Differences in ocular adverse events associated with phosphodiesterase-5 inhibitors: a real-world pharmacovigilance study.

OBJECTIVE: Our study aims to characterize the ocular safety profiles of phosphodiesterase type 5 (PDE5) inhibitors and explore the differences among different PDE5 inhibitors. METHODS: We analyzed reports on ocular adverse events associated with sildenafil, vardenafil and tadalafil submitted to the FDA Adverse Event Reporting System (FAERS) database from the first quarter of 2004 to the first quarter of 2023. Disproportionality analysis was conducted to evaluate reporting risk profiles. RESULTS: Among 61,211 reports qualifying for analysis, 5,127 involved sildenafil, 832 vardenafil, and 3,733 tadalafil. All PDE5 inhibitors showed increased reporting odds ratios (ROR) for ocular adverse events, with vardenafil highest (ROR 4.47) followed by sildenafil and tadalafil. Key ocular adverse events included cyanopsia, optic ischemic neuropathy, visual field defects, unilateral blindness and blindness. Sildenafil showed the highest disproportionality for cyanopsia (ROR 1148.11) while vardenafil and tadalafil showed the highest disproportionality for optic ischemic neuropathy. Time-to-onset analysis also revealed significant differences, with sildenafil having a later median time-to-onset compared to vardenafil and tadalafil. CONCLUSIONS: This comprehensive pharmacovigilance study reveals distinct patterns of ocular adverse events associated with PDE5 inhibitors. These findings contribute to a better understanding of the safety profiles of PDE5 inhibitors and may guide healthcare professionals in clinical decision-making.

Large DNA fragment knock-in and sequential gene editing in Plasmodium falciparum: a preliminary study using suicide-rescue-based CRISPR/Cas9 system.

CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.

Plasmodium immunotherapy combined with gemcitabine has a synergistic inhibitory effect on tumor growth and metastasis in murine Lewis lung cancer models.

Objective: Our previous studies have demonstrated that Plasmodium immunotherapy (infection) has antitumor effects in mice. However, as a new form of immunotherapy, this therapy has a weakness: its specific killing effect on tumor cells is relatively weak. Therefore, we tested whether Plasmodium immunotherapy combined with gemcitabine (Gem), a representative chemotherapy drug, has synergistic antitumor effects. Methods: We designed subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) models to test the antitumor effect of Plasmodium chabaudi ASS (Pc) infection in combination with Gem treatment and explored its underlying mechanisms. Results: We found that both Pc infection alone and Gem treatment alone significantly inhibited tumor growth in the subcutaneous model, and combination therapy was more effective than either monotherapy. Monotherapy only tended to prolong the survival of tumor-bearing mice, while the combination therapy significantly extended the survival of mice, indicating a significant synergistic effect of the combination. In the mechanistic experiments, we found that the combination therapy significantly upregulated E-cadherin and downregulated Snail protein expression levels, thus inhibiting epithelial-mesenchymal transition (EMT) of tumor cells, which may be due to the blockade of CXCR2/TGF-ß-mediated PI3K/Akt/GSK-3ß signaling pathway. Conclusion: The combination of Pc and Gem plays a synergistic role in inhibiting tumor growth and metastasis, and prolonging mice survival in murine lung cancer models. These effects are partially attributed to the inhibition of EMT of tumor cells, which is potentially due to the blockade of CXCR2/TGF-ß-mediated PI3K/Akt/GSK-3ß/Snail signaling pathway. The clinical transformation of Plasmodium immunotherapy combined with Gem for lung cancer is worthy of expectation.

Circular RNA DHTKD1 targets miR­338­3p/ETS1 axis to regulate the inflammatory response in human bronchial epithelial cells.

