Publications by authors named "Sanjai Kumar"

96 Publications

CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with infection.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305;

CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47 mice infected with , which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the ANKA () murine model. In vitro we tested the effect of anti-CD47 mAb on infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of -infected RBCs. Infection of C57BL/6 mice with is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8 T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.
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http://dx.doi.org/10.1073/pnas.1907653118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980459PMC
March 2021

Babesiosis Occurrence Among United States Medicare Beneficiaries, Ages 65 and Older, During 2006-2017: Overall and by State and County of Residence.

Open Forum Infect Dis 2021 Feb 15;8(2):ofaa608. Epub 2020 Dec 15.

Food and Drug Administration, Silver Spring, Maryland, USA.

Background: Human babesiosis is a mild-to-severe parasitic infection that poses health concerns especially in older and other at-risk populations. The study objective was to assess babesiosis occurrence among US Medicare beneficiaries, ages 65 and older, during 2006-2017.

Methods: Our retrospective claims-based study used Medicare databases. Babesiosis cases were identified using recorded diagnosis codes. The study estimated rates (per 100 000 beneficiary-years) overall, by year, diagnosis month, demographics, and state and county of residence.

Results: Nationwide, 19 469 beneficiaries had babesiosis recorded, at a rate of 6 per 100 000 person-years, ranging from 4 in 2006 to 9 in 2017 ( < .05). The highest babesiosis rates by state were in the following: Massachusetts (62), Rhode Island (61), Connecticut (51), New York (30), and New Jersey (19). The highest rates by county were in the following: Nantucket, Massachusetts (1089); Dukes, Massachusetts (236); Barnstable, Massachusetts (213); and Dutchess, New York (205). Increasing rates, from 2006 through 2017 ( < .05), were identified in multiple states, including states previously considered nonendemic. New Hampshire, Maine, Vermont, Pennsylvania, and Delaware saw rates increase by several times.

Conclusions: Our 12-year study shows substantially increasing babesiosis diagnosis trends, with highest rates in well established endemic states. It also suggests expansion of babesiosis infections in other states and highlights the utility of real-world evidence.
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http://dx.doi.org/10.1093/ofid/ofaa608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875327PMC
February 2021

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Arch Virol 2021 Jan 30;166(1):139-155. Epub 2020 Oct 30.

School of Biological Sciences, National Institute of Science Education & Research, Bhubaneswar, HBNI, Jatni, Khurda, Odisha, 752050, India.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca imaging that TRPV1 regulates Ca influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.
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http://dx.doi.org/10.1007/s00705-020-04852-8DOI Listing
January 2021

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Sci Rep 2020 06 12;10(1):9598. Epub 2020 Jun 12.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.
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http://dx.doi.org/10.1038/s41598-020-66273-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293334PMC
June 2020

Transient receptor potential ankyrin1 channel is endogenously expressed in T cells and is involved in immune functions.

Biosci Rep 2019 09 20;39(9). Epub 2019 Sep 20.

School of Biological Sciences, National Institute of Science Education and Research, HBNI, Bhubaneswar, Jatni, Khurda 752050, Odisha, India

Transient receptor potential channel subfamily A member 1 (TRPA1) is a non-selective cationic channel, identified initially as a cold sensory receptor. TRPA1 responds to diverse exogenous and endogenous stimuli associated with pain and inflammation. However, the information on the role of TRPA1 toward T-cell responses remains scanty. data suggest that TRPA1 can play an important role in the T-cell activation process. In this work, we explored the endogenous expression of TRPA1 and its function in T cells. By reverse transcription polymerase chain reaction (RT-PCR), confocal microscopy and flow cytometry, we demonstrated that TRPA1 is endogenously expressed in primary murine splenic T cells as well as in primary human T cells. TRPA1 is primarily located at the cell surface. TRPA1-specific activator namely allyl isothiocyanate (AITC) increases intracellular calcium ion (Ca) levels while two different inhibitors namely A-967079 as well as HC-030031 reduce intracellular Ca levels in T cells; TRPA1 inhibition also reduces TCR-mediated calcium influx. TRPA1 expression was found to be increased during αCD3/αCD28 (TCR) or Concanavalin A (ConA)-driven stimulation in T cells. TRPA1-specific inhibitor treatment prevented induction of cluster of differentiation 25 (CD25), cluster of differentiation 69 (CD69) in ConA/TCR stimulated T cells and secretion of cytokines like tumor necrosis factor (TNF), interferon γ (IFN-γ), and interleukin 2 (IL-2) suggesting that endogenous activity of TRPA1 may be involved in T-cell activation. Collectively these results may have implication in T cell-mediated responses and indicate possible role of TRPA1 in immunological disorders.
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http://dx.doi.org/10.1042/BSR20191437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753326PMC
September 2019

Persistence of Infection in Humans.

