Bone Marrow Is a Major Parasite Reservoir inPlasmodium vivaxInfection

ABSTRACTPlasmodium vivaxcauses heavy burdens of disease across malarious regions worldwide. MatureP. vivaxasexual and transmissive gametocyte stages occur in the blood circulation, and it is often assumed that accumulation/sequestration in tissues is not an important phase in their development. Here, we present a systematic study ofP. vivaxstage distributions in infected tissues of nonhuman primate (NHP) malaria models as well as in blood from human infections. In a comparative analysis of the transcriptomes ofP. vivaxandPlasmodium falciparumblood-stage parasites, we found a conserved cascade of stage-specific gene expression despite the greatly different gametocyte maturity times of these two species. Using this knowledge, we validated a set of conserved asexual- and gametocyte-stage markers both by quantitative real-time PCR and by antibody assays of peripheral blood samples from infected patients and NHP (Aotussp.). Histological analyses ofP. vivaxparasites in organs of 13 infected NHP (AotusandSaimirispecies) demonstrated a major fraction of immature gametocytes in the parenchyma of the bone marrow, while asexual schizont forms were enriched to a somewhat lesser extent in this region of the bone marrow as well as in sinusoids of the liver. These findings suggest that the bone marrow is an important reservoir for gametocyte development and proliferation of malaria parasites.IMPORTANCEPlasmodium vivaxmalaria continues to cause major public health burdens worldwide. Yet, significant knowledge gaps in the basic biology and epidemiology ofP. vivaxmalaria remain, largely due to limited available tools for research and diagnostics. Here, we present a systematic examination of tissue sequestration duringP. vivaxinfection. Studies of nonhuman primates and malaria patients revealed enrichment of developing sexual stages (gametocytes) and mature replicative stages (schizonts) in the bone marrow and liver, relative to those present in peripheral blood. Identification of the bone marrow as a majorP. vivaxtissue reservoir has important implications for parasite diagnosis and treatment.

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Immune Response and Microbiota Profiles during Coinfection with Plasmodium vivax and Soil-Transmitted Helminths

mBio ◽

10.1128/mbio.01705-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Alice V. Easton ◽

Mayra Raciny-Aleman ◽

Victor Liu ◽

Erica Ruan ◽

Christian Marier ◽

...

Keyword(s):

Gut Microbiota ◽

Plasmodium Vivax ◽

Peripheral Blood ◽

Parasite Burden ◽

Helminth Parasite ◽

Blood Samples ◽

Content Type ◽

Soil Transmitted Helminths ◽

Cell Counts ◽

Cytokine Levels

ABSTRACT The role of the gut microbiota during coinfection with soil-transmitted helminths (STH) and Plasmodium spp. is poorly understood. We examined peripheral blood and fecal samples from 130 individuals who were either infected with Plasmodium vivax only, coinfected with P. vivax and STH, infected with STH alone, or not infected with either P. vivax or STH. In addition to a complete blood count (CBC) with differential, transcriptional profiling of peripheral blood samples was performed by transcriptome sequencing (RNA-Seq), fecal microbial communities were determined by 16S rRNA gene sequencing, and circulating cytokine levels were measured by bead-based immunoassays. Differences in blood cell counts, including an increased percentage of neutrophils, associated with a transcriptional signature of neutrophil activation, were driven primarily by P. vivax infection. P. vivax infection was also associated with increased levels of interleukin 6 (IL-6), IL-8, and IL-10; these cytokine levels were not affected by STH coinfection. Surprisingly, P. vivax infection was more strongly associated with differences in the microbiota than STH infection. Children infected with only P. vivax exhibited elevated Bacteroides and reduced Prevotella and Clostridiaceae levels, but these differences were not observed in individuals coinfected with STH. We also observed that P. vivax parasitemia was higher in the STH-infected population. When we used machine learning to identify the most important predictors of the P. vivax parasite burden (among P. vivax-infected individuals), bacterial taxa were the strongest predictors of parasitemia. In contrast, circulating transforming growth factor β (TGF-β) was the strongest predictor of the Trichuris trichiura egg burden. This study provides unexpected evidence that the gut microbiota may have a stronger link with P. vivax than with STH infection. IMPORTANCE Plasmodium (malaria) and helminth parasite coinfections are frequent, and both infections can be affected by the host gut microbiota. However, the relationship between coinfection and the gut microbiota is unclear. By performing comprehensive analyses on blood/stool samples from 130 individuals in Colombia, we found that the gut microbiota may have a stronger relationship with the number of P. vivax (malaria) parasites than with the number of helminth parasites infecting a host. Microbiota analysis identified more predictors of the P. vivax parasite burden, whereas analysis of blood samples identified predictors of the helminth parasite burden. These results were unexpected, because we expected each parasite to be associated with greater differences in its biological niche (blood for P. vivax and the intestine for helminths). Instead, we find that bacterial taxa were the strongest predictors of P. vivax parasitemia levels, while circulating TGF-β levels were the strongest predictor of helminth parasite burdens.

