Cytomegalovirus Infection Facilitates the Costimulation of CD57+CD28- CD8 T Cells in HIV Infection and Atherosclerosis via the CD2-LFA-3 Axis.

CD8 T cells are emerging as important mediators in atherosclerosis and cardiovascular disease (CVD). Immune activation may play a particular role in people with HIV (PWH) who are at an increased risk of CVD, even after controlling for known CVD risk factors. Latent CMV infection is associated with increased CVD risk for both PWH and people without HIV, and human CMV-specific CD4 and CD8 T cells are enriched for an immunosenescent phenotype. We previously showed that CMV coinfection in PWH promotes vascular homing and activation of inflammatory CD4 T cells through the CD2-LFA-3 axis. However, the role of CD2/LFA3 costimulation of CD8 T cells in PWH with CMV has yet to be described. In the present study, we demonstrate that CD2 expression on CX3CR1+CD57+CD28- inflammescent CD8 T cells is increased on cells from CMV-seropositive PWH. In vitro CD2/LFA-3 costimulation enhances TCR-mediated activation of these inflammatory CD8 memory T cells. Finally, we show that LFA-3 is highly expressed in aortas of SIV-infected rhesus macaques and in atherosclerotic plaques of people without HIV. Our findings are consistent with a model in which CMV infection enhances CD2 expression on highly proinflammatory CD8 T cells that can then be stimulated by LFA-3 expressed in the vasculature, even in the absence of CD28 costimulation. This model, in which CMV infection exacerbates toxic cytokine and granzyme production by CD8 T cells within the vasculature, highlights a potential therapeutic target in atherosclerosis development and progression, especially for PWH.

New trichrome stains identify cysts of Colpodella sp. (Apicomplexa) and Bodo caudatus.

Colpodella species are free-living close relatives of apicomplexans that were recently reported to cause red blood cell infection in an immunocompromised human host and in a tick-borne human infection resulting in neurological symptoms. Unambiguous identification of the life cycle stages of Colpodella sp. using routine stains for light microscopy will aid rapid diagnosis in infections. Similarly, cells in culture and environmental samples can be rapidly identified by staining. Staining protocols are currently unavailable for cell detection by light microscopy. In this study, we investigated the feasibility of performing routine staining techniques for light microscopy for differentiating Colpodella sp. (ATCC 50594) and Bodo caudatus cysts in Hay medium cultures. We tested different basic and acidic dyes alone and in combination and also utilized a commercial trichrome staining protocol. The nonspecific fluorescent dye Calcofluor white was also evaluated. Staining times, dye concentrations, use of tap or distilled water rinses, use of a mordant and inclusion, or omission of decolorizers after staining were evaluated. We compared the intensity of color, clarity of morphological features, and cytoplasmic structures detected after staining. We report a new trichrome staining technique that allowed clear identification and differentiation of cyst stages of Colpodella sp. and B. caudatus. Immature Colpodella sp. cysts were identified as having an irregular, dual-colored (demilune), dark blue-purple and white appearance. Mature Colpodella sp. cysts stained dark red-blue and were identified in four-way mitotic division, while cysts of B. caudatus in diprotist or monoprotist (ATCC 30905) cultures were detected as spherical and red-pink in appearance.

Genome to phenome tools: In vivo and in vitro transfection of Crassostrea virginica hemocytes.

The sequencing of the Crassostrea virginica genome has brought back the interest for gene delivery and editing methodologies. Here, we report the expression in oyster hemocytes of two heterologous expression vectors under the CMV promoter delivered with dendrimers. Expression was monitored using confocal microscopy, flow cytometry, and immunofluorescence assay. C. virginica hemocytes were able to express the green fluorescence protein and Crassostrea gigas vascular endothelial growth factor under CMV viral promoter both in vivo and in vitro. These results provide the bases for interrogating the genome and adapting genome editing methodologies.

Trafficking and Association of Plasmodium falciparum MC-2TM with the Maurer's Clefts.

In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus.

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for "Dermo" disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 µg of pPmMOE[MOE1]:GFP and 25.0 × 106 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

RhopH3, rhoptry gene conserved in the free-living alveolate flagellate Colpodella sp. (Apicomplexa).

In this study, we investigated morphological, immunological and molecular characteristics of Colpodella sp. (American Type Culture Collection 50594) in a diprotist culture containing Bodo caudatus as prey using Plasmodium rhoptry specific antibodies and oligonucleotide primers targeting Plasmodium falciparum rhoptry genes. In culture, Colpodella sp. attached to its prey using the apical end with attachment lasting for approximately 20 min while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella sp. Encystment of Colpodella sp. was observed following feeding. Indirect immunofluorescence assay (IFA) and confocal microscopy using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella sp. Bodo caudatus from diprotist and monoprotist (ATCC 30395) cultures showed weak background reactivity. Giemsa staining permitted differentiation of both protists. Genomic DNA isolated from the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers targeting the P. falciparum rhoptry genes RhopH3, RhopH1/Clag3.2 and RAMA. Primers targeting exon 7 of the P. falciparum RhopH3 gene amplified an approximately 2 kb DNA fragment from the diprotist DNA template. DNA sequence and BLAST search analysis of the amplified product from diprotist DNA identified the RhopH3 gene demonstrating that the RhopH3 gene is conserved in Colpodella sp.

From the raw bar to the bench: Bivalves as models for human health.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa).

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and ß-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying "doughnut-shaped" vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.