Pathogens 2019 Jul 17;8(3). Epub 2019 Jul 17.

Yale School of Public Health and Yale School of Medicine, New Haven, CT 06520 USA.

Persistent infection is a characteristic feature of babesiosis, a worldwide, emerging tick-borne disease caused by members of the genus Persistence of infection in reservoir hosts increases the probability of survival and transmission of these pathogens. Laboratory tools to detect in red blood cells include microscopic detection using peripheral blood smears, nucleic acid detection (polymerase chain reaction and transcription mediated amplification), antigen detection, and antibody detection. , the major cause of human babesiosis, can asymptomatically infect immunocompetent individuals for up to two years. Chronically infected blood donors may transmit the pathogen to another person through blood transfusion. Transfusion-transmitted babesiosis causes severe complications and death in about a fifth of cases. Immunocompromised patients, including those with asplenia, HIV/AIDS, malignancy, or on immunosuppressive drugs, often experience severe disease that may relapse up to two years later despite anti- therapy. Persistent infection is promoted by immune evasive strategies and impaired host immune mechanisms. The health burden of persistent and recrudescent babesiosis can be minimized by development of novel therapeutic measures, such as new anti-parasitic drugs or drug combinations, improved anti-parasitic drug duration strategies, or immunoglobulin preparations; and novel preventive approaches, including early detection methods, tick-avoidance, and blood donor screening.
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http://dx.doi.org/10.3390/pathogens8030102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789900PMC
July 2019

Antibody-Dependent, Gamma Interferon-Independent Sterilizing Immunity Induced by a Subunit Malaria Vaccine.

Infect Immun 2019 10 19;87(10). Epub 2019 Sep 19.

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the circumsporozoite protein (CSP)-based RTS,S/AS01 vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from (rCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4 and CD8 T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4 T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4 T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.
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http://dx.doi.org/10.1128/IAI.00236-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759290PMC
October 2019

P38 and JNK Mitogen-Activated Protein Kinases Interact With Chikungunya Virus Non-structural Protein-2 and Regulate TNF Induction During Viral Infection in Macrophages.

Front Immunol 2019 12;10:786. Epub 2019 Apr 12.

School of Biological Sciences, National Institute of Science Education and Research, HBNI, Bhubaneswar, India.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is endemic in different parts of the globe. The host macrophages are identified as the major cellular reservoirs of CHIKV during infection and this virus triggers robust TNF production in the host macrophages, which might be a key mediator of virus induced inflammation. However, the molecular mechanism underneath TNF induction is not understood yet. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV to address the above-mentioned question. It was observed that CHIKV induces both p38 and JNK phosphorylation in macrophages in a time-dependent manner and p-p38 inhibitor, SB203580 is effective in reducing infection even at lower concentration as compared to the p-JNK inhibitor, SP600125. However, inhibition of p-p38 and p-JNK decreased CHIKV induced TNF production in the host macrophages. Moreover, CHIKV induced macrophage derived TNF was found to facilitate TCR driven T cell activation. Additionally, it was noticed that the expressions of key transcription factors involved mainly in antiviral responses (p-IRF3) and TNF production (p-c-jun) were induced significantly in the CHIKV infected macrophages as compared to the corresponding mock cells. Further, it was demonstrated that CHIKV mediated TNF production in the macrophages is dependent on p38 and JNK MAPK pathways linking p-c-jun transcription factor. Interestingly, it was found that CHIKV nsP2 interacts with both p-p38 and p-JNK MAPKs in the macrophages. This observation was supported by the protein-protein docking analysis which illustrates the specific amino acids responsible for the nsP2-MAPKs interactions. A strong polar interaction was predicted between Thr-180 (within the phosphorylation lip) of p38 and Gln-273 of nsP2, whereas, no such polar interaction was predicted for the phosphorylation lip of JNK which indicates the differential roles of p-p38 and p-JNK during CHIKV infection in the host macrophages. In summary, for the first time it has been shown that CHIKV triggers robust TNF production in the host macrophages via both p-p38 and p-JNK/p-c-jun pathways and the interaction of viral protein, nsP2 with these MAPKs during infection. Hence, this information might shed light in rationale-based drug designing strategies toward a possible control measure of CHIKV infection in future.
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http://dx.doi.org/10.3389/fimmu.2019.00786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473476PMC
August 2020

