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Toxoplasma gondii is an intracellular protozoan parasite which can infect most warm-blooded animals and humans. Among the different mouse models, C57BL/6 mice are more susceptible to T. gondii infection compared to BALB/c mice, and this increased susceptibility has been attributed to various factors, including T-cell responses. Dendritic cells (DCs) are the most prominent type of antigen-presenting cells and regulate the host immune response, including the response of T-cells. However, differences in the DC responses of these mouse strains to T. gondii infection have yet to be characterized. In this study, we cultured bone marrow-derived DCs (BMDCs) from BALB/c and C57BL/6 mice. These cells were infected with T. gondii. The activation of the BMDCs was assessed based on the expression of cell surface markers and cytokines. In the BMDCs of both mouse strains, we detected significant increases in the expression of cell surface T-cell co-stimulatory molecules (major histocompatibility complex (MHC) II, CD40, CD80, and CD86) and cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-12p40, IL-1β, and IL-10) from 3 h post-T. gondii infection. The expression of MHC II, CD40, CD80, CD86, IFN-γ, IL-12p40, and IL-1β was significantly higher in the T. gondii-infected BMDCs obtained from the C57BL/6 mice than in those from the BALB/c mice. These findings indicate that differences in the activation status of the BMDCs in the BALB/c and C57BL/6 mice may account for their differential susceptibility to T. gondii.

BACKGROUND Diced cartilage glue (DG) grafts have been widely used in dorsal augmentation but can induce dorsal irregularities. The authors evaluated the postoperative feasibility of a crushed septal cartilage-covered diced cartilage glue (CCDG) graft. METHODS The medical records of 38 patients who underwent dorsal augmentation rhinoplasty with an open approach were retrospectively reviewed. DG graft was used in 18 patients (47.4%), and CCDG graft was used in 20 patients (52.6%). Surgical outcomes were assessed by comparing anthropometric data on facial photographs and satisfaction questionnaires on aesthetic outcomes and palpable irregularities on nasal dorsum before and after surgery. RESULTS Both groups showed successful aesthetic outcomes. Dorsal height, radix height, and tip projection were all increased postoperatively in both groups. Tip rotation did not significantly increase (p > 0.05). Both groups showed similar outcomes in terms of aesthetic satisfaction but a significant difference in palpable irregularity. CCDG graft group showed significantly better (p = 0.04) satisfaction with dorsal irregularities (4.15 ± 0.75) than the DG graft group (3.56 ± 0.92). CCDG graft group also showed significantly better mean values (p = 0.048) in the degree of irregularity by two surgeons (3.85 ± 0.65) than the DG graft group (3.25 ± 0.97). No patient had significant complaints about irregular dorsum, and none of them underwent a revision rhinoplasty. CONCLUSION CCDG graft can be a complementary option for avoiding postoperative irregular dorsum complications. LEVEL OF EVIDENCE IV This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Sirtuin 1 (SIRT1) regulates cellular processes by deacetylating non-histone targets, including transcription factors and intracellular signalling mediators; thus, its abnormal activation is closely linked to the pathophysiology of several diseases. However, its function in Toxoplasma gondii infection is unclear. We found that SIRT1 contributes to autophagy activation via the AMP-activated protein kinase (AMPK) and PI3K/AKT signalling pathways, promoting anti-Toxoplasma responses. Myeloid-specific Sirt1−/− mice exhibited an increased cyst burden in brain tissue compared to wild-type mice following infection with the avirulent ME49 strain. Consistently, the intracellular survival of T. gondii was markedly increased in Sirt1-deficient bone-marrow-derived macrophages (BMDMs). In contrast, the activation of SIRT1 by resveratrol resulted in not only the induction of autophagy but also a significantly increased anti-Toxoplasma effect. Notably, SIRT1 regulates the FoxO-autophagy axis in several human diseases. Importantly, the T. gondii-induced phosphorylation, acetylation, and cytosolic translocation of FoxO1 was enhanced in Sirt1-deficient BMDMs and the pharmacological inhibition of PI3K/AKT signalling reduced the cytosolic translocation of FoxO1 in BMDMs infected with T. gondii. Further, the CaMKK2-dependent AMPK signalling pathway is responsible for the effect of SIRT1 on the FoxO3a-autophagy axis and for its anti-Toxoplasma activity. Collectively, our findings reveal a previously unappreciated role for SIRT1 in Toxoplasma infection.