Asthma is a chronic inflammatory airway disease and the airway epithelium is involved in airway inflammation and innate immunity. However, whether circular RNA (circRNA) is involved in the pathogenesis of asthma remains unclear. The present study aimed to determine the functions and molecular mechanisms of circRNA targeting dehydrogenase E1 (circDHTKD1) in the inflammation response of human bronchial epithelial cells. BEAS-2B cells were stimulated with lipopolysaccharide (LPS) to establish a model of in vitro airway inflammation. Cell viability was assessed using Cell Counting Kit-8 assay. CircDHTKD1 was characterised by nucleocytoplasmic isolation and Sanger sequencing. The RNA expression levels of circDHTKD1, microRNA (miR)-338-3p and potential ERK pathway downstream genes were evaluated by reverse transcription-quantitative polymerase chain reaction. Western blot analysis was performed to measure associated protein levels. The levels of inflammatory cytokines were detected by ELISA. The interaction between circDHTKD1 and miR-338-3p was confirmed by dual-luciferase reporter assay. circDHTKD1 expression was significantly upregulated by LPS treatment, whereas miR-338-3p expression was decreased. Furthermore, circDHTKD1 directly targeted miR-338-3p, which negatively regulated expression of E26 transformation specific-1 (ETS1). Inflammatory cytokine and ETS1 expression levels decreased following transfection with small interfering RNA targeting circDHTKD1 or miR-338-3p mimics. In addition, co-transfection with miR-338-3p inhibitor reversed the effects caused by circDHTKD1 knockdown. The knockdown of ETS1 in LPS-induced BEAS-2B cells resulted in decreased cytokine production and inhibition of the ERK signalling pathway. Overall, these results suggested that the knockdown of circDHTKD1 alleviated the LPS-induced production of inflammatory cytokines and activation of the ERK pathway in BEAS-2B cells through the miR-338-3p/ETS1 axis. In summary, circDHTKD1 exacerbated LPS-triggered inflammation responses in BEAS-2B cells by regulating ETS1 expression by interacting with miR-338-3p, suggesting that circDHTKD1 may serve as a potential therapeutic target against asthma.

Application of Monocyte-to-Albumin Ratio and Neutrophil Percentage-to-Hemoglobin Ratio on Distinguishing Non-Small Cell Lung Cancer Patients from Healthy Subjects.

Objective: This study aims at assessing the potential benefits of observation of monocyte-to-albumin ratio (MAR) and neutrophil percentage-to-hemoglobin ratio (NPHR) in the detection of non-small cell lung cancer (NSCLC). Methods: This study retrospectively involved 195 NSCLC patients and 204 healthy volunteers. The correlations between the clinicopathological characteristics of NSCLC and the two ratios including MAR and NPHR were assessed. The diagnostic efficiency of NSCLC patients by MAR and NPHR, alone or in combination with carcinoembryonic antigen (CEA), was assessed by receiver operating characteristic (ROC) curve. The risk factors for NSCLC were analyzed with binary logistic regression. Results: Compared to healthy controls, the levels of MAR and NPHR in NSCLC patients were elevated. MAR and NPHR were related to clinicopathologic characteristics and increased significantly along with the progression of NSCLC. The area under the curve (AUC) for 95% confidence interval (95% CI) of MAR and NPHR in the diagnosis of NSCLC was 0.812 (0.769-0.854) and 0.724 (0.675-0.774), respectively. The combination of MAR, NPHR, and CEA achieved the highest diagnostic utility compared to each individually or combined markers (AUC, 0.86; 95% CI, 0.824-0.896; sensitivity, 72.8%; specificity, 87.3%). Further analysis showed that MAR combined with NPHR presented the potential to detect early-stage (IA-IIB) NSCLC (AUC, 0.794; 95% CI, 0.743-0.845; sensitivity, 55.1%; specificity, 87.7%). The result indicated that MAR and NPHR might be risk factors for NSCLC. Conclusion: MAR and NPHR could be novel and effective auxiliary indexes in the detection of NSCLC, especially when combined with CEA.

