The role and influence of bacteria on the growth of Blastocystis sp. subtype 3.

Blastocystis sp. is an enteric protistan parasite that affects individuals worldwide with gastrointestinal symptoms such as abdominal discomfort, diarrhea, and flatulence. However, its pathogenicity is controversial due to its presence among asymptomatic individuals. Blastocystis sp. subtype 3 (ST3) is the most prevalent subtype among humans that have been associated with irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, and colorectal cancer. Axenization of the parasite has been shown to impede its growth thus revealing the importance of accompanying bacteria in ensuring Blastocystis sp. survival. This study aims to identify the influence of accompanying bacteria on the growth of Blastocystis sp. ST3. Blastocystis sp. cultures were treated with Meropenem, Vancomycin, and Amoxicillin-Clavulanic acid (Augmentin). Bacteria-containing supernatant of antibiotic-treated and control cultures were isolated and identified through 16 s rRNA sequencing. Morphological changes of antibiotic-treated Blastocystis sp. ST3 were also observed. The cultures treated with meropenem and augmentin exhibited opposing effects with reduced growth of isolates from symptomatic patients and a significant increase in asymptomatic isolates. Whereas, vancomycin-treated cultures had no difference in the growth of Blastocystis sp. ST3 isolates from symptomatic and asymptomatic patients. Isolates from symptomatic and asymtomatic patients had 6 and 2 distinct bacterial species identified with Proteus mirabilis as the common bacteria among both types of isolates. Morphologically, Blastocystis sp. ST3 cultures exposed to meropenem and augmentin demonstrated an increase in pre-cystic forms. These findings demonstrate the effects of accompanying bacteria on the growth of Blastocystis sp. ST3 that could translate into clinical manifestations observed among Blastocystis sp.-infected patients.

Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity.

Whilst the influence of intestinal microbiota has been shown in many diseases such as irritable bowel syndrome, colorectal cancer, and aging, investigations are still scarce on its role in altering the nature of other infective organisms. Here we studied the association and interaction of Blastocystis sp. and human intestinal microbiota. In this study, we investigated the gut microbiome of Blastocystis sp.-free and Blastocystis sp. ST3-infected individuals who are symptomatic and asymptomatic. We tested if the expression of phenotype and pathogenic characteristics of Blastocystis sp. ST3 was influenced by the alteration of its accompanying microbiota. Blastocystis sp. ST3 infection alters bacterial composition. Its presence in asymptomatic individuals showed a significant effect on microbial richness compared to symptomatic ones. Inferred metagenomic findings suggest that colonization of Blastocystis sp. ST3 could contribute to the alteration of microbial functions. For the first time, we demonstrate the influence of bacteria on Blastocystis sp. pathogenicity. When Blastocystis sp. isolated from a symptomatic individual was co-cultured with bacterial suspension of Blastocystis sp. from an asymptomatic individual, the parasite demonstrated increased growth and reduced potential pathogenic expressions. This study also reveals that Blastocystis sp. infection could influence microbial functions without much effect on the microbiota diversity itself. Our results also demonstrate evidence on the influential role of gut microbiota in altering the characteristics of the parasite, which becomes the basis for the contradictory findings on the parasite's pathogenic role seen across different studies. Our study provides evidence that asymptomatic Blastocystis sp. in a human gut can be triggered to show pathogenic characteristics when influenced by the intestinal microbiota.

Presence of Polyketide Synthase (PKS) Gene and Counterpart Virulence Determinants in Klebsiella pneumoniae Strains Enhances Colorectal Cancer Progression In-Vitro.

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression.

Three Members of Transmembrane-4-Superfamily, TM4SF1, TM4SF4, and TM4SF5, as Emerging Anticancer Molecular Targets against Cancer Phenotypes and Chemoresistance.