Toxoplasmosis diagnosis predominantly relies on serology testing via enzyme-linked immunosorbent assay (ELISA), but these results are highly variable. Consequently, various antigens are being evaluated to improve the sensitivity and specificity of toxoplasmosis serological diagnosis. Here, we generated Toxoplasma gondii virus-like particles displaying AMA1 of T. gondii and evaluated their diagnostic potential. We found that AMA1 VLPs were highly sensitive and reacted with the sera acquired from mice infected with either T. gondii ME49 or RH strains. The overall IgG and IgM antibody responses elicited by AMA1 VLPs were substantially higher than those induced by the conventionally used T. gondii lysate antigen (TLA). Importantly, AMA1 VLPs were capable of detecting parasitic infection with T. gondii RH and ME49 as early as 1 week post-infection, even when mice were exposed to low infectious doses (5 × 103 and 10 cysts, respectively). AMA1 VLPs also did not cross-react with the immune sera acquired from Plasmodium berghei-infected mice. Compared to TLA, stronger antibody responses were induced by AMA1 VLPs when tested using T. gondii-infected human sera. The sensitivities and specificities of the two antigens were substantially different, with AMA1 VLPs demonstrating over 90% sensitivity and specificity, whereas these values were in the 70% range for the TLA. These results indicated that AMA1 VLPs can detect infections of both T. gondii ME49 and RH at an early stage of infection caused by very low infection doses in mice, and these could be used for serological diagnosis of human toxoplasmosis.

Fas‐associated factor 1 (FAF1) has gained a reputation as a member of the FAS death‐inducing signalling complex. However, the role of FAF1 in the immunity response is not fully understood. Here, we report that, in the human retinal pigment epithelial (RPE) cell line ARPE‐19 cells, FAF1 expression level was downregulated by Toxoplasma gondii infection, and PI3K/AKT inhibitors reversed T. gondii‐induced FAF1 downregulation. In silico analysis for the FAF1 promoter sequence showed the presence of a FOXO response element (FRE), which is a conserved binding site for FOXO1 transcription factor. In accordance with the finding, FOXO1 overexpression potentiated, whereas FOXO1 depletion inhibited intracellular FAF1 expression level. We also found that FAF1 downregulation by T. gondii is correlated with enhanced IRF3 transcription activity. Inhibition of PI3K/AKT pathway with specific inhibitors had no effect on the level of T. gondii‐induced IRF3 phosphorylation but blocked IRF3 nuclear import and ISGs transcription. These results suggest that T. gondii can downregulate host FAF1 in PI3K/AKT/FOXO1‐dependent manner, and the event is essential for IRF3 nuclear translocation to active the transcription of ISGs and thereby T. gondii proliferation.

Aim: To investigate the anticancer mechanisms of silver nanoparticles (AgNPs) in colorectal cancer. Methods: Anticancer effects of AgNPs were determined in colorectal cancer HCT116 cells and xenograft mice using cellular and molecular methods. Results: AgNPs induced mitochondrial reactive oxygen species production, mitochondrial dysfunction and endoplasmic reticulum (ER) stress responses through NOX4 and led to HCT116 cell apoptosis. Pretreatment with DPI or 4-PBA significantly inhibited mitochondrial reactive oxygen species production, apoptosis, ER stress response, NOX4 expression and mitochondrial dysfunction in AgNP-treated HCT116 cells. AgNPs also significantly suppressed HCT116 cell-based xenograft tumor growth in nude mice by inducing apoptosis and ER stress responses. Conclusion: AgNPs exert anticancer effects against colorectal cancer via ROS- and ER stress-related mitochondrial apoptosis pathways.

We aimed to explore the population dynamics of snail in 3 sites of the White Nile in Sudan. More specifically, we aimed to investigate the annual patterns of snail populations that act as intermediate hosts of schistosomes and monthly snail infection rates and ecological characteristics presumably related to snail populations. We collected snails for 1 year monthly at 3 different shore sites in the vicinity of El Shajara along the White Nile river in Khartoum State, Sudan. In addition, we measured air and water temperatures, water turbidities, vegetation coverages, and water depths and current speeds. Most of the collected snails were Biomphalaria pfeifferi and Bulinus truncatus. The population densities of snails and their infection rates varied across survey sites. The collected snails liberated S. mansoni and S. haematobium cercariae as well as Amphistome and Echinostome cercariae. Infected snails were found during March-June. The ecological characteristics found to be associated with the absence of snails population were: high turbidity, deep water, low vegetation coverage (near absence of vegetation), high water temperature, and high current speed. To our knowledge, this is the first longitudinal study of the snail population and ecological characteristics in the main basin of the White Nile river.