Sensitive real-time PCR detection of Plasmodium falciparum parasites in whole blood by erythrocyte membrane protein 1 gene amplification.

Malar J 2019 Apr 2;18(1):116. Epub 2019 Apr 2.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Background: Malaria remains a global public health problem responsible for 445,000 deaths in 2016. While microscopy remains the mainstay of malaria diagnosis, highly sensitive molecular methods for detection of low-grade sub-microscopic infections are needed for surveillance studies and identifying asymptomatic reservoirs of malaria transmission.

Methods: The Plasmodium falciparum genome sequence was analysed to identify high copy number genes that improve P. falciparum parasite detection in blood by RT-PCR. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)-specific primers were evaluated for P. falciparum detection in hospital-based microscopically positive dried blood spots and field-acquired whole blood from asymptomatic individuals from Ghana.

Results: PfEMP1 outperformed the Pf18S sequence for amplification-based P. falciparum detection. PfEMP1 primers exhibited sevenfold higher sensitivity compared to Pf18S primers for parasite genomic DNA. Probit analysis established a 95% detection threshold of 9.3 parasites/mL for PfEMP1 compared to 98.2 parasites/mL for Pf18S primers. The PfEMP1 primers also demonstrated superior clinical sensitivity, identifying 100% (20/20) of dried blood spot samples and 70% (69/98) of asymptomatic individuals as positive versus 55% (11/20) and 54% (53/98), respectively, for Pf18S amplification.

Conclusions: These results establish PfEMP1 as a novel amplification target for highly sensitive detection of both acute infections from filter paper samples and submicroscopic asymptomatic low-grade infections.
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http://dx.doi.org/10.1186/s12936-019-2743-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444846PMC
April 2019

Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes.

Sci Rep 2018 08 1;8(1):11568. Epub 2018 Aug 1.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.

The Plasmodium life cycle within the mosquito involves the gamete, zygote, motile ookinete, and the oocyst stage that supports sporogony and sporozoite formation. We mapped the P. falciparum transcriptome as the parasite progresses through the oocyst stage of development on days 2, 4, 6, and 8 post-P. falciparum infectious blood meal. Through these genomic studies, we identified 212 novel transmission stage biomarkers including genes that are developmentally expressed at a single time point and genes that are pan-developmentally expressed at all four time points in P. falciparum oocysts. Validation of a small subset of genes at the transcriptional and translational level resulted in identification of a signature of genes/proteins that can detect parasites within the mosquito as early as day 2 post-infectious blood meal and can be used to distinguish early versus late stage P. falciparum oocyst development in the mosquito. Currently, circumsporozoite protein (CSP), which is detectable only after day 7 post-infection, is the only marker used for detection of P. falciparum infection in mosquitoes. Our results open the prospect to develop a non-CSP based detection assay for assessment of P. falciparum infection in mosquitoes and evaluate the effect of intervention measures on malaria transmission in an endemic setting.
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http://dx.doi.org/10.1038/s41598-018-29969-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070505PMC
August 2018

TCRβ-expressing macrophages induced by a pathogenic murine malaria correlate with parasite burden and enhanced phagocytic activity.

PLoS One 2018 25;13(7):e0201043. Epub 2018 Jul 25.

Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States of America.

Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11bhighCD14+F4/80+ macrophages that express TCRβ. This population expands dramatically during a Plasmodium berghei ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRβ transcript and protein levels in macrophages in wildtype versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRβ on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vβ usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRβ and peripheral parasitemia. In addition, presence of TCRβ on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRβ expression by the macrophage. Expansion of TCRβ-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201043PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059462PMC
January 2019

Highly Efficient Cell-Penetrating Probes of Protein Arginine Deiminases for Functional Proteomics.