Additional diagnostic value of the monocyte to red blood cell count ratio and the product of lymphocyte count and albumin concentration in lung cancer management.

The present study aimed to evaluate the potential of the monocyte to red blood cell count ratio (MRR), the neutrophil to red blood cell count ratio (NRR), the lymphocyte to red blood cell count ratio (LRR) and the product of lymphocyte count and albumin concentration (LA) for the diagnosis of lung cancer. The cases of 216 patients with newly diagnosed lung cancer and 184 healthy volunteers were retrospectively analysed. The MRR and NRR were found to be higher in patients with lung cancer compared with those in healthy controls, while the LRR and LA were lower. The receiver operating characteristic curve analysis revealed that of the four markers, the MRR and LA yielded a higher area under the curve (AUC) (MRR: AUC, 0.810; 95% CI, 0.768-0.847; and LA: AUC, 0.721; 95% CI, 0.674-0.764). The combination of MRR, LA, carcinoembryonic antigen (CEA) and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) achieved the highest diagnostic value when compared with other single or combined markers (AUC, 0.882; 95% CI, 0.846-0.912; sensitivity, 81.9%; specificity, 81.0%). As the disease progressed, the MRR tended to increase, while LA exhibited a decreasing trend. Binary logistic regression analysis revealed an increase in the MRR, as well as in CEA and CYFRA21-1 concentrations, and a decrease in the LA, which could all be possible risk factors for lung cancer. Differences in the MRR and LA between patients with early stage (IA-IIIA) lung cancer and healthy controls were observed. Further analysis revealed that the MRR also exhibited the potential to detect early stage (IA-IIIA) lung cancer in the model. The present findings demonstrated that the MRR and LA may be used as auxiliary biomarkers for the diagnosis of lung cancer and could partly indicate disease progression.

Exogenous melatonin enhances tomato heat resistance by regulating photosynthetic electron flux and maintaining ROS homeostasis.

Heat stress reduces plant growth and reproduction and increases agricultural risks. As a natural compound, melatonin modulates broad aspects of the responses of plants to various biotic and abiotic stresses. However, regulation of the photosynthetic electron transfer, reactive oxygen species (ROS) homeostasis and the redox state of redox-sensitive proteins in the tolerance to heat stress induced by melatonin remain largely unknown. The oxygen evolution complex activity on the electron-donating side of photosystem II (PSII) is inhibited, and the electron transfer process from QA to QB on the electron-accepting side of PSII is inhibited. In this case, heat stress decreased the chlorophyll content, carbon assimilation rate, PSII activity, and the proportion of light absorbed by tomato seedlings during electron transfer. The ROS burst led to the breakdown of the PSII core protein. However, exogenous melatonin increased the net photosynthetic rate by 11.3% compared with heat stress, substantially reducing the restriction of photosynthetic systems induced by heat stress. Additionally, melatonin reduces the oxidative damage to PSII by balancing electron transfer on the donor, reactive center, and acceptor sides. Melatonin was used under heat stress to increase the activity of the antioxidant enzyme and preserve ROS equilibrium. In addition, redox proteomics also showed that melatonin controls the redox levels of proteins involved in photosynthesis, and stress and defense processes, which enhances the expression of oxidative genes. In conclusion, melatonin via controlling the photosynthetic electron transport and antioxidant, melatonin increased tomato heat stress tolerance and aided plant growth.

Preclinical Study of Plasmodium Immunotherapy Combined with Radiotherapy for Solid Tumors.