This study aimed to investigate whether mass drug administration (MDA) intervention has an equivalent effect on reducing the prevalence and intensity of Schistosoma haematobium infection regardless of the baseline values. A repeated cross-sectional survey was performed targeting students of 12 primary schools in Al Jabalain and El Salam districts of White Nile State, Sudan, at both 1 week before and 8 months after the MDA. Prior to the baseline survey, school-aged children in Al Jabalain had received MDA interventions twice in 4 years, while those in El Salam had not. The baseline prevalence was 9.1% in Al Jabalain and 35.2% in El Salam, which were reduced to 1.8% and 5.5% at 8 months after the MDA, respectively. The corresponding reduction rates were 80.3% and 84.4%, not significant difference between both districts. However, changes in the geometric mean intensity (GMI) of egg counts were significantly different between both districts. The baseline GMIs were 14.5 eggs per 10 ml of urine (EP10) in Al Jabalain and 18.5 EP10 in El Salam, which were reduced to 7.1 and 11.2 EP10 after treatment, respectively. The corresponding reduction rates were 51.0% and 39.5%. In conclusion, MDA interventions were found to bring about similar relative reduction in prevalence regardless of the baseline value; however, the relative reduction in infection intensity was more salient in the district with a low baseline value for both prevalence and intensity. This clearly points to the importance of repeated MDA interventions in endemic areas, which will eventually contribute to schistosomiasis elimination.

Dendritic cell is one of the first innate immune cell to encounter T. gondii after the parasite crosses the host intestinal epithelium. T. gondii requires intact DC as a carrier to infiltrate into host central nervous system (CNS) without being detected or eliminated by host defense system. The mechanism by which T. gondii avoids innate immune defense of host cell, especially in the dendritic cell is unknown. Therefore, we examined the role of host PI3K/AKT signaling pathway activation by T. gondii in dendritic cell. T. gondii infection or T. gondii excretory/secretory antigen (TgESA) treatment to the murine dendritic cell line DC2.4 induced AKT phosphorylation, and treatment of PI3K inhibitors effectively suppressed the T. gondii proliferation but had no effect on infection rate or invasion rate. Furthermore, it is found that T. gondii or TgESA can reduce H2O2-induced intracellular reactive oxygen species (ROS) as well as host endogenous ROS via PI3K/AKT pathway activation. While searching for the main source of the ROS, we found that NADPH oxidase 4 (NOX4) expression was controlled by T. gondii infection or TgESA treatment, which is in correlation with previous observation of the ROS reduction by identical treatments. These findings suggest that the manipulation of the host PI3K/AKT signaling pathway and NOX4 expression is an essential mechanism for the down-regulation of ROS, and therefore, for the survival and the proliferation of T. gondii.

Purpose External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. Materials and methods ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. Results AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. Conclusion AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

The reinfection rate of schistosomiasis after mass drug administration (MDA) has not been documented in Sudan. We aimed to explore the transmission dynamics of urogenital schistosomiasis after MDA, targeting school-aged children in the White Nile State of Sudan, assessing the prevalence, reinfection rate, and incidence. A single dose of praziquantel (40 mg/kg) was administered to 1951 students in five primary schools from January to February 2018 immediately after a baseline survey, and follow-up surveys were performed at 2 weeks and 6 months after treatment. We examined Schistosoma haematobium eggs by centrifugation methods. The overall reinfection rate at 6 months after treatment was 9.8% (95% confidence interval: 0.5–17.4%). By school, the reinfection rate was highest in the Al Hidaib school, whose prevalence was highest at baseline. The reinfection rate was significantly higher in high-infection areas than low-infection areas (p = 0.02). Of the prevalence at 6 months in high-infection areas, 41% of cases were due to reinfection. MDA interventions are decided upon and undertaken at the district level. A more targeted treatment strategy should be developed with a particular focus on tracking high-risk groups, even within a school or a community.

