Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

A systematic review and meta-analysis of changes in interleukin-8 levels in malaria infection.

The roles of interleukin-8 (IL-8) in malaria are inconsistent and unclear. This study synthesised evidence for differences in IL-8 levels in patients with malaria of various levels of severity. Relevant studies were searched in Scopus, MEDLINE, Embase, CENTRAL and PubMed from inception to 22 April 2022. Pooled mean differences (MDs) and 95% confidence intervals (CIs) were estimated using the random effects model. Of 1083 articles retrieved from the databases, 34 were included for syntheses. The meta-analysis revealed increased IL-8 levels in individuals with uncomplicated malaria compared with those without malaria (P = 0.04; MD, 25.57 pg/mL; 95% CI, 1.70 to 49.43 pg/mL; I2, 99.53, 4 studies; 400 uncomplicated malaria, 204 uninfected controls). The meta-analysis revealed comparable levels of IL-8 between the two groups (P = 0.10; MD, 74.46 pg/mL; 95% CI, -15.08 to 164.0 pg/mL; I2, 9.03; 4 studies; 133 severe malaria cases, 568 uncomplicated malaria cases). The study found evidence of increased IL-8 levels in individuals with malaria compared with those without malaria. However, no differences were found in IL-8 levels between patients with severe and non-severe malaria. Further research is needed to investigate the IL-8 cytokine levels in patients with malaria of different levels of severity.

Distinct cytokine profiles in malaria coinfections: A systematic review.

BACKGROUND: Few data exist on the distinct cytokine profiles of individuals with malaria coinfections and other diseases. This study focuses on data collation of distinct cytokine profiles between individuals with malaria coinfections and monoinfections to provide evidence for further diagnostic or prognostic studies. METHODS: We searched five medical databases, including Embase, MEDLINE, PubMed, Ovid, and Scopus, for articles on cytokines in malaria coinfections published from January 1, 1983 to May 3, 2022, after which the distinct cytokine patterns between malaria coinfection and monoinfection were illustrated in heat maps. RESULTS: Preliminary searches identified 2127 articles, of which 34 were included in the systematic review. Distinct cytokine profiles in malaria coinfections with bacteremia; HIV; HBV; dengue; filariasis; intestinal parasites; and schistosomiasis were tumor necrosis factor (TNF), interferon (IFN)-γ, IFN-α, interleukin (IL)-1, IL-1 receptor antagonist (Ra), IL-4, IL-7, IL-12, IL-15, IL-17; TNF, IL-1Ra, IL-4, IL-10, IL-12, IL-18, CCL3, CCL5, CXCL8, CXCL9, CXCL11, granulocyte colony-stimulating factor (G-CSF); TNF, IFN-γ, IL-4, IL-6, IL-10, IL-12, CCL2; IFN-γ, IL-1, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, CCL2, CCL3, CCL4, G-CSF; IL-1Ra, IL-10, CXCL5, CXCL8, CXCL10; TNF, IL-2, IL-4, IL-6, IL-10; and TNF, IFN-γ, IL-4, IL-5, IL-10, transforming growth factor-ß, CXCL8, respectively. CONCLUSION: This systematic review provides information on distinct cytokine profiles of malaria coinfections and malaria monoinfections. Further studies should investigate whether specific cytokines for each coinfection type could serve as essential diagnostic or prognostic biomarkers for malaria coinfections.

Transforming Growth Factor-ß Concerning Malarial Infection and Severity: A Systematic Review and Meta-Analysis.