There are six members of the transmembrane 4 superfamily (TM4SF) that have similar topology and sequence homology. Physiologically, they regulate tissue differentiation, signal transduction pathways, cellular activation, proliferation, motility, adhesion, and angiogenesis. Accumulating evidence has demonstrated, among six TM4SF members, the regulatory roles of transmembrane 4 L6 domain family members, particularly TM4SF1, TM4SF4, and TM4SF5, in cancer angiogenesis, progression, and chemoresistance. Hence, targeting derailed TM4SF for cancer therapy has become an emerging research area. As compared to others, this review aimed to present a focused insight and update on the biological roles of TM4SF1, TM4SF4, and TM4SF5 in the progression, metastasis, and chemoresistance of various cancers. Additionally, the mechanistic pathways, diagnostic and prognostic values, and the potential and efficacy of current anti-TM4SF antibody treatment were also deciphered. It also recommended the exploration of other interactive molecules to be implicated in cancer progression and chemoresistance, as well as potential therapeutic agents targeting TM4SF as future perspectives. Generally, these three TM4SF members interact with different integrins and receptors to significantly induce intracellular signaling and regulate the proliferation, migration, and invasion of cancer cells. Intriguingly, gene silencing or anti-TM4SF antibody could reverse their regulatory roles deciphered in different preclinical models. They also have prognostic and diagnostic value as their high expression was detected in clinical tissues and cells of various cancers. Hence, TM4SF1, TM4SF4, and TM4SF5 are promising therapeutic targets for different cancer types preclinically and deserve further investigation.

Natural products from medicinal plants in Asia and the Pacific for RNA viruses: Hercules' fifth labour.

CONTEXT: The emergence of zoonotic viruses in the last decades culminating with COVID-19 and challenges posed by the resistance of RNA viruses to antiviral drugs requires the development of new antiviral drugs. OBJECTIVE: This review identifies natural products isolated from Asian and Pacific medicinal plants with in vitro and in vivo antiviral activity towards RNA viruses and analyses their distribution, molecular weights, solubility and modes of action. MATERIALS AND METHODS: All data in this review was compiled from Google Scholar, PubMed, Science Direct, Web of Science, ChemSpider, PubChem and library search from 1961 to 2022. RESULTS: Out of about 350 molecules identified, 43 phenolics, 31 alkaloids, and 28 terpenes were very strongly active against at least one type of RNA virus. These natural products are mainly planar and amphiphilic, with a molecular mass between 200 and 400 g/mol and target viral genome replication. Hydroxytyrosol, silvestrol, lycorine, tylophorine and 12-O-tetradecanoylphorbol 13-acetate with IC50 below 0.01 µg/mL and selectivity index (S.I.) above 100 have the potential to be used for the development of anti-RNA virus leads. DISCUSSION AND CONCLUSIONS: The medicinal plants of Asia and the Pacific are a rich source of natural products with the potential to be developed as lead for the treatment of RNA viral infections.

Distinct Phenotypic Variation of Blastocystis sp. ST3 from Urban and Orang Asli Population-An Influential Consideration during Sample Collection in Surveys.

Blastocystis sp. is a globally distributed protozoan parasite with uncertain pathogenicity. Phenotypic variation in Blastocystis sp. suggests its adaptation; however, the phenotypic features of Blastocystis sp. ST3 from a distinct source of isolation is unknown. Blastocystis sp. isolated from individuals in urban and orang asli (indigenous population in Selangor, Malaysia) settlements were studied for phenotypic characteristics such as growth profile, morphology, ultrastructure, and resistance to harsh conditions. Subsequently, pathogenic potentials, such as in protease activity and the ability to stimulate the proliferation of cancer cells, were assessed. Higher parasite counts with granular and apoptotic forms were found in Blastocystis sp. from orang asli individuals. Cells with fuzzy coats and amoebic structures which seemingly implicate increased interaction with bacteria were seen predominantly in urban symptomatic persons. Also, Blastocystis sp. from orang asli isolates resisted harsh environments, suggesting longer co-adaptation to the hosts. Urban and orang asli symptomatic isolates possessed a predominance of only cysteine protease, whereas all the asymptomatic isolates showed significantly higher cysteine, serine, or aspartic protease activity. However, only solubilized antigen from urban symptomatic isolates showed significant stimulation of cancer cell proliferation. For the first time, our findings demonstrate significant phenotypic variation in a single subtype, ST3 of Blastocystis sp., isolated from urban and orang asli populations that are known to have distinct gut microbial compositions. The outcome emphasizes the importance of identifying people's locations and lifestyles during sample collection before forming conclusions on the prevailing data and implicating subtypes to pathogenicity. The environment plays a significant role in Blastocystis sp. infection.