Immune checkpoint blockade therapy (ICB) is ineffective against cold tumors and, although it is effective against some hot tumors, drug resistance can occur. We have developed a Plasmodium immunotherapy (PI) that can overcome these shortcomings. However, the specific killing effect of PI on tumor cells is relatively weak. Radiotherapy (RT) is known to have strong specific lethality to tumor cells. Therefore, we hypothesized that PI combined with RT could produce synergistic antitumor effects. We tested our hypothesis using orthotopic and subcutaneous models of mouse glioma (GL261, a cold tumor) and a subcutaneous model of mouse non-small cell lung cancer (NSCLC, LLC, a hot tumor). Our results showed that, compared with each monotherapy, the combination therapy more significantly inhibited tumor growth and extended the life span of tumor-bearing mice. More importantly, the combination therapy could cure approximately 70 percent of glioma. By analyzing the immune profile of the tumor tissues, we found that the combination therapy was more effective in upregulating the perforin-expressing effector CD8+ T cells and downregulating the myeloid-derived suppressor cells (MDSCs), and was thus more effective in the treatment of cancer. The clinical transformation of PI combined with RT in the treatment of solid tumors, especially glioma, is worthy of expectation.

Subsequent malaria enhances virus-specific T cell immunity in SIV-infected Chinese rhesus macaques.

BACKGROUND: Coinfection with HIV and Plasmodium parasites is fairly common, but the sequence of infection with these two pathogens and their impact on disease progression are poorly understood. METHODS: A Chinese rhesus macaque HIV and Plasmodium coinfection model was established to compare the impact of pre-existing and subsequent malaria on the progression of SIV infection. RESULTS: We found that a pre-existing malaria caused animals to produce a greater number of CD4+CCR5+ T cells for SIV replication, resulting in higher viral loads. Conversely, subsequent malaria induced a substantially larger proportion of CD4+CD28highCD95high central memory T cells and a stronger SIV-specific T cell response, maintained the repertoire diversity of SIV-specific T cell receptors, and generated new SIV-specific T cell clonotypes to trace SIV antigenic variation, resulting in improved survival of SIV-infected animals. CONCLUSION: The complex outcomes of this study may have important implications for research on human HIV and malaria coinfection. The infection order of the two pathogens (HIV and malaria parasites) should be emphasized. Video abstract.

Preparation of Decoquinate Solid Dispersion by Hot-Melt Extrusion as an Oral Dosage Form Targeting Liver-Stage Plasmodium Infection.

Liver-stage Plasmodium in humans is an early stage of malarial infection. Decoquinate (DQ) has a potent multistage antimalarial activity. However, it is practically water insoluble. In this study, the hot-melt extrusion (HME) approach was employed to prepare solid dispersions of DQ to improve oral bioavailability. The DQ dispersions were homogeneous in an aqueous suspension that contained most DQ (>90%) in the aqueous phase. Soluplus, a solubilizer, was found compatible with DQ in forming nanoparticle formulations during the HME process. Another excipient HPMC AS-126 was also proven to be suitable for making DQ nanoparticles through HME. Particle size and antimalarial activity of HME DQ suspensions remained almost unchanged after storage at 4°C for over a year. HME DQ was highly effective at inhibiting Plasmodium infection in vitro at both the liver stage and blood stage. HME DQ at 3 mg/kg by oral administration effectively prevented Plasmodium infection in mice inoculated with Plasmodium berghei sporozoites. Orally administered HME DQ at 2,000 mg/kg to mice showed no obvious adverse effects. HME DQ at 20 mg/kg orally administered to rats displayed characteristic distributions of DQ in the blood with most DQ in the blood cells, revealing the permeability of HME DQ into the cells in relation to its antimalarial activity. The DQ dispersions may be further developed as an oral formulation targeting Plasmodium infection at the liver stage.

Decoquinate liposomes: highly effective clearance of Plasmodium parasites causing severe malaria.