Transforming growth factor-ß (TGF-ß) is important in the pathophysiology of malaria, but its role in acute and severe malaria is largely unknown. As a result, this study used a meta-analysis approach to investigate the difference in TGF-ß levels between several groups of malaria patients and healthy controls. The systematic review protocol was registered at PROSPERO (ID: CRD42022318864). From inception to 7 March 2022, studies that reported TGF-ß levels in patients with uncomplicated and healthy controls and patients with severe and uncomplicated malaria were searched in PubMed, Scopus and Embase. The assessment of the quality of the included studies was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. Qualitative and quantitative syntheses were performed to narratively describe and quantitatively pool the mean difference (MD) in TGF-ß levels between uncomplicated malaria and healthy controls, and between severe and uncomplicated malaria, using a random-effects model. A total of 1027 relevant articles were identified, and 13 studies were included for syntheses. The meta-analysis results show 233 patients with uncomplicated malaria and 239 healthy controls. Patients with uncomplicated malaria (233 cases) had lower mean TGF-ß levels than healthy controls (239 cases; p < 0.01, pooled MD = −14.72 pg/mL, 95% confidence interval (95% CI) = −20.46 to 8.99 pg/mL, I2 = 98.82%, seven studies). The meta-analysis found no difference in mean TGF-ß levels between patients with severe malaria (367 cases) and patients with uncomplicated malaria (180 cases; p = 0.11, pooled MD = −6.07 pg/mL, 95% CI = −13.48 to 1.35 pg/mL, I2 = 97.73%, six studies). The meta-analysis demonstrated decreased TGF-ß levels in patients with uncomplicated malaria compared to healthy controls. In addition, no difference in TGF-ß levels was found between patients with severe and uncomplicated malaria. More research is needed to determine whether TGF-ß levels could be a candidate marker for malarial infection or disease severity.

Elevation of serum interleukin-1ß levels as a potential indicator for malarial infection and severe malaria: a meta-analysis.

BACKGROUND: Interleukin (IL)-1ß is a proinflammatory cytokine that has a role in disease-related inflammation, including malaria. However, reports on the effect of IL-1ß on malaria severity are inconsistent. Therefore, meta-analyses to compare differences in IL-1ß levels between patients with severe malaria, patients with uncomplicated malaria and healthy controls were performed. METHODS: The PRISMA standards were used to perform a systematic review and meta-analysis. A search of PubMed, Scopus, EMBASE and reference lists was conducted for articles providing data on IL-1ß levels between patients with severe malaria, patients with uncomplicated malaria and healthy controls between January 1988 and March 2022, using a combination of search terms. The quality of all studies included in this review was determined using the Strengthening the Reporting of Observational Studies in Epidemiology statement: guidelines for reporting observational studies. The evidence was synthesized quantitatively and qualitatively. The differences in IL-1 levels across participant groups were recounted narratively for qualitative synthesis. For quantitative synthesis, the mean difference in IL-1ß levels across groups of participants was calculated using a random effects meta-analysis. The publication bias was assessed using funnel plots, Egger's test and a contour-enhanced funnel plot. RESULTS: A total of 1281 articles were discovered, and the 17 that satisfied the inclusion criteria were included for syntheses. The meta-analysis results using data from 555 cases of severe malaria and 1059 cases of uncomplicated malaria showed that severe malaria had a higher mean of IL-1ß levels than uncomplicated malaria (P < 0.01, pooled mean difference: 1.92 pg/mL, 95% confidence interval: 0.60-3.25 pg/mL, I2: 90.41%, 6 studies). The meta-analysis results using data from 542 cases of uncomplicated malaria and 455 healthy controls showed no difference in mean IL-1ß levels between the two groups (P = 0.07, pooled mean difference: 1.42 pg/mL, 95% confidence interval: - 0.1-2.94 pg/mL, I2: 98.93%, 6 studies). CONCLUSION: The results from the meta-analysis revealed that IL-1ß levels were higher in patients with severe malaria than in patients with uncomplicated malaria; however, IL-1ß levels were similar in patients with uncomplicated malaria and healthy controls. Based on the limitations of the number of studies included in the meta-analysis and high levels of heterogeneity, further studies are needed to conclude that differences in IL-1ß levels can be useful for monitoring the malaria severity.

Low Interleukin-12 Levels concerning Severe Malaria: A Systematic Review and Meta-Analysis.