Isolation and Characterization of Werneria Chromene and Dihydroxyacidissimol from Burkillanthus malaccensis (Ridl.) Swingle

The secondary metabolites of endemic plants from the Rutaceae family, such as Burkillanthusmalaccensis (Ridl.) Swingle from the rainforest of Malaysia, has not been studied. Burkillanthusmalaccensis (Ridl.) Swingle may produce antibacterial and antibiotic-potentiating secondary metabolites. Hexane, chloroform, and methanol extracts of leaves, bark, wood, pericarps, and endocarps were tested against bacteria by broth microdilution assay and their antibiotic-potentiating activities. Chromatographic separations of hexane extracts of seeds were conducted to investigate effective phytochemicals and their antibacterial activities. Molecular docking studies of werneria chromene and dihydroxyacidissiminol against SARS-CoV-2 virus infection were conducted using AutoDock Vina. The methanol extract of bark inhibited the growth of Staphylococcusaureus, Escherichiacoli, and Pseudomonasaeruginosa with the minimum inhibitory concentration of 250, 500, and 250 µg/mL, respectively. The chloroform extract of endocarps potentiated the activity of imipenem against imipenem-resistant Acinetobacterbaumannii. The hexane extract of seeds increased the sensitivity of P. aeruginosa against ciprofloxacin and levofloxacin. The hexane extract of seeds and chloroform extract of endocarps were chromatographed, yielding werneria chromene and dihydroxyacidissiminol. Werneria chromene was bacteriostatic for P.aeruginosa and P.putida, with MIC/MBC values of 1000 > 1000 µg/mL. Dihydroxyacidissiminol showed the predicted binding energies of −8.1, −7.6, −7.0, and −7.5 kcal/mol with cathepsin L, nsp13 helicase, SARS-CoV-2 main protease, and SARS-CoV-2 spike protein receptor-binding domain S-RBD. Burkillanthusmalaccensis (Ridl.) Swingle can be a potential source of natural products with antibiotic-potentiating activity and that are anti-SARS-CoV-2.

Evaluation of immature platelet fraction as a marker of dengue fever progression.

OBJECTIVES: Progression of dengue is often associated with thrombocytopenia resulting from viral-induced bone marrow suppression and immune-mediated peripheral platelet consumption. Immature platelet fraction (IPF), which can be measured using a haematology analyser, is a precursor indicating platelet formation in the bone marrow. This study evaluated the trend of IPF as an early recovery indicator of platelets in dengue patients with thrombocytopenia, and its relationship with severe dengue in conjunction with reticulocyte count. METHODS: Hospitalized patients with dengue were enrolled and followed-up daily until discharge. Blood investigations included daily full blood counts and IPF measured using a haematology analyser. RESULTS: In total, 287 patients with confirmed dengue were enrolled in this study, 25 of whom had severe dengue. All patients had a decreasing trend in platelet count in the first week of illness, concomitant with an increasing trend in the percentage of immature platelets to total platelets (IPF%) for more than 3 days prior to platelet recovery. IPF% was significantly increased in patients with severe dengue compared with patients with non-severe dengue on days 3-5 after the onset of fever. Reticulocyte count increased significantly in patients with severe dengue on day 5. CONCLUSIONS: IPF can be utilized as an early recovery indicator of platelets in patients with dengue and thrombocytopenia.

Experimental exposure of Burkholderia pseudomallei crude culture filtrate upregulates PD-1 on T lymphocytes.