BACKGROUND: Severe malaria caused by Plasmodium falciparum leads to most malaria-related deaths globally. Decoquinate (DQ) displays strong activity against multistage infection by Plasmodium parasites. However, the development of DQ as an oral dosage form for the treatment of malaria at the blood stage has not been successful. In this study, liposome formulations of DQ were created for intravenous (IV) injection to suppress Plasmodium berghei, a parasite that causes severe malaria in mice. METHODS: DQ liposomes were prepared by conventional ethanol injection method with slight modifications and encapsulation efficiency evaluated by the well-established centrifugation method. Potency of the DQ liposomes against P. falciparum was assessed in vitro using freshly isolated human red blood cells. The efficacy of the DQ liposomes was examined in the mouse model of severe malaria. RESULTS: The DQ liposomes were around 150 nm in size and had the encapsulation efficiency rates > 95%. The freshly prepared and lyophilized liposomes were stable after storage at - 20 °C for 6 months. The liposomes were shown to have excellent activity against P. falciparum in vitro with DQ IC50 0.91 ± 0.05 nM for 3D7 (chloroquine sensitive strain) and DQ IC50 1.33 ± 0.14 nM for Dd2 (multidrug resistant strain), which were 18- and 14-fold more potent than artemisinin, respectively. Mice did not have any signs of toxicity after receiving high dose of the liposomes (DQ 500 mg/kg per mouse) by IV injection. In the mouse model of severe malaria, the liposomes had impressive efficacy against P. berghei with DQ ED50 of 0.720 mg/kg. CONCLUSION: The DQ liposomes prepared in this study were stable for long term storage and safe for IV injection in mammalian animals. The newly created liposome formulations had excellent activity against Plasmodium infection at the blood-stage, which encourages their application in the treatment of severe malaria.

Plasmodium infection prevents recurrence and metastasis of hepatocellular carcinoma possibly via inhibition of the epithelial­mesenchymal transition.

Postoperative recurrence causes a high mortality rate among patients with hepatocellular carcinoma (HCC). The current study aimed to determine the effects of Plasmodium infection on HCC metastasis and recurrence. The antitumor effects of Plasmodium infection were determined using two murine orthotopic HCC models: The non­resection model and the resection model. Tumour tissues derived from tumour­bearing mice treated with or without Plasmodium infection were harvested 15 days post­tumour inoculation. The expression levels of biomarkers related to epithelial­mesenchymal transition (EMT) and molecules associated with CC­chemokine receptor 10 (CCR10)­mediated PI3K/Akt/GSK­3ß/Snail signalling were identified using reverse transcription­quantitative PCR and western blotting. The results demonstrated that Plasmodium infection significantly suppressed the progression, recurrence and metastasis of HCC in the two mouse models. The expression levels of E­cadherin were significantly higher in the Plasmodium­treated group compared with that in the control group, whereas the expression levels of Vimentin and Snail were significantly lower in the Plasmodium­treated group. Furthermore, Plasmodium infection inhibited the activation of Akt and GSK­3ß in the tumour tissues by downregulating the expression levels of CCR10 and subsequently suppressing the accumulation of Snail, which may contribute to the suppression of EMT and the prevention of tumour recurrence and metastasis. In conclusion, the results of the present study demonstrated that Plasmodium infection inhibited the recurrence and metastasis and improved the prognosis of HCC by suppressing CCR10­mediated PI3K/Akt/GSK­3ß/Snail signalling and preventing the EMT. These results may be important for the development of novel therapies for HCC recurrence and metastasis, especially for patients in the perioperative period.

Plasmodium infection inhibits triple negative 4T1 breast cancer potentially through induction of CD8+ T cell-mediated antitumor responses in mice.