DNA replication and sister chromatid cohesion 1 (DSCC1) combines with chromosome transmission-fidelity protein 18 (CTF18) to form a CTF18-DSCC1-CTF8 (CTF18-1-8) module, which in combination with CTF18-replication factor C (RFC) acts as a proliferating cell nuclear antigen (PCNA) loader during DNA replication-associated processes. It was found that DSCC1 was overexpressed in tumor tissues from patients with colon cancer and that the survival probability of patients with colon cancer was lower when the expression of cytosolic DSCC1 was higher in tumor regions (P=0.047). By using DSCC1- or CTF18-knockdown cell lines (HCT116-shDSCC1 or HCT116-shCTF18, respectively), it was confirmed that DSCC1-knockdown inhibits cell proliferation and invasion, but that CTF18-knockdown does not. Tumors in mice xenografted with shDSCC1 cells were significantly smaller compared with those in mice in the mock group or those xenografted with shCTF18 cells. The shDSCC1 cells were highly sensitive to γ-irradiation and other DNA replication inhibitory treatments, resulting in low cell viability. The present results suggested that DSCC1 is the most important component in the CTF18-1-8 module for CTF18-RFC and is highly relevant to the growth and metastasis of colon cancer cells, and, therefore, it may be a potential therapeutic target for colon cancer treatment.

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Background: Human trichomoniasis is one of the most common sexually transmitted infections; however, its pathogenesis remains unclear. Here, we investigated the role of the endoplasmic reticulum (ER) in apoptosis induction by T. vaginalis in human cervical epithelial SiHa cellsMethods: We evaluated the cytotoxicity, apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response, and Bcl-2 family protein expressions using LDH assay, immunocytochemistry, flow cytometry, JC-1 dye staining, and western blotting.Results: T. vaginalis induced LDH-dependent cytotoxicity, mitochondrial ROS production, and apoptosis in SiHa cells, parasite burden- and infection time-dependently. T. vaginalis also induced ER stress response and mitochondrial dysfunction, such as MMP depolarization and imbalance in levels of Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-Acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA, ER stress inhibitor) significantly alleviated apoptosis, ROS production, mitochondrial dysfunction, and ER stress response in a dose-dependent manner. These data suggested that SiHa cell apoptosis is affected by ROS and ER stress after T. gondii infection. In addition, T. vaginalis induced ASK1 and JNK phosphorylation in SiHa cells, however 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis, and ER stress response in SiHa cells, dose-dependently.Conclusions: T. vaginalis induces mitochondrial apoptosis via ROS and parasite-mediated ER stress via the IRE1/ASK1/JNK/Mcl-1 pathways, and also induces ER stress response directly and mitochondrial ROS-dependently in human cervical epithelial SiHa cells, thus, T. vaginalis induces apoptosis via ROS and ER stress through ER-mitochondria crosstalk in human cervical epithelial cells. These results expand our understanding of the molecular mechanisms underlying the pathogenesis of human trichomoniasis.

Background Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. Methods FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. Results Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1β, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. Conclusions This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells. Graphical Abstract

Microbial adhesion is critical for parasitic infection and colonization of host cells. To study the host&ndash, parasite interaction in vitro, we established a flow cytometry-based assay to measure the adherence of Trichomonas vaginalis to epithelial cell line SiHa. SiHa cells and T. vaginalis were detected as clearly separated, quantifiable populations by flow cytometry. We found that T. vaginalis attached to SiHa cells as early as 30 min after infection and the binding remained stable up to several hours, allowing for analysis of drug treatment efficacy. Importantly, NADPH oxidase inhibitor DPI treatment induced the detachment of T. vaginalis from SiHa cells in a dose-dependent manner without affecting host cell viability. Thus, this study may provide an understanding for the potential development of therapies against T. vaginalis and other parasite infections.

Microbial adhesion is critical for parasitic infection and colonization of host cells. To study the host–parasite interaction in vitro, we established a flow cytometry-based assay to measure the adherence of Trichomonas vaginalis to epithelial cell line SiHa. SiHa cells and T. vaginalis were detected as clearly separated, quantifiable populations by flow cytometry. We found that T. vaginalis attached to SiHa cells as early as 30 min after infection and the binding remained stable up to several hours, allowing for analysis of drug treatment efficacy. Importantly, NADPH oxidase inhibitor DPI treatment induced the detachment of T. vaginalis from SiHa cells in a dose-dependent manner without affecting host cell viability. Thus, this study may provide an understanding for the potential development of therapies against T. vaginalis and other parasite infections.