Burkholderia pseudomallei is the causative agent for melioidosis. Because of its intracellular nature, the bacterium is capable of replicating within a plethora of eukaryotic cell lines. B. pseudomallei can remain dormant within host cells without symptoms for years, causing recrudescent infections. Here, we investigated the pathogenesis mechanism behind the suppression of T cell responses by B. pseudomallei . Peripheral blood mononuclear cells (1×106 cells/well) isolated by Ficoll Paque (Sigma-Aldrich) density gradient centrifugation were incubated with optimized concentrations of bacterial crude culture filtrate antigens (CFAs) (10 ug ml-1) and heat-killed bacteria [1 : 10 multiplicity of infection (m.o.i.)]. Following incubation, cells were investigated for surface expression of coinhibitory molecules by flow cytometry. We found that B. pseudomallei induced the upregulation of programmed death 1 (PD-1), a molecule responsible for T cell exhaustion, on T cells in vitro following exposure to crude CFAs of B. pseudomallei . This upregulation of PD-1 probably contributes to poor immune surveillance and disease pathogenesis.

PLGA nanoparticles loaded with Gallic acid- a constituent of Leea indica against Acanthamoeba triangularis.

Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Resistance towards metronidazole in Blastocystis sp.: A pathogenic consequence.

Blastocsytis sp. is a protozoan parasite that has been linked to common gastrointestinal illnesses. Metronidazole, the first line therapy, was reported to show frequent inefficacy. Previously, Blastocystis sp. isolated from different population showed varying metronidazole resistance. However, the effect of metronidazole treatment on pathogenic potentials of Blastocystis sp. isolated from different populations, which is known to have different gut environment, is unclear. This study investigates the in vitro effect of metronidazole on the pathogenic potentials of Blastocystis sp. isolated from urban and orang asli individuals. Blastocystis sp. ST 3 isolated from symptomatic and asymptomatic individuals were treated with a range of metronidazole concentration. The parasites' growth characteristics, apoptotic rate, specific protease activity and the ability to proliferate cancer cells were analyzed upon treatment with 0.001 mg/l metronidazole. The study demonstrates that Blastocystis sp. isolates showed increase in the parasite numbers especially the amoebic forms (only in urban isolates) after treating with metronidazole at the concentration of 0.001 mg/ml. High number of cells in post-treated isolates coincided with increase of apoptosis. There was a significant increase in cysteine protease of Blastocystis sp. isolates upon treatment despite the initial predominance of serine protease in asymptomatic isolates. Metronidazole resistant Blastocystis sp. also showed significant increase in cancer cell proliferation. Resistance to metronidazole did not show significant different influence on the pathogenicity between Blastocystis sp. isolated from urban and orang asli individual. However, an increase in parasite numbers, higher amoebic forms, cysteine protease and ability to proliferate cancer cells implicates a pathogenic role. The study provides evidence for the first time, the effect of metronidazole towards enhancing pathogenic potentials in Blastocystis sp. when isolated from different gut environment. This necessitates the need for reassessment of metronidazole treatment modalities.

Preparation of Poly (dl-Lactide-co-Glycolide) Nanoparticles Encapsulated with Periglaucine A and Betulinic Acid for In Vitro Anti-Acanthamoeba and Cytotoxicity Activities.

Poly (dl-lactide-co-glycolide) (PLGA) microspheres were synthesized as delivery system for the natural anti-parasitic compounds, Periglaucine A (PGA) and Betulinic acid (BA). Periglaucine A and Betulinic acid were encapsulated in PLGA nanoparticles by single emulsion method with an average particle size of approximately 100⁻500 nm. Periglaucine A and Betulinic acid encapsulation efficiency was observed to be 90% and 35% respectively. Anti-Acanthamoeba property of Periglaucine A and Betulinic acid remained intact after encapsulation. PGA-PLGA and BA-PLGA nanoparticles demonstrated inhibition in viability of Acanthamoeba triangularis trophozoites by 74.9%, 59.9%, 49.9% and 71.2%, 52.2%, 88% respectively at concentration of 100 µg/mL, 50 µg/mL and 25 µg/mL. Cytotoxicity of PGA-PLGA and BA-PLGA nanoparticles has been evaluated against lung epithelial cell line and showed dose dependent cytotoxicity value of IC50 2 µg/mL and 20 µg/mL respectively. Futher, increased viability was observed in lung epithelial cell line in higher doses of synthesized polymeric nanoparticles. Results indicate that poly (dl-lactide-co-glycolide) (PLGA) nanoparticles could be exploratory delivery systems for natural products to improve their therapeutic efficacy.