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Disruption of GAS6-MER Pathway Leads to Defects in Physiological Clearance of Expelled Nuclei from Erythroblasts by Bone Marrow Macrophages

Blood ◽

10.1182/blood.v112.11.499.499 ◽

2008 ◽

Vol 112 (11) ◽

pp. 499-499

Author(s):

Linda Kadi ◽

Laurent Burnier ◽

Rocco Sugamele ◽

Peter Carmeliet ◽

Greg Lemke ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Fetal Liver ◽

Specific Gene ◽

Positive Staining ◽

Morphological Examination ◽

Double Positive ◽

Blood Island

Abstract Late in erythropoiesis, nuclei are expelled from erythroblasts and 2×1011 anucleated new red blood cells are daily delivered in the peripheral blood. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as an “eat me” signal for their engulfment by macrophages located in the blood island. The two PS opsonins, milk-fatglobule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors αvβ5/αvβ3 and TAM (TYRO3, AXL and MER), are involved in the phagocytosis of apoptotic cells, but their role in the phagocytosis of expelled nuclei from erythroblasts is not determined. Because fetal liver and bone marrow macrophages do not express MFG-E8, the GAS6-MER pathway might constitute a crucial pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from spleens of phlebotomized mice, and studied nuclei internalization capacity of bone marrow derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), AXL (AXL−/−) or TYRO3 (TYRO3−/−), or lacking MER kinase domain (MERkd). Released nuclei were identified by flow cytometry according to their size and their double positive staining for the erythroid lineage marker Ter119 and Annexin V for PS. Purity of the preparation was checked by morphological examination of cytospin preparations. In vitro phagocytosis assays show that GAS6−/− BMDM cleared 30% less nuclei than wild-type (WT) BMDM. We observed a slight decrease of internalization capacity for AXL−/− BMDM, whereas TYRO3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, MER deficiency nearly abolished nuclei phagocytosis. AXL−/−/TYRO3−/− and AXL−/−/MERkd BMDM were tested and did not show any cumulative effects when compared to WT and single knockouts. We also investigated the signalling pathway downstream of MER in BMDM. In particular, we assessed the expression of the activated form of Rac1, which is crucial for the cytoskeletal reorganization in phagocytosis. Activation of Rac1 after the initiation of the phagocytosis was delayed for 45 minutes in MERkd as compared to WT BMDM. In vivo, we found an accumulation of nuclei in MERkd mice 4 days post phlebotomy, when erythropoiesis is increased in response to anemia. Nuclei circulated in the blood of MERkd mice at a level of 0.08 ± 0.042 G/L and were identified on peripheral blood smears of these mice whereas they were undetectable in the blood of WT mice. We demonstrated an increase of a double labelled Ter119/AnnexinV population corresponding to nuclei in BM (2-fold) and spleen (1.5-fold) of MERkd mice as compared to WT mice. The augmentation of this double labelled population in the MERkd mice translated the phenotype of splenomegaly of these mice. Hematocrit and reticulocyte levels were comparable between WT and MERkd as previously reported (JCI118:583–596, 2008). Thus, MER was critical for in vitro phagocytosis of nuclei from erythroblasts whereas the role of AXL and TYRO3 appeared to be negligible. GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and MER receptor on BMDM, allowing nuclei clearance. In vivo, the absence of MER caused an accumulation of nuclei in BM and spleen and their appearance in circulating blood due to their inefficient elimination during erythropoietic response to anemia. In conclusion, we postulate that GAS6 and its receptor MER were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island, through Rac1 activation.