We previously reported that Plasmodium infection promotes antitumor immunity in a murine Lewis lung cancer. In this study, we investigated the effects of Plasmodium infection on the tumor inhibition and antitumor CD8+ T cell responses in a murine triple negative breast cancer (TNBCA) model. The results showed that Plasmodium infection significantly inhibited tumor growth, and increased the survival rate of the tumor-bearing mice. Both effector and memory CD8+ T cells were increased in peripheral blood and tumor-draining lymph node (DLN) in the infected mice. The co-stimulatory (CD40L, GITR and OX-40) and co-inhibitory (PD-1, CTLA-4, TIM-3, LAG3) immune checkpoints were up-regulated on CD8+ T cells in infected mice. Importantly, Py induced remarkable effects on the infiltration of CD8+ T cells in the tumor and granzym B+ CD8+ T cells in tumor-bearing mice while not in tumor-free mice. In summary, the results suggested that the effects of Plasmodium infection on murine 4T1 breast cancer might be related to the induction of CD8+ T cell-mediated antitumor immune responses. This finding may provide a novel strategy for the treatment of triple negative breast cancer.

Plasmodium infection inhibits tumor angiogenesis through effects on tumor-associated macrophages in a murine implanted hepatoma model.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in China. The lack of an effective treatment for this disease results in a high recurrence rate in patients who undergo radical tumor resection, and the 5-year survival rate of these patients remains low. Our previous studies demonstrated that Plasmodium infection provides a potent antitumor effect by inducing innate and adaptive immunity in a murine Lewis lung carcinoma (LLC) model. METHODS: This study aimed to investigate the inhibitory effect of Plasmodium infection on hepatocellular carcinoma in mice, and various techniques for gene expression analysis were used to identify possible signal regulation mechanisms. RESULTS: We found that Plasmodium infection efficiently inhibited tumor progression and prolonged survival in tumor-bearing mice, which served as a murine implanted hepatoma model. The inhibition of tumor progression by Plasmodium infection was related to suppression of tumor angiogenesis within the tumor tissue and decreased infiltration of tumor-associated macrophages (TAMs). Further study demonstrated that matrix metalloprotease 9 (MMP-9) produced by TAMs contributed to tumor angiogenesis in the tumor tissue and that the parasite-induced reduction in MMP-9 expression in TAMs resulted in the suppression of tumor angiogenesis. A mechanistic study revealed that the Plasmodium-derived hemozoin (HZ) that accumulated in TAMs inhibited IGF-1 signaling through the PI3-K and MAPK signaling pathways and thereby decreased the expression of MMP-9 in TAMs. CONCLUSIONS: Our study suggests that this novel approach of inhibiting tumor angiogenesis by Plasmodium infection is of high importance for the development of new therapies for cancer patients. Video abstract.

Experimental Treatment of SIV-Infected Macaques via Autograft of CCR5-Disrupted Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem cell (HSC)-based gene therapy targeting CCR5 represents a promising way to cure human immunodeficiency virus type 1 (HIV-1) infection. Yet the preclinical animal model with transplantation of autologous CCR5-ablated HSCs remains to be optimized. In this study, four Chinese rhesus macaques of simian immunodeficiency virus (SIV) chronic infection were given long-term antiretroviral therapy (ART), during which peripheral CD34+ hematopoietic stem and progenitor cells (HSPCs) were purified and infected with CCR5-specific CRISPR/Cas9 lentivirus (three monkeys) or GFP lentivirus (one monkey). After non-myeloablative conditioning, the CCR5-modified or GFP-labeled HSPCs were autotransplanted to four recipients, and ART was withdrawn following engraftment. All of the recipients survived the process of transplantation. The purified CD34+ HSPCs harbored an undetectable level of integrated SIV DNA. The efficiency of CCR5 disruption in HSPCs ranges from 6.5% to 15.6%. Animals experienced a comparable level of hematopoietic reconstuction and displayed a similar physiological homeostasis Despite the low-level editing of CCR5 in vivo (0.3%-1%), the CCR5-disrupted cells in peripheral CD4+ Effector Memory T cell (TEM) subsets were enriched 2- to 3-fold after cessation of ART. Moreover, two of the three treated monkeys displayed a delayed viral rebound and a moderately recovered immune function 6 months after ART withdrawal. This study highlights the importance of improving the CCR5-editing efficacy and augmenting the virus-specific immunity for effective treatment of HIV-1 infection.