Exacerbated symptoms in Blastocystis sp.-infected patients treated with metronidazole: two case studies.

Blastocystis sp. is a gastrointestinal (GI) protozoan parasite reported to cause non-specific GI symptoms including diarrhea, flatulence, abdominal pain, and nausea. Complete eradication of Blastocystis sp. is rather challenging even with the drug of choice, i.e., metronidazole. Here, we report on two Blastocystis sp.-infected individuals, who presented increased parasite load and exacerbated symptoms upon treatment with the usual recommended dosage and regime of metronidazole. The two studies uniquely demonstrate for the first time a cyst count as high as fivefold more than the original cyst count before treatment and show an exacerbation of GI symptoms despite treatment. The study provides additional support in recognizing metronidazole resistance in Blastocystis sp. and its consequences towards the pathogenicity of the parasite.

Estimation of the Burden of Serious Human Fungal Infections in Malaysia.

Fungal infections (mycoses) are likely to occur more frequently as ever-increasingly sophisticated healthcare systems create greater risk factors. There is a paucity of systematic data on the incidence and prevalence of human fungal infections in Malaysia. We conducted a comprehensive study to estimate the burden of serious fungal infections in Malaysia. Our study showed that recurrent vaginal candidiasis (>4 episodes/year) was the most common of all cases with a diagnosis of candidiasis (n = 501,138). Oesophageal candidiasis (n = 5850) was most predominant among individuals with HIV infection. Candidemia incidence (n = 1533) was estimated in hospitalized individuals, some receiving treatment for cancer (n = 1073), and was detected also in individuals admitted to intensive care units (ICU) (n = 460). In adults with asthma, allergic bronchopulmonary aspergillosis (ABPA) was the second most common respiratory mycoses noticed (n = 30,062) along with severe asthma with fungal sensitization (n = 39,628). Invasive aspergillosis was estimated in 184 cases undergoing anti-cancer treatment and 834 ICU cases. Cryptococcal meningitis was diagnosed in 700 subjects with HIV/AIDS and Pneumocystis jirovecii pneumonitis (PCP) in 1286 subjects with underlying HIV disease. The present study indicates that at least 590,214 of the Malaysian population (1.93%) is affected by a serious fungal infection annually. This problem is serious enough to warrant the further epidemiological studies to estimate the burden of human fungal infections in Malaysia.

Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 µg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection.

Exacerbation of colon carcinogenesis by Blastocystis sp.

Colorectal cancer (CRC) is one the most commonly diagnosed cancers worldwide and the number is increasing every year. Despite advances in screening programs, CRC remains as the second leading cause of cancer deaths in the United States. Oxidative stress plays an important role in the molecular mechanisms of colorectal cancer (CRC) and has been shown to be associated with Blastocystis sp., a common intestinal microorganism. In the present study, we aimed to identify a role for Blastocystis sp. in exacerbating carcinogenesis using in vivo rat model. Methylene blue staining was used to identify colonic aberrant crypt foci (ACF) and adenomas formation in infected rats whilst elevation of oxidative stress biomarker levels in the urine and serum samples were evaluated using biochemical assays. Histological changes of the intestinal mucosa were observed and a significant number of ACF was found in Blastocystis sp. infected AOM-rats compared to the AOM-controls. High levels of urinary oxidative indices including advanced oxidative protein products (AOPP) and hydrogen peroxide were observed in Blastocystis sp. infected AOM-rats compared to the uninfected AOM-rats. Our study provides evidence that Blastocystis sp. has a significant role in enhancing AOM-induced carcinogenesis by resulting damage to the intestinal epithelium and promoting oxidative damage in Blastocystis sp. infected rats.