Authors:
Sanjai Kumar

Chembiochem 2018 09 23;19(17):1806-1809. Epub 2018 Jul 23.

Department of Chemistry and Biochemistry, Queens College-CUNY, 65-30 Kissena Blvd., Queens, NY, 11367, USA.

Protein arginine deiminases (PADs) have recently emerged at the forefront of drug-discovery programs for several human disorders. Despite this, a precise understanding of their functional roles in human biology remains to be fully elucidated. This report highlights a recent development of next-generation activity-based PAD probes that are highly efficient, cell active, and metabolically stable. This discovery should rapidly expedite functional assignments of PAD biology in both normal and diseased cells, thereby leading to the development of PAD-targeted therapeutics in the near future.
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http://dx.doi.org/10.1002/cbic.201800257DOI Listing
September 2018

VIPER regulates naive T cell activation and effector responses: Implication in TLR4 associated acute stage T cell responses.

Sci Rep 2018 05 8;8(1):7118. Epub 2018 May 8.

School of Biological Sciences, National Institute of Science Education and Research, HBNI, Bhubaneswar, Jatni, Khurda, 752050, Odisha, India.

Naive T cells are known to express the modest level of TLR4 while it is known to go down during TCR activation. However, information towards the requirement of TLR4 signaling during TCR or mitogenic activation of naive wild-type T cells remains scanty. Here we have investigated the endogenous functional expression of TLR4 in naive mice T cells during TCR and mitogenic stimulation in presence of VIPER peptide (VP), an established inhibitor of TLR4 signaling. As expected we found that TLR4 expression goes down during TCR and mitogenic activation. Interestingly, we observed that VP treatment restores TLR4 expression on those activated T cells. Moreover, VP was found to regulate such activation of naive T cell as evident by reduction of CD25, CD69 expression, effector cytokines (IL-2, IFN-γ, TNF) production, T cell proliferation and down-regulation of T cell activation-dependent Fas (CD95), FasL (CD95L) expression. Together, our current observation highlights a possible requirement of TLR4 responses in T cells, which might have possible implication towards the pathogenic acute phase activation of naive T cells.
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http://dx.doi.org/10.1038/s41598-018-25549-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940837PMC
May 2018

TCRβ Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection.

Infect Immun 2018 07 21;86(7). Epub 2018 Jun 21.

Laboratory of Mucosal Pathogens and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccine Research and Review, Food and Drug Administration, Silver Spring, Maryland, USA

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαβ combinatorial immunoreceptor in humans and mice. Here, we report that a ANKA murine malaria infection induces expansion of TCRβ expressing CD11b Ly6G neutrophils in the spleen during the early phase of infection. Measurement of TCRβ transcript and protein levels of neutrophils in wild-type versus nude and knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRβ and peripheral blood parasite burden. In addition, TCRβ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRβ neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.
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http://dx.doi.org/10.1128/IAI.00899-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013678PMC
July 2018

Induction of immunity following vaccination with a chemically attenuated malaria vaccine correlates with persistent antigenic stimulation.

Clin Transl Immunology 2018 11;7(4):e1015. Epub 2018 Apr 11.

Institute for Glycomics Griffith University Gold Coast QLD Australia.

Objectives: Blood stage malaria parasites attenuated with seco-cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction.

Methods: Mice were vaccinated with chemically attenuated parasites. PCR and adoptive transfer studies were used to determine the presence of parasites and antigen . In other experiments, parasitised red blood cells were attenuated and RNA and antigen expression studied.

Results: We show that blood transferred from vaccinated mice into naïve mice activates T cells and induces complete protective immunity in the recipient mice strongly suggesting that there is persistence of parasite antigen postvaccination. This is supported by the presence of parasite RNA in vaccinated mice and both RNA and antigen expression in cultures treated with CPI drugs . In addition, drugs that block parasite growth also prevent the induction of immunity in vaccinated mice, indicating that some growth of attenuated parasites is required for immune induction.

Conclusions: Attenuated parasites persist at submicroscopic levels in the blood of mice postvaccination with the ability to activate T cells and induce ongoing protective immune responses.
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http://dx.doi.org/10.1002/cti2.1015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894007PMC
April 2018

Superior real-time polymerase chain reaction detection of Babesia microti parasites in whole blood utilizing high-copy BMN antigens as amplification targets.