This study compared the anthelminthic effects of three different brands of praziquantel being used in Sudan against Schistosoma haematobium (S. haematobium) infection. We enrolled 1,286 schoolchildren from six primary schools and examined their urine samples for eggs of S. haematobium at the baseline survey and follow-up two weeks after administering the medication. The schoolchildren were divided into three groups based on the three brands of praziquantel (different material production), with two school children for one brand. The overall baseline prevalence of S. haematobium infection was 15.5%. Two weeks after treatment with brands A, B, and C of praziquantel, cure rates were 87.1%, 82.4% and 83.8% respectively, and the egg-reduction rates were 69.0%, 81.0% and 70.6% respectively. There was no statistically significant difference in cure rates and egg-reduction rates between the three brands. We conclude that the three different commercial brands of praziquantel used in Sudan have similar anthelminthic effects on S. haematobium., Graphical Abstract

Trichomonas vaginalis is an extracellular protozoan parasite known to cause vaginitis and urethritis...

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.

Rodents are important reservoirs of diseases affecting people and livestock, and are major sources of parasite contamination of agricultural products. We surveyed the infection status of intestinal helminths in 2 species of field mice, Apodemus agrarius and A. peninsulae, captured in the agricultural fields of Gangwon-do and Chungcheongnam-do, Korea. Total 83 mice (57 A. agrarius and 26 A. peninsulae) were collected in 2 surveyed areas, and the intestines of each mouse were opened with scissors, and then intestinal contents were examined with microscope. Total 6 species of intestinal helminth were detected in 61 (73.5%) out of 83 mice examined. Four species of nematode, i.e., Nippostrongylus brasiliensis, Aspiculuris tetraptera, Heterakis spp. and ascarid, were found in 40 (48.2%), 14 (16.9%), 11 (13.3%) and 13 (15.7%) mice respectively. One species of cestode, Hymenolepis diminuta and 1 unidentified egg were also detected in the intestines of 14 (16.9%) and 1 (1.2%) mice, respectively. Conclusively, this study identified 5 helminth species in the gastrointestinal tracts of wild rodents captured in some areas in central and northern Korea, and N. brasiliensis was the most prevalent (dominant) species rather than zoonotic ones.

The conversion of tachyzoites into bradyzoites is a way for Toxoplasma gondii to establish a chronic and asymptomatic infection and achieve lifelong persistence in the host. The bradyzoites form tissue cysts in the retina, but not much is known about the horizontal distribution of the cysts or their interactions with glial cells in the retina. A chronic ocular toxoplasmosis model was induced by per oral administration of T. gondii Me49 strain cysts to BALB/c mice. Two months after the infection, retinas were flat-mounted and immunostained to detect cysts, ganglion cells, Müller cells, astrocytes, and microglial cells, followed by observation under fluorescence and confocal microscope. The horizontal distribution showed a rather clustered pattern, but the clusters were not restricted to certain location of the retina. Axial distribution was confined to the inner retina, mostly in ganglion cell layer or the inner plexiform layer. Both ganglion cells, a type of retinal neurons, and Müller cells, predominant retinal glial cells, could harbor cysts. The cysts were spatially separated from astrocytes, the most abundant glial cells in the ganglion cell layer, while close spatial distribution of microglial cells was observed in two thirds of retinal cysts. In this study, we demonstrated that the retinal cysts were not evenly distributed horizontally and were confined to the inner retina axially. Both neurons and one type of glial cells could harbor cysts, and topographic analysis of other glial cells suggests role of microglial cells in chronic ocular toxoplasmosis.

