The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8⁺ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4⁺ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a⁺ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses. In efforts to define the potential role of innate immune effector mechanisms in influencing the course of SIV infection during the acute infection period, our lab utilized the in vivo daily administration of 20 mg/kg orally of a compound called Tofacitinib (a Janus kinase 3 inhibitor) to a group of 15 rhesus macaques starting at day −6 and until day 28 post intravenous SIVmac239 infection. An additional group of 16 similarly SIV infected rhesus macaques served as a placebo control. This drug targets the JAK/STAT pathway that is utilized by cells including the NK cell lineage, a major cell of the innate immune system. The dosage utilized was based on extensive previous PK studies that resulted in a marked depletion of the NK cells. Of interest while such drug administration had no effect on plasma viral loads during acute infection, such drug administration led to significant increases in plasma and gastro-intestinal tissues (GIT) viral loads during chronic infection. A series of phenotypic/functional studies were performed to determine the mechanisms for this delayed effect and the correlates identified. These data are the first to document the effect of JAK-3 inhibitor during acute SIV infection with implications for HIV vaccine design.

Extracellular vesicles (EVs) released from pathogenic protozoans play crucial roles in host–parasite communication and disease pathogenesis. Naegleria fowleri is a free-living protozoan causing primary amoebic meningoencephalitis, a fatal disease in the central nervous system. This study aims to explore the roles of N. fowleri-derived EVs (Nf-EVs) in host–pathogen interactions using the THP-1 cell line as a model. The Nf-EVs were isolated from the N. fowleri trophozoite culture supernatant using sequential centrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy. The functional roles of Nf-EVs in the apoptosis and immune response induction of THP-1 monocytes and macrophages were examined by flow cytometry, quantitative PCR, and ELISA. Results showed that Nf-EVs displayed vesicles with bilayer membrane structure approximately 130–170 nm in diameter. The Nf-EVs can be internalized by macrophages and induce macrophage responses by induction of the expression of costimulatory molecules CD80, CD86, HLA-DR, and CD169 and the production of cytokine IL-8. However, Nf-EVs did not affect the apoptosis of macrophages. These findings illustrate the potential role of Nf-EVs in mediating the host immune cell activation and disease pathogenesis.

In infectious diseases, extracellular vesicles (EVs) released from a pathogen or pathogen-infected cells can transfer pathogen-derived biomolecules, especially proteins, to target cells and consequently regulate these target cells. For example, malaria is an important tropical infectious disease caused by Plasmodium spp. Previous studies have identified the roles of Plasmodium falciparum-infected red blood cell-derived EVs (Pf-EVs) in the pathogenesis, activation, and modulation of host immune responses. This study investigated the proteomic profiles of Pf-EVs isolated from four P. falciparum strains. We also compared the proteomes of EVs from (i) different EV types (microvesicles and exosomes) and (ii) different parasite growth stages (early- and late-stage). The proteomic analyses revealed that the human proteins carried in the Pf-EVs were specific to the type of Pf-EVs. By contrast, most of the P. falciparum proteins carried in Pf-EVs were common across all types of Pf-EVs. As the proteomics results revealed that Pf-EVs contained invasion-associated proteins, the effect of Pf-EVs on parasite invasion was also investigated. Surprisingly, the attenuation of parasite invasion efficiency was found with the addition of Pf-MVs. Moreover, this effect was markedly increased in culture-adapted isolates compared with laboratory reference strains. Our evidence supports the concept that Pf-EVs play a role in quorum sensing, which leads to parasite growth-density regulation.

Context.—: Spurious platelet counts from automated methods have been reported in patients with abnormal red blood cells. However, there is no specific study regarding performance of platelet counts by automated methods in patients with thalassemia. Objective.—: To investigate the performance of automated platelet counts, including impedance (PLT-I) and optical fluorescent (PLT-O and PLT-F) methods, and compare them with the international reference method (IRM) for platelet counting in patients with thalassemia. Design.—: Two hundred forty-nine thalassemia specimens from various subtypes were examined. PLT-I, PLT-O, and PLT-F from a Sysmex XN analyzer were evaluated and compared against the IRM. Demographic data, platelet counts, and red blood cell parameters are shown. Comparability between evaluated methods and IRM, as well as test characteristics, is presented. Factors involving inaccurate PLT-I were analyzed. Results.—: Specimens with platelet counts ranging from 31 × 103/μL to 932 × 103/μL were included. Most patients were patients with thalassemia major. Correlation between PLT-I and IRM was lower than that of the other methods in overall patients. PLT-O and PLT-F were correlated to IRM when classifying patients according to clinically significant platelet ranges. All automated methods had acceptable sensitivities; however, specificity of PLT-I was low for diagnosis of thrombocytopenia. High RDW-CV (red blood cell distribution width–coefficient of variation) was an independent factor of inaccurate PLT-I measurement. Conclusions.—: Among the evaluated methods, PLT-I was the method least correlated to IRM, with PLT-O and PLT-F comparable to IRM in patients with thalassemia. Optical platelet counts and careful blood smear examination are recommended alternative platelet counting methods, depending on the clinical setting.