Transfusion 2018 08 17;58(8):1924-1932. Epub 2018 Apr 17.

Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland.

Background: Babesiosis is a zoonotic disease transmitted to humans by the bite of infected ticks and caused by apicomplexan parasites, most commonly Babesia microti. Additionally, blood and blood products collected from asymptomatically infected blood donors may cause transfusion-transmitted infections in recipients. Highly sensitive molecular assays that detect parasite nucleic acid are needed for laboratory diagnosis and to identify and defer clinically silent but parasitemic blood donors.

Study Design And Methods: Here we report the development and analytical and clinical characterization of a real-time polymerase chain reaction (RT-PCR)-based assay for the detection of B. microti genomic DNA in whole blood. We evaluate the detection of Babesia parasites using two separate targets, the traditional18S ribosomal subunit gene (Bm18S) and members of the abundant BMN family of seroreactive antigens (BmBMN).

Results: Analytical sensitivity determination using a probit analysis demonstrated an analytical sensitivity of 30.9 parasites/mL for 18S amplification and 10.0 parasites/mL for BMN amplification The BMN primer set also demonstrates superior sensitivity for serial dilution panels prepared from clinically diagnosed Babesia-infected blood samples, generally detecting 10-fold more dilute nucleic acid.

Conclusions: Cumulatively, our data demonstrate that RT-PCR detection of the BMN family of seroreactive antigens reflects a sensitive and superior assay for the detection of B. microti in whole blood samples.
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http://dx.doi.org/10.1111/trf.14642DOI Listing
August 2018

Community-acquired and transfusion-transmitted babesiosis are increasing: why and what to do?

Transfusion 2018 03;58(3):617-619

Yale School of Public Health, New Haven, CT.

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http://dx.doi.org/10.1111/trf.14518DOI Listing
March 2018

Reply to Lisette et al.

J Infect Dis 2018 03;217(6):1012-1013

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Silver Spring, Maryland.

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http://dx.doi.org/10.1093/infdis/jix673DOI Listing
March 2018

Synthesis, Crystal Structure, and Photoluminescent Properties of 3,3',4,4'-Tetraethyl-5,5'-divinyl-2,2'-bipyrrole Derivatives.

Molecules 2017 Oct 26;22(11). Epub 2017 Oct 26.

Department of Chemistry and Biochemistry, Queens College, Queens, NY 11367, USA.

Photoluminescent divinylbipyrroles were synthesized from 3,3',4,4'-tetraetyl-2,2'-bipyrrole-5,5'-dicarboxaldehyde and activated methylene compounds via aldol condensation. For mechanistic clarity, molecular structures of Meldrum's acid- and 1,3-dimethylbarbituric acid-derived divinylbipyrroles were determined by single-crystal X-ray diffraction. Photoluminescent properties of the synthesized divinylbipyrroles in dichloromethane were found to be dependent on the presence of electron withdrawing groups at the vinylic terminal. The divinylbipyrroles derived from malononitrile, Meldrum's acid, and 1,3-dimethylbarbituric acid showed fluorescent peaks at 553, 576, and 602 nm respectively. Computational studies indicated that the alkyl substituents on the bipyrrole 3 and 3' positions increased energy level of the highest occupied molecular orbital (HOMO) compared to the unsubstituted derivatives and provided rationale for the bathochromic shift of the ultraviolet-visible (UV-Vis) spectra compared to the previously reported analogs.
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http://dx.doi.org/10.3390/molecules22111816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150395PMC
October 2017

A Novel Gametocyte Biomarker for Superior Molecular Detection of the Plasmodium falciparum Infectious Reservoirs.

J Infect Dis 2017 12;216(10):1264-1272

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases.

Background: Complete malaria eradication and optimal use of transmission-reducing interventions require knowledge of submicroscopic infectious reservoirs among asymptomatic individuals. Even submicroscopic levels of Plasmodium falciparum gametocytes can infect mosquitoes and promote onward transmission. Most efforts to identify gametocyte carriers use polymerase chain reaction amplification of the gametocyte-specific transcript Pfs25.

Methods: To expand the repertoire of biomarkers available for superior gametocyte detection, we compared the gene expression profiles of gametocytes and asynchronous blood-stage P. falciparum parasites by microarray technology. This allowed the identification of 56 molecules abundantly expressed in the gametocyte stage of the parasite. The analytical sensitivity for gametocyte detection was evaluated for 25 genes with the highest expression levels.