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Introduction: CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+ B-cell malignancies. All the FDA-approved CART19, including tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, and lisocabtagene maraucel use an anti-CD19 single-chain variable fragments (scFv) derived from the FMC63 antibody that binds to a CD19 epitope that is located in the membrane-distal portion of CD19. While this CART19 products are very effective in the clinic, the majority of patients still fails treatment or eventually relapses due to several mechanisms of resistance, including CAR T cell dysfunction in the immunosuppressive microenvironment. Novel strategies to enhance the activity of CART cells are critically needed. We and others recently demonstrated that modifications of the binding region of the CAR (scFv) (Singh N., Nat Med, 2021) can drastically change the interaction between the CAR T cell and the cancer cells, potentially improving the anti-tumor effect. In this study, we aimed to develop a novel anti-CD19 CAR that binds to a membrane-proximal domain of CD19 with the goal of improving CART functions. Moreover, we hypothesized that such a CART product would be active against B-cell acute lymphoblastic leukemia (B-ALL) blasts that present aberrant expression of the CAR19(FMC63) on the surface as we described in two pediatric B-ALL patients relapsed after CART19 (CTL019) at our Institution (Ruella M., Nat Med, 2018). Methods and Results: To these goals, we screened a chicken immune scFv library against the CD19 extracellular domain. We selected an scFv clone (1218) that was not competing with FMC63 for CD19 binding in competition enzyme immunoassays and real-time interaction analysis. The chicken 1218 scFv was humanized by CDR-grafting to human germline genes and backmutations (h1218 scFv) and was confirmed to be specific for CD19 in cell microarray assays and flow cytometry analyses. In order to define the epitope that was recognized by the 1218 scFv, we performed mutagenesis assays replacing several residues of human CD19 with monkey CD19 residues showed that, as compared to FMC63, the epitope of the h1218 scFv is localized in a membrane-proximal location shown by three-dimensional modeling (Teplyakov A., Proteins, 2018) (Fig. 1A). Key residues needed for the binding of the h1218 scFv were identified as T51, S53, E55, K59 and K63, while the membrane-distal amino acid H218 is recognized as a key residue for FMC63 binding. Therefore, we designed a novel CAR construct using the h1218 scFv, a 4-1BB costimulatory and CD3z signaling domains, using a lentiviral backbone. This CAR(h1218) construct was very active in vitro in human T cells demonstrating cytotoxicity, cytokine release, and proliferation upon encounter with CD19+ leukemic and lymphoma cells (NALM6, RAJI, and OCI-Ly18) (data not shown). Moreover, CART19(h1218) showed enhanced long-term (12 days) tumor control as compared to CART19(FMC63) against wild type NALM6 (data not shown). In vivo, CART19(h1218) cells (1.5x10 6 CAR+ cells/mouse) demonstrated tumor clearance against both Raji (CD19+ B-cell lymphoma) and Nalm6 (CD19+ B-cell leukemia) xenografts (Fig. 1B). Remarkably, in a more challenging stress- test model with lower CART doses (0.75x10 6 CAR+ cells/mouse) CART19(h1218) demonstrated stronger tumor control against Nalm6 cells as compared to CART19(FMC63) (Fig. 1C). Furthermore, our group recently described the occurrence of a B-ALL relapse characterized by the expression of the CAR19 itself on the cell surface due to accidental transduction of leukemic cells during manufacturing. The CART19(h1218) product but not CART19(FMC63) was able to recognize and kill CAR19(FMC63)-expressing leukemic cells in vitro at varying E:T ratios. Of note, in a long-term (12 days) in vitro killing assay using a low, challenging E:T ratio of 0.125:1 we observed potent and prolonged tumor control (Fig. 1D). Conclusions: These results indicate that the development of a CAR binder able to recognize an alternative and membrane-proximal epitope of CD19 might lead to improved anti-leukemia and lymphoma activity and can recognize CAR19(FMC63)+ blasts that are not recognized by CART19(FMC63). This novel CART19(h1218) product will be tested in phase I-II clinical trials for patients with B-cell malignancies. Figure 1 Figure 1. Disclosures Patel: AbClon Inc.: Research Funding. Kim: Abclon Inc.: Current Employment. Lee: AbClon Inc.: Research Funding. Guruprasad: AbClon Inc.: Research Funding. Kim: AbClon Inc.: Current Employment. Choi: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Park: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Cui: AbClon Inc.: Current Employment. Yoon: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Research Funding. Kim: AbClon Inc.: Research Funding. Hwang: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Chung: AbClon Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ruella: Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding.

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

User specific preamble transmission and timing advance command mechanisms are widely adopted for cellular uplink synchronization. However, the signaling overheads are scaled with the number of users and become prohibitive in massive connectivity usage scenario. In this paper, a kind of cooperative synchronization method is discussed resorting to cell specific signals. More specifically, the cluster of synchronous cells transmit time advanced signals cooperatively, and users estimate individual timing advances based on the reception time.