=7.5x10-21). Our results indicate that increased inflammation in AIA rats compared to healthy control is accompanied by an increased SIRT1 activity in both PBMCs and liver, which could be decreased under GC treatment.

The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells.

BACKGROUND: Human cytomegalovirus (HCMV) establishes a persistent life-long infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission in humans and we hypothesized that mammary epithelial cells could be one of the main cellular targets of HCMV infection. METHODS: The infectivity of primary human mammary epithelial cells (HMECs) was assessed following infection with the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infection on expression of p53 and retinoblastoma proteins, telomerase activity and oncogenic pathways (c-Myc, Akt, Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agar assay. CTH cells (CMV Transformed HMECs) were detected in prolonged cultures of infected HMECs. Tumor formation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non coding RNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsies of patients with breast cancer using qualitative and quantitative PCR. RESULTS: We found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs in vitro. The clinical strain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcripts and proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastoma and p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras, the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted in the development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cells when injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTH cells the presence of a HCMV signature corresponding to a sequence of the long noncoding RNA4.9 (lncRNA4.9) gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSG mice injected with CTH cells and in biopsies of patients with breast cancer using qualitative and quantitative PCR. CONCLUSIONS: Our data indicate that key molecular pathways involved in oncogenesis are activated in HCMV-DB-infected HMECs that ultimately results in the transformation of HMECs in vitro with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 genomic sequence and give rise to fast growing triple-negative tumors in NSG mice. A similar lncRNA4.9 genomic sequence was detected in tumor biopsies of patients with breast cancer.

Counteracting Akt Activation by HIV Protease Inhibitors in Monocytes/Macrophages.

Akt signaling plays a central role in many biological processes that are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. The persistence of latent reservoirs in successfully treated patients, mainly located in macrophages and latently infected resting CD4+ T cells, remains a major obstacle in HIV-1 eradication. We assessed the in vitro effects of an HIV protease inhibitor (PI) and a non-nucleoside reverse transcriptase inhibitor (NNRTI) on HIV-1 Nef-induced Akt activation in macrophages and on HIV-1 reactivation in U1 monocytoid cells. Ex vivo, we investigated the impact of combination antiretroviral therapy (cART) on Akt activation, as measured by flow cytometry, and on the viral reservoir size, quantified by qPCR, in monocytes and autologous resting CD4+ T cells from HIV-infected individuals (Trial registration: NCT02858414). We found that, in myeloid cells, both Akt activation and HIV-1 reactivation were inhibited by PI but not by NNRTI in vitro. Our results indicate that cART decreases Akt activation and reduces the size of the HIV reservoir in both monocytes and resting CD4+ T cells. Our study indicates that Akt activation could play a role in HIV reservoir formation, indicating that drugs which target Akt could be efficient for limiting its size in aviremic chronically infected patients.

Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

HIV-1 in Pakistan: Where we stand? Where we will go?

Adherence to Islamic beliefs and being home to more than 190 million Muslims made many to believe that Pakistan was protected from human immunodeficiency virus or acquired immunodeficiency syndrome (HIV/AIDS). More than 30 years of HIV-1 epidemic, the reality is totally different now. HIV/AIDS is not only becoming a major health concern of Pakistan, but also in several other Muslim-majority countries like Malaysia, Iran and Indonesia having prevalence rates of 0·4%, 0·2% and 0·3%, respectively. While in most parts of the world, HIV-1 infections have decreased or stabilised. However, the countries where HIV-1 prevalence is increased by 25-35% has Muslim majority. The high-risk populations in these countries are drug users and immoral sexual behaviours that include practices forbidden in Islam.

Limited HIV-1 Reactivation in Resting CD4+ T cells from Aviremic Patients under Protease Inhibitors.

A latent viral reservoir that resides in resting CD4+ T cells represents a major barrier for eradication of HIV infection. We test here the impact of HIV protease inhibitor (PI) based combination anti-retroviral therapy (cART) over nonnucleoside reverse transcriptase inhibitor (NNRTI)-based cART on HIV-1 reactivation and integration in resting CD4+ T cells. This is a prospective cohort study of patients with chronic HIV-1 infection treated with conventional cART with an undetectable viremia. We performed a seven-year study of 47 patients with chronic HIV-infection treated with cART regimens and with undetectable plasma HIV-1 RNA levels for at least 1 year. Of these 47 patients treated with cART, 24 were treated with a PI-based regimen and 23 with a NNRTI-based regimen as their most recent treatment for more than one year. We evaluated the HIV-1 reservoir using reactivation assay and integrated HIV-1 DNA, respectively, in resting CD4+ T cells. Resting CD4+ T cells isolated from PI-treated patients compared to NNRTI-treated patients showed a limited HIV-1 reactivation upon T-cell stimulation (p = 0·024) and a lower level of HIV-1 integration (p = 0·024). Our study indicates that PI-based cART could be more efficient than NNRTI-based cART for limiting HIV-1 reactivation in aviremic chronically infected patients.

Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.

Eradication of HIV-1 from the macrophage reservoir: an uncertain goal?