Results: One candidate, Pfg17, exhibited superior analytical sensitivity against a panel of gametocyte-spiked whole blood, detecting 10 gametocytes/mL; in comparison, Pfs25 detected only 25.3 gametocytes/mL. Pfg17 also exhibited superior clinical sensitivity, identifying 19.1% more samples from blood-film microscopy-negative Ghanaian children and 40% more samples from asymptomatic adults as gametocyte positive.

Conclusions: Cumulatively, our results suggest Pfg17 is an excellent biomarker for detecting asymptomatic infectious reservoirs otherwise missed by the most sensitive molecular method available. Our study has also improved the repertoire of transmission-stage antigens available for evaluation as candidate vaccines.
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http://dx.doi.org/10.1093/infdis/jix442DOI Listing
December 2017

Highly Multiplex Real-Time PCR-Based Screening for Blood-Borne Pathogens on an OpenArray Platform.

J Mol Diagn 2017 07 13;19(4):549-560. Epub 2017 Jun 13.

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland. Electronic address:

Molecular diagnostics are increasingly used in the blood bank industry. A device that can combine simultaneous detection of multiple targets with the flexibility of inclusion of emerging pathogens is desirable for testing blood products. A highly multiplexed blood-borne pathogen panel (BBPP) using dual-label probe chemistry (TaqMan assays) was developed for simultaneous detection and discrimination of 17 viral pathogens in human plasma samples and 13 bacterial and protozoan pathogens in human blood samples on the OpenArray platform. The custom BBPP OpenArray plate was tested for specificity and analytical sensitivity with purified nucleic acids from each pathogen and with pathogen-spiked human blood and plasma samples. The results of analytical validation of known samples yielded decision trees for identification of coded samples: pathogens spiked in human plasma or whole blood. Results from coded samples demonstrated no false positives among the plasma or whole blood specimens. Samples not detected were at the lower limit of the detectible range or qualified for retesting as indeterminate. Further demonstration of the performance of the BBPP OpenArray was achieved with clinical samples from a blood donor testing organization. Ninety-five percent of virus-positive samples were correctly identified. These results show that a high-throughput OpenArray PCR platform can be expanded and adapted for higher discrimination and newly emerging agents, enabling consideration for development as a next-generation device for testing blood products.
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http://dx.doi.org/10.1016/j.jmoldx.2017.03.004DOI Listing
July 2017

A no film slot blot for the detection of developing P. falciparum oocysts in mosquitoes.

PLoS One 2017 21;12(4):e0174229. Epub 2017 Apr 21.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

Non-microscopy-based assays for sensitive and rapid detection of Plasmodium infection in mosquitoes are needed to allow rapid and high throughput measurement of transmission intensity and malaria control program effectiveness. Here, we report on a modified enhanced chemiluminescence-based slot blot assay for detection of Plasmodium falciparum (Pf) circumsporozite protein (PfCSP) expressed on parasite oocysts developing inside the mosquito midgut. This modified assay has several novel features that include eliminating the need for exposure to autoradiography (AR) film, as well as utilizing a novel high affinity anti-CSP antibody, and optimizing assay procedures resulting in significant reduction in the time required to perform the assay. The chemiluminescent signal for the detection of PfCSP in mosquito samples was captured digitally utilizing the C-Digit blot scanner that, allowed the detection of 0.01 pg of recombinant P. falciparum CSP and as few as 0.02 P. falciparum oocysts in a little over two hours. The earlier ECL-SB detected rCSP and oocysts and took approximately 5 h to perform. Whole mosquito lysates from both high and low prevalence-infected mosquito populations were prepared and evaluated for PfCSP detection on the ECL-SB by both AR film and digital data capture and analysis. There was a 100% agreement between the AR film and the C-Digit scanner methods for PfCSP detection in randomly sampled mosquitoes. This novel "No Film" Slot Blot assay obviates the need for AR film exposure and development and significantly reduces the assay time enabling widespread use in field settings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400236PMC
September 2017

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites.

PLoS One 2016 2;11(12):e0166814. Epub 2016 Dec 2.

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States.

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166814PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135057PMC
June 2017

Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples.