Although many studies have investigated the role of interleukin (IL)-12 cytokine in the pathogenesis of severe malaria, these studies were based on a limited number of participants, possibly affecting their outcomes. We analyzed the difference in IL-12 levels between patients with severe and uncomplicated malaria through a meta-analysis. A systematic review was conducted following the Cochrane Handbook for Systematic Reviews of Interventions and was reported according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement. Systematic literature searches were performed between 20 February and 2 March, 2022 in PubMed, Scopus, and Embase to identify studies reporting IL-12 levels in patients with severe and uncomplicated malaria. The quality of included studies was determined using the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. The pooled mean difference (MD) in IL-12 between patients with severe and uncomplicated malaria was estimated using the DerSimonian-Laird method for the random-effects model. Altogether, 1885 potentially relevant articles were identified, and 10 studies enrolling 654 patients with severe malaria and 626 patients with uncomplicated malaria were included in the meta-analysis. Patients with severe malaria had lower mean IL-12 levels than those with uncomplicated malaria (p = 0.01, MD: -33.62, 95% confidence interval [CI]: -58.79 to -8.45, I2: 99.29%, 10 studies). In conclusion, decreased IL-12 levels might significantly contribute to the development of severe malaria. As most published literature demonstrated the role of IL-12 in animal models, human studies are required to understand the mechanisms involved in low IL-12 levels in patients with severe malaria.

Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation.

Background: Curcumin is an active compound derived from turmeric, Curcuma longa, and is known for its benefits to human health. The amoebicidal activity of curcumin against Acanthamoeba triangularis was recently discovered. However, a physiological change of intracellular pathways related to A. triangularis encystation mechanism, including autophagy in the surviving amoeba after curcumin treatment, has never been reported. This study aims to investigate the effect of curcumin on the survival of A. triangularis under nutrient starvation and nutrient-rich condition, as well as to evaluate the A. triangularis encystation and a physiological change of Acanthamoeba autophagy at the mRNA level. Methods: In this study, A. triangularis amoebas were treated with a sublethal dose of curcumin under nutrient starvation and nutrient-rich condition and the surviving amoebas was investigated. Cysts formation and vacuolization were examined by microscopy and transcriptional expression of autophagy-related genes and other encystation-related genes were evaluated by real-time PCR. Results: A. triangularis cysts were formed under nutrient starvation. However, in the presence of the autophagy inhibitor, 3-methyladenine (3-MA), the percentage of cysts was significantly reduced. Interestingly, in the presence of curcumin, most of the parasites remained in the trophozoite stage in both the starvation and nutrient-rich condition. In vacuolization analysis, the percentage of amoebas with enlarged vacuole was increased upon starvation. However, the percentage was significantly declined in the presence of curcumin and 3-MA. Molecular analysis of A. triangularis autophagy-related (ATG) genes showed that the mRNA expression of the ATG genes, ATG3, ATG8b, ATG12, ATG16, under the starvation with curcumin was at a basal level along the treatment. The results were similar to those of the curcumin-treated amoebas under a nutrient-rich condition, except AcATG16 which increased later. On the other hand, mRNA expression of encystation-related genes, cellulose synthase and serine proteinase, remained unchanged during the first 18 h, but significantly increased at 24 h post treatment. Conclusion: Curcumin inhibits cyst formation in surviving trophozoites, which may result from its effect on mRNA expression of key Acanthamoeba ATG-related genes. However, further investigation into the mechanism of curcumin in A. triangularis trophozoites arrest and its association with autophagy or other encystation-related pathways is needed to support the future use of curcumin.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Effects of the interaction between a clip domain serine protease and a white spot syndrome virus protein on phenoloxidase activity.

Clip domain serine proteinases participate in invertebrate innate immunity by acting as crucial enzymes in the signaling cascade involved in shrimp immunity. To functionally characterize its role in Fenneropenaeus merguiensis, FmclipSP cDNA was cloned and characterized. The FmclipSP gene comprised 1353 bp with an open reading frame of 1110 bp and encoded 369 amino acids. The protein contained clip and serine protease domains. FmClipSP mRNA is highly expressed in hemocytes, and its expression was significantly upregulated by bacterial or viral pathogen challenge. Furthermore, FmClipSP recombinant protein (rFmClipSP) was produced and possessed protease activity, stimulating prophenoloxidase activity. Additionally, rFmClipSP exhibited antibacterial activity against pathogens and nonpathogens. ELISA results demonstrated the binding ability of rFmClipSP to a recombinant protein of VP28 (rVP28). Interestingly, the binding significantly inhibited prophenoloxidase activity. Altogether, we partially characterized the function of FmclipSP and demonstrated its association with VP28. This study indicates the importance of clipSP as a component of F. merguiensis innate immunity. However, the role of clipSP in crustaceans remains unclear and requires further investigation.