SIV infection aggravates malaria in a Chinese rhesus monkey coinfection model.

BACKGROUND: The co-occurrence of human immunodeficiency virus (HIV) infection and malaria in humans in endemic areas raises the question of whether one of these infections affects the course of the other. Although epidemiological studies have shown the impact of HIV infection on malaria, the mechanism(s) are not yet fully understood. Using a Chinese rhesus macaque coinfection model with simian immunodeficiency virus (SIV) and Plasmodium cynomolgi (Pc) malaria, we investigated the effect of concurrent SIV infection on the course of malaria and the underlying immunological mechanism(s). METHODS: We randomly assigned ten Chinese rhesus monkeys to two groups based on body weight and age. The SIV-Pc coinfection animals (S + P group) were infected intravenously with SIVmac251 eight weeks prior to malaria infection, and the control animals (P group) were infected intravenously with only Pc-infected red blood cells. After malaria was cured with chloroquine phosphate, we also initiated a secondary malaria infection that lasted 4 weeks. We monitored body weight, body temperature and parasitemia, measured SIV viral loads, hemoglobin and neopterin, and tracked the CD4+, CD8+, and CD4+ memory subpopulations, Ki67 and apoptosis by flow cytometry. Then, we compared these parameters between the two groups. RESULTS: The animals infected with SIV prior to Pc infection exhibited more severe malaria symptoms characterized by longer episodes, higher parasitemia, more severe anemia, greater body weight loss and higher body temperature than the animals infected with Pc alone. Concurrent SIV infection also impaired immune protection against the secondary Pc challenge infection. The coinfected animals showed a reduced B cell response to Pc malaria and produced lower levels of Pc-specific antibodies. In addition, compared to the animals subjected to Pc infection alone, the animals coinfected with SIV and Pc had suppressed total CD4+ T cells, CD4+CD28highCD95high central memory T cells, and CD4+CD28lowCD95- naïve T cells, which may result from the imbalanced immune activation and faster CD4+ T cell turnover in coinfected animals. CONCLUSIONS: SIV infection aggravates malaria physiologically and immunologically in Chinese rhesus monkeys. This nonhuman primate SIV and Pc malaria coinfection model might be a useful tool for investigating human HIV and malaria coinfection and developing effective therapeutics.

4-Aryl Pyrrolidines as Novel Orally Efficacious Antimalarial Agents. Part 2: 2-Aryl-N-(4-arylpyrrolidin-3-yl)acetamides.

Malaria is caused by infection from the Plasmodium parasite and kills hundreds of thousands of people every year. Emergence of new drug resistant strains of Plasmodium demands identification of new drugs with novel chemotypes and mechanisms of action. As a follow up to our evaluation of 4-aryl-N-benzylpyrrolidine-3-carboxamides as novel pyrrolidine-based antimalarial agents, we describe herein the structure-activity relationships of the reversed amide homologues 2-aryl-N-(4-arylpyrrolidin-3-yl)acetamides. Unlike their carboxamide homologues, acetamide pyrrolidines do not require a third chiral center to be potent inhibitors of P. falciparum and have good pharmacokinetic properties and improved oral efficacy in a mouse model of malaria. Compound (-)-32a (CWHM-1552) has an in vitro IC50 of 51 nM in the P. falciparum 3D7 assay and an in vivo ED90 of <10 mg/kg/day and ED99 of 30 mg/kg/day in a murine P. chabaudi model. Remarkably, the absolute stereochemical preference for this acetamide series (3S,4R) is opposite of that determined for the homologous carboxamide series. Lead compounds for this class have modest affinities for the hERG channel and inhibit CYP 3A4. Additional optimization is needed in order to eliminate these undesired properties from this otherwise promising series of antimalarial compounds.

Plasmodium infection inhibits the expansion and activation of MDSCs and Tregs in the tumor microenvironment in a murine Lewis lung cancer model.