J Microbiol Methods 2017 01 9;132:76-82. Epub 2016 Nov 9.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA. Electronic address:

Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat. Here we describe standardized steps for developers to follow for the culture and quantification of three organisms used to spike human whole blood to create mock specimens. The three organisms chosen for this study were the Live Vaccine Strain (LVS) of Francisella tularensis, surrogate for a potential biothreat pathogen, Escherichia coli, a representative Gram-negative bacterium and Babesia microti (Franca) Reichenow Peabody strain, representing a protozoan parasite. Mock specimens were prepared with blood from both healthy donors and donors with nonspecific symptoms including fever, malaise, and flu-like symptoms. There was no significant difference in detection results between the two groups for any pathogen. Testing of the mock samples was compared on two platforms, Target Enriched Multiplex-PCR (TEM-PCR™) and singleplex real-time PCR (RT-PCR). Results were reproducible on both platforms. The reproducibility demonstrated by obtaining the same results between two testing methods and between healthy and symptomatic mock specimens, indicates the standardized methods described for creating the mock specimens are valid and effective for evaluating diagnostic devices.
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http://dx.doi.org/10.1016/j.mimet.2016.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224703PMC
January 2017

Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake.

Sci Signal 2016 05 31;9(430):ra55. Epub 2016 May 31.

Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.
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http://dx.doi.org/10.1126/scisignal.aac8035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150984PMC
May 2016

Adverse neuropsychiatric effects of antimalarial drugs.

Expert Opin Drug Saf 2016 Jul 18;15(7):903-10. Epub 2016 Apr 18.

a Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases , Center for Biologics Evaluation and Research, Food and Drug Administration , Silver Spring , MD , USA.

Introduction: Antimalarial drugs are the primary weapon to treat parasite infection, save lives, and curtail further transmission. Accumulating data have indicated that at least some antimalarial drugs may contribute to severe neurological and/or psychiatric side effects which further complicates their use and limits the pool of available medications.

Areas Covered: In this review article, we summarize published scientific studies in search of evidence of the neuropsychiatric effects that may be attributed to the commonly used antimalarial drugs administered alone or in combination. Each individual drug was used as a search term in addition to keywords such as neuropsychiatric, adverse events, and neurotoxicity.

Expert Opinion: Accumulating data based on published reports over several decades have suggested that among the major commonly used antimalarial drugs, only mefloquine exhibited clear indications of serious neurological and/or psychiatric side effects. A more systematic approach to assess the neuropsychiatric adverse effects of new or repurposed antimalarial drugs on their safety, tolerability and efficacy phases of clinical studies and in post-marketing surveillance, is needed to ensure that these life-saving tools remain available and can be prescribed with appropriate caution and medical judgment.
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http://dx.doi.org/10.1080/14740338.2016.1175428DOI Listing
July 2016

A comparison of Plasmodium falciparum circumsporozoite protein-based slot blot and ELISA immuno-assays for oocyst detection in mosquito homogenates.

Malar J 2015 Nov 14;14:451. Epub 2015 Nov 14.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA.

Background: The infectivity of Plasmodium gametocytes is typically determined by microscopically examining the midguts of mosquitoes that have taken a blood meal containing potentially infectious parasites. Such assessments are required for the development and evaluation of transmission-reducing interventions (TRI), but are limited by subjectivity, technical complexity and throughput. The detection of circumsporozoite protein (CSP) by enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescent slot-blot (ECL-SB) may be used as objective, scalable alternatives to microscopy for the determination of infection prevalence.

Methods: To compare the performance of the CSP ELISA and ECL-SB for the detection of mosquito infection, four groups of Anopheles stephensi mosquitoes were infected with cultured Plasmodium falciparum gametocytes. At day-8 post-infection (PI), parasite status was determined by microscopy for a sample of mosquitoes from each group. At days 8 and 10 PI, the parasite status of separate mosquito samples was analysed by both CSP ELISA and ECL-SB.

Results: When mosquito samples were analysed 8 days PI, the ECL-SB determined similar infection prevalence to microscopy; CSP ELISA lacked the sensitivity to detect CSP in all infected mosquitoes at this early time point. When mosquitoes were analysed 48 h later (10 days PI) both assays performed as well as microscopy for infection detection.