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Detection of tumor specific gene expression in bone marrow and peripheral blood from patients with small cell lung carcinoma

Cancer ◽

10.1002/cncr.11108 ◽

2003 ◽

Vol 97 (4) ◽

pp. 1057-1062 ◽

Cited By ~ 4

Author(s):

Masato Shingyoji ◽

Yuichi Takiguchi ◽

Reiko Watanabe ◽

Kenzo Hiroshima ◽

Ken Motoori ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Lung Carcinoma ◽

Peripheral Blood ◽

Small Cell Lung Carcinoma ◽

Small Cell ◽

Specific Gene ◽

Small Cell Lung ◽

Cell Lung Carcinoma ◽

Specific Gene Expression

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Long-Term Remission in a Juvenile Myelomonocytic Leukemia Patient After Graft Rejection of Unrelated Bone Marrow Transplantation

Blood ◽

10.1182/blood.v120.21.4962.4962 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4962-4962

Author(s):

Kimiyoshi Sakaguchi ◽

Hiroyoshi Takahashi ◽

Daisuke Shimizu ◽

Shuichi Okada ◽

Tsutomu Ogata

Keyword(s):

Bone Marrow ◽

Graft Rejection ◽

Peripheral Blood ◽

Conditioning Regimen ◽

Gene Mutations ◽

Bone Marrow Aspiration ◽

Juvenile Myelomonocytic Leukemia ◽

Specific Gene ◽

Wbc Count ◽

Myelomonocytic Leukemia

Abstract Abstract 4962 Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy and mixed myeloproliferative and myelodysplastic disorder. Although cure in most cases requires hematopoietic stem cell transplantation (HSCT), the major cause of treatment failure is relapse. However, in some cases, symptoms improve without treatment. We report a case of a patient with JMML who sustained remission after graft rejection of an unrelated bone marrow transplantation (UBMT). Case An 18-month-old girl presented with marked splenomegaly and hemorrhagic diathesis. Laboratory blood tests revealed the following: white blood cell (WBC) count 12. 2 × 109/L, monocytes 22. 0%, hemoglobin 7. 6 g/dL, platelets 10. 0 × 109/L, and fetal hemoglobin 12. 8%. A bone marrow aspirate revealed a hypercellular marrow with mild dysplastic changes and 4. 4% blast cells. The BCR–ABL fusion gene was not detected. Following a diagnosis of JMML, she subsequently developed respiratory failure due to leukemic infiltration of the lungs, and was referred to our hospital. On admission, she developed severe thrombocytopenia due to splenic sequestration of platelets, and she needed frequent transfusions. She received chemotherapy with cytarabine and 6-mercaptopurine. Pulmonary leukemic infiltration improved, but transfusion frequency could not be reduced. After she had undergone splenectomy, platelet transfusion was not needed. When her clinical condition had improved, KRAS mutation was investigated by bone marrow aspiration, and the KRAS 13G>D mutation was detected. Five months after diagnosis, she was transplanted with major mismatch blood type, HLA-A 1-allele mismatch, from an unrelated female donor. The conditioning regimen consisted of busulfan (BU; 16 mg/kg), fludarabine (Flu; 120 mg/m2), and cyclophosphamide (CY; 120 mg/kg). Short-term methotrexate and tacrolimus (FK506) were administered for the prevention of graft-versus-host disease. The level of infused donor marrow cells was 1. 18