BACKGROUND: A major challenge in the development of effective cancer immunotherapy is the ability of tumors and their microenvironment to suppress immune cells through immunosuppressive cells such as myeloid -derived suppressor cells and regulatory T cells. We previously demonstrated that Plasmodium infection promotes innate and adaptive immunity against cancer in a murine Lewis lung cancer model but its effects on immunosuppressive cells in the tumor microenvironment are unknown. METHODS: Whole Tumors and tumor-derived sorted cells from tumor-bearing mice treated with or without plasmodium infected red blood cells were harvested 17 days post tumor implantation and analyzed using QPCR, western blotting, flow cytometry, and functional assays. Differences between groups were analyzed for statistical significance using Student's t-test. RESULTS: Here we found that Plasmodium infection significantly reduced the proportions of MDSCs and Tregs in the lung tumor tissues of the treated mice by downregulating their recruiting molecules and blocking cellular activation pathways. Importantly, CD8+ T cells isolated from the tumors of Plasmodium-treated mice exhibited significantly higher levels of granzyme B and perforin and remarkably lower levels of PD-1. CONCLUSION: We reveal for the first time, the effects of Plasmodium infection on the expansion and activation of MDSCs and Tregs with a consequent elevation of CD8+T cell-mediated cytotoxicity within the tumor microenvironment and hold great promise for the development of effective immunotherapeutic strategies.

4-Aryl Pyrrolidines as a Novel Class of Orally Efficacious Antimalarial Agents. Part 1: Evaluation of 4-Aryl- N-benzylpyrrolidine-3-carboxamides.

Identification of novel chemotypes with antimalarial efficacy is imperative to combat the rise of Plasmodium species resistant to current antimalarial drugs. We have used a hybrid target-phenotype approach to identify and evaluate novel chemotypes for malaria. In our search for drug-like aspartic protease inhibitors in publicly available phenotypic antimalarial databases, we identified GNF-Pf-4691, a 4-aryl- N-benzylpyrrolidine-3-carboxamide, as having a structure reminiscent of known inhibitors of aspartic proteases. Extensive profiling of the two terminal aryl rings revealed a structure-activity relationship in which relatively few substituents are tolerated at the benzylic position, but the 3-aryl position tolerates a range of hydrophobic groups and some heterocycles. Out of this effort, we identified (+)-54b (CWHM-1008) as a lead compound. 54b has EC50 values of 46 and 21 nM against drug-sensitive Plasmodium falciparum 3D7 and drug-resistant Dd2 strains, respectively. Furthermore, 54b has a long half-life in mice (4.4 h) and is orally efficacious in a mouse model of malaria (qd; ED99 ∼ 30 mg/kg/day). Thus, the 4-aryl- N-benzylpyrrolidine-3-carboxamide chemotype is a promising novel chemotype for malaria drug discovery.

Granuloma formation in the liver is relatively delayed, although sustained, in BCG­infected mice co­infected with Plasmodium.

The purpose of the present study was to examine the effects of Plasmodium on the process of granuloma formation in Bacille Calmette­Guerin (BCG)­infected mice. Female six­week­old BALB/c mice were co­infected with BCG and Plasmodium. The liver index, pathological alterations and quantity of granulomas in the mice were observed when the mice were co­injected with BCG and Plasmodium. The expression of inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry and reverse transcription­polymerase chain reaction (RT­PCR) analysis. In addition, the expression of interleukin (IL)­10 in liver tissues was observed by RT­PCR. Following co­infection with BCG and Plasmodium, the swelling of the liver had been slowly restored to normal, and the time required to allow granulomas to subside had prolonged. In addition, the expression of iNOS increased, while the expression of IL­10 gradually decreased in Plasmodium­infected mice. It was concluded that the use of Plasmodium relatively delayed granuloma formation in livers of BCG­infected mice. In addition, iNOS and IL­10 are involved in this pathogenesis.