FmLC6: An ultimate dual-CRD C-type lectin from Fenneropenaeus merguiensis mediated its roles in shrimp defense immunity towards bacteria and virus.

C-type lectins are a member of pattern recognition receptors (PRRs) that can interact with pathogen-associated molecular patterns of invading microorganisms by using their conserved motifs in carbohydrate recognition domain (CRD). The binding can trigger various immune responses in both direct and indirect mechanisms. Hereby, an ultimate C-type lectin with dual CRDs each of which containing a different motif was identified from hepatopancreas of Fenneropenaeus merguiensis (mentioned as FmLC6). The full-length cDNA of FmLC6 consisted of 1148 bp comprising one 1005 bp open reading frame (ORF) encoding a signal peptide and a mature protein of 317 residues. FmLC6 was composed of two CRDs with a highly conserved QPD (Gln-Pro-Asp) motif and one variant EPQ (Glu-Pro-Gln) motif for illustrating the carbohydrate binding affinity. The transcription of FmLC6 was detected only in hepatopancreas of normal shrimp. After injection with pathogens or immunostimulants, the expression of FmLC6 was significantly up-regulated and reached the highest level at 12 h post-injection except with lipoteichoic acid challenge. The FmLC6 expression was severely suppressed by knockdown based-silencing. This gene silencing with co-injection by Vibrio parahaemolyticus caused increasing in cumulative mortality and reduction of the median lethal time. Purified recombinant proteins of an entire ORF and two individual CRDs of FmLC6 produced in Escherichia coli could induce a broad spectrum of microbial agglutination with calcium dependence. The agglutination induced by rFmLC6, rCRD1 and rCRD2 was suppressed by galactose plus mannose, galactose and mannose, respectively which this event was confirmed by the inhibition of hemagglutination. All three recombinant proteins possessed ability to inhibit the bacterial growth with a dose-response. Purified rFmLC6 could bind directly to white spot syndrome virus particles and also its recombinant proteins including VP15, VP39A and VP28 with different affinity. Altogether, these results indicate that FmLC6 acts as a PRR to recognize invading microorganisms and leads to mediating the immune response to cooperation in pathogenic elimination via the binding, agglutination and antimicrobial activity.

An alternative function of C-type lectin comprising low-density lipoprotein receptor domain from Fenneropenaeus merguiensis to act as a binding receptor for viral protein and vitellogenin.

A diversity of C-type lectins (CTLs) was coming reported and they are known to participate in invertebrate innate immunity by act as pattern recognition receptor (PRR). In the present study, a unique CTL containing low-density lipoprotein receptor (LDLR) domain from Fenneropenaeus merguiensis (designated as FmLdlr) was cloned. Its sequence contained a single LDLR domain and one carbohydrate recognition domain (CRD) with a QAP motif putative for galactose-specific binding. The expression of FmLdlr was detected only in hemocytes of healthy shrimp. Its expression was significantly up-regulated by Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenge. The knockdown by FmLdlr dsRNA resulted in severe gene down-regulation. The gene silencing with pathogenic co-inoculation led to reduction of the median lethal time and increasing in the cumulative mortality including the remained WSSV in WSSV co-challenge group. Recombinant proteins of FmLdlr and two domains could agglutinate various bacterial strains which LDLR domain revealed the lowest activity. Only FmLdlr and CRD could enhance phagocytosis and encapsulation by hemocytes. Both FmLdlr and CRD except LDLR domain exhibited the antibacterial activity by inhibiting the growth of pathogenic V. parahaemolyticus in cultured medium and disk diffusion assay. Only FmLdlr and CRD could bind to WSSV proteins, envelope VP28, tegument VP39A and also capsid VP15, which FmLdlr had the higher binding affinity than that of CRD. Altogether, we concluded that FmLdlr contributed in shrimp immune defense through the main action of CRD in capable of bacterial agglutination, enhancing the phagocytosis and encapsulation, antimicrobial activity and binding to viral proteins. Interestingly, ELISA approach revealed that LDLR domain displayed the highest binding affinity to vitellogenin than whole molecule and CRD. We signified a new function of FmLdlr that it might presumably act as a receptor for vitellogenin transportation in hemolymph during vitellogenesis of shrimp.