Human immunodeficiency virus type 1 (HIV-1) establishes latency in resting memory CD4+ T cells and cells of myeloid lineage. In contrast to the T cells, cells of myeloid lineage are resistant to the HIV-1 induced cytopathic effect. Cells of myeloid lineage including macrophages are present in anatomical sanctuaries making them a difficult drug target. In addition, the long life span of macrophages as compared to the CD4+ T cells make them important viral reservoirs in infected individuals especially in the late stage of viral infection where CD4+ T cells are largely depleted. In the past decade, HIV-1 persistence in resting CD4+ T cells has gained considerable attention. It is currently believed that rebound viremia following cessation of combination anti-retroviral therapy (cART) originates from this source. However, the clinical relevance of this reservoir has been questioned. It is suggested that the resting CD4+ T cells are only one source of residual viremia and other viral reservoirs such as tissue macrophages should be seriously considered. In the present review we will discuss how macrophages contribute to the development of long-lived latent reservoirs and how macrophages can be used as a therapeutic target in eradicating latent reservoir.

The eEF1A Proteins: At the Crossroads of Oncogenesis, Apoptosis, and Viral Infections.

Eukaryotic translation elongation factors 1 alpha, eEF1A1 and eEF1A2, are not only translation factors but also pleiotropic proteins that are highly expressed in human tumors, including breast cancer, ovarian cancer, and lung cancer. eEF1A1 modulates cytoskeleton, exhibits chaperone-like activity and also controls cell proliferation and cell death. In contrast, eEF1A2 protein favors oncogenesis as shown by the fact that overexpression of eEF1A2 leads to cellular transformation and gives rise to tumors in nude mice. The eEF1A2 protein stimulates the phospholipid signaling and activates the Akt-dependent cell migration and actin remodeling that ultimately favors tumorigenesis. In contrast, inactivation of eEF1A proteins leads to immunodeficiency, neural and muscular defects, and favors apoptosis. Finally, eEF1A proteins interact with several viral proteins resulting in enhanced viral replication, decreased apoptosis, and increased cellular transformation. This review summarizes the recent findings on eEF1A proteins indicating that eEF1A proteins play a critical role in numerous human diseases through enhancement of oncogenesis, blockade of apoptosis, and increased viral pathogenesis.

Dysregulated serum IL-23 and SIRT1 activity in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

Sirtuin 1 (Sirt1) is a class III histone deacetylase (HDAC) that modulates gene expression and is involved in the regulation of proinflammatory cytokines. Interleukin-23 (IL-23) is produced by activated macrophages and dendritic cells and could fuel the progression of rheumatoid arthritis (RA). The goal of our study was to evaluate serum IL-23 levels and both Sirt1 activity and expression in peripheral blood mononuclear cells (PBMCs) in patients with RA compared to healthy controls (HC) and to determine the relationship between Sirt1 activity/expression and IL-23 levels. We assessed apoptosis in PBMCs of RA patients and its association with Sirt1 expression and serum IL-23. Serum IL-23 levels were increased in RA patients in comparison with controls. We found a positive correlation between the levels of serum IL-23 and serum IL-6 in RA patients. Decreased cytoplasmic Sirt1 activity was observed in RA patients with severe disease compared to HC. The expression of Sirt1 protein was significantly decreased in PBMCs of RA patients compared to HC using western blotting. Serum IL-23 levels correlated positively with the cytoplasmic Sirt1 activity in RA patients. Apoptosis rate of PBMCs isolated from RA patients was increased compared to HC and correlated negatively with the expression of Sirt1 protein and serum IL-23 levels. Levels of serum IL-23 and Sirt1 activity and expression were disturbed in RA parallel to increased PBMC apoptosis. Our findings might provide the rationale for the development of new therapeutic approaches in RA.

Sirtuin 1 activity in peripheral blood mononuclear cells of patients with osteoporosis.

BACKGROUND: Sirtuin 1 (SIRT1) is a class III histone deacetylase that may play a critical role in several biological functions, including lifespan, stress, and inflammation. Our main objective was to evaluate SIRT1 activity in peripheral blood mononuclear cells (PBMCs) in patients with osteoporosis and to analyze the relationship between the SIRT 1 activity and markers of inflammation and bone remodelling. MATERIAL AND METHODS: We performed a prospective monocentric study of patients with osteoporosis and measured the nuclear and cytoplasmic activities of SIRT1 in PBMCs. Levels of proinflammatory cytokines were assessed in culture supernatants of PBMCs isolated from the osteoporosis patients. The level of serum C-terminal cross-linking telopeptide of type I collagen (CTX), a marker of bone resorption, was measured in the serum of osteoporosis patients. RESULTS: Sixteen women with osteoporosis were included. A statistically significant correlation between the cytoplasmic and nuclear SIRT 1 activities was found in PBMCs of patients with osteoporosis. Although non-significant, we observed a negative trend between nuclear SIRT 1 activity and the rate of serum CTX and a positive trend between IL-6 and CTX levels in patients with osteoporosis. CONCLUSIONS: This study shows that the cytoplasmic and nuclear SIRT 1 activities are measurable in circulating PBMCs of patients with osteoporosis and that these 2 activities are correlated. The potential role of inflammation in bone resorption in patients with osteoporosis was also studied.