Conclusions: Whilst microscopical examination of mosquito guts is of great value when quantification of parasite burden is required, ECL-SB and CSP ELISA are suitable alternatives at day 10 PI when infection prevalence is the desired endpoint, although CSP ELISA is not suitable at day 8 PI. These results are important to groups considering large-scale implementation of TRI.
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http://dx.doi.org/10.1186/s12936-015-0954-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647817PMC
November 2015

Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

PLoS One 2015 27;10(10):e0141141. Epub 2015 Oct 27.

National Cancer Institute, Bethesda, MD, 20892, United States of America.

Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141141PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624717PMC
June 2016

Babesiosis Occurrence among the Elderly in the United States, as Recorded in Large Medicare Databases during 2006-2013.

PLoS One 2015 15;10(10):e0140332. Epub 2015 Oct 15.

Food and Drug Administration, Silver Spring, Maryland, United States of America.

Background: Human babesiosis, caused by intraerythrocytic protozoan parasites, can be an asymptomatic or mild-to-severe disease that may be fatal. The study objective was to assess babesiosis occurrence among the U.S. elderly Medicare beneficiaries, ages 65 and older, during 2006-2013.

Methods: Our retrospective claims-based study utilized large Medicare administrative databases. Babesiosis occurrence was ascertained by recorded ICD-9-CM diagnosis code. The study assessed babesiosis occurrence rates (per 100,000 elderly Medicare beneficiaries) overall and by year, age, gender, race, state of residence, and diagnosis months.

Results: A total of 10,305 elderly Medicare beneficiaries had a recorded babesiosis diagnosis during the eight-year study period, for an overall rate of about 5 per 100,000 persons. Study results showed a significant increase in babesiosis occurrence over time (p<0.05), with the largest number of cases recorded in 2013 (N = 1,848) and the highest rates (per 100,000) in five Northeastern states: Connecticut (46), Massachusetts (45), Rhode Island (42), New York (27), and New Jersey (14). About 75% of all cases were diagnosed from May through October. Babesiosis occurrence was significantly higher among males vs. females and whites vs. non-whites.

Conclusion: Our study reveals increasing babesiosis occurrence among the U.S. elderly during 2006-2013, with highest rates in the babesiosis-endemic states. The study also shows variation in babesiosis occurrence by age, gender, race, state of residence, and diagnosis months. Overall, our study highlights the importance of large administrative databases in assessing the occurrence of emerging infections in the United States.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140332PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607449PMC
June 2016

HLA-DR peptide occupancy can be regulated with a wide variety of small molecules.

Hum Vaccin Immunother 2016 03;12(3):593-8

a Department of Molecular Medicine , Morsani College of Medicine, University of South Florida , Tampa , FL , USA.

HLA-DR is the most commonly expressed and likely the most medically important human MHC class II, antigen presenting protein. In a normal immune response, HLA-DR binds to antigenic peptide and the HLA-DR/peptide complex binds to a T-cell receptor, thus contributing to T-cell activation and stimulation of an immune response against the antigen. When foreign antigen is not present, HLA-DR binds endogenous peptide which, under normal conditions does not stimulate an immune response. In most cases, the human peptide is CLIP, but a certain percentage of HLA-DR molecules will be present at the cell surface with other human peptides. We have recently shown that cell surface, CLIP/HLA-DR ratios are a measure of peptide heterogeneity, and in particular, changes in CLIP/HLA-DR ratios represent changes in the occupancy of HLA-DR by other, endogenous peptides. For example, treatment of cells with the HDAC inhibitor, Entinostat, leads to an upregulation of Cathepsin L1 and replacement of Cathepsin L1 senstitive peptides with HLA-DR binding, Cathepsin L1 resistant peptides, an alteration that can be at least partially assessed via assessment of CLIP/HLA-DR cell surface ratios. Here we assay for CLIP/HLA-DR ratios following treatment of immortalized B-cells with a variety of common drugs, almost all of which indicate significant changes in the CLIP/HLA-DR ratios. Furthermore, the CLIP/HLA-DR ratio changes parallel the impact of the drug panoply on cell viability, suggesting that alterations in the HLA-DR peptidome are governed by a variety of mechanisms, rather than exclusively dependent on a dedicated peptide loading process. These results raise questions about how FDA approved drugs may affect the immune response, and whether any of these drugs could be useful as vaccine adjuvants?
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http://dx.doi.org/10.1080/21645515.2015.1089370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4964726PMC
March 2016