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro.

OBJECTIVE: To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro. METHODS: Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment. RESULTS: Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h. CONCLUSIONS: Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Experimental Persistent Infection of BALB/c Mice with Small-Colony Variants of Burkholderia pseudomallei Leads to Concurrent Upregulation of PD-1 on T Cells and Skewed Th1 and Th17 Responses.

BACKGROUND: Burkholderia pseudomallei (B. pseudomallei), the causative agent of melioidosis, is a deadly pathogen endemic across parts of tropical South East Asia and Northern Australia. B. pseudomallei can remain latent within the intracellular compartment of the host cell over prolonged periods of time, and cause persistent disease leading to treatment difficulties. Understanding the immunological mechanisms behind persistent infection can result in improved treatment strategies in clinical melioidosis. METHODS: Ten-day LD50 was determined for the small-colony variant (SCV) and its parental wild-type (WT) via intranasal route in experimental BALB/c mice. Persistent B. pseudomallei infection was generated by administrating sub-lethal dose of the two strains based on previously determined LD50. After two months, peripheral blood mononuclear cells (PBMCs) and plasma were obtained to investigate host immune responses against persistent B. pseudomallei infection. Lungs, livers, and spleens were harvested and bacterial loads in these organs were determined. RESULTS: Based on the ten-day LD50, the SCV was ~20-fold less virulent than the WT. The SCV caused higher bacterial loads in spleens compared to its WT counterparts with persistent B. pseudomallei infection. We found that the CD4+ T-cell frequencies were decreased, and the expressions of PD-1, but not CTLA-4 were significantly increased on the CD4+ and CD8+ T cells of these mice. Notably, persistent infection with the SCV led to significantly higher levels of PD-1 than the WT B. pseudomallei. Plasma IFN-γ, IL-6, and IL-17A levels were elevated only in SCV-infected mice. In addition, skewed plasma Th1 and Th17 responses were observed in SCV-infected mice relative to WT-infected and uninfected mice. CONCLUSION: B. pseudomallei appears to upregulate the expression of PD-1 on T cells to evade host immune responses, which likely facilitates bacterial persistence in the host. SCVs cause distinct pathology and immune responses in the host as compared to WT B. pseudomallei.

Blastocystis sp. subtype 3 triggers higher proliferation of human colorectal cancer cells, HCT116.

Blastocystis sp. is a commonly found intestinal microorganism and was reported to cause many nonspecific gastrointestinal symptoms. Various subtypes have been previously reported, and the pathogenicity of different subtypes of Blastocystis is unclear and remains as a controversial issue. A recent study has shown that the Blastocystis antigen isolated from an unknown subtype could facilitate the proliferation of colon cancer cells. Current study was conducted to compare the effect of solubilized antigen isolated from five different subtypes of Blastocystis on colon cancer cells, HCT116. A statistically significant proliferation of these cells was observed when exposed to 1.0 µg/ml solubilized antigen isolated from subtype 3 Blastocystis (37.22%, p < 0.05). Real-time polymerase chain reaction demonstrated the upregulation of Th2 cytokines especially transforming growth factor beta in subtype 3-treated cancer cells (p < 0.01, 3.71-fold difference). Of interest, subtype 3 Blastocystis antigen also caused a significantly higher upregulation of cathepsin B (subtypes 1 and 2, p < 0.01; subtypes 4 and 5, p < 0.001; 6.71-fold difference) which lead to the postulation that it may enhance the exacerbation of existing colon cancer cells by weakening the cellular immune response. The dysregulation of IFN-γ and p53 expression also suggest Blastocystis as a proponent of carcinogenesis. Therefore, it is very likely for subtype 3 Blastocystis to have higher pathogenic potential as it caused an increased propagation of cancer cells and substantial amount of inflammatory reaction compared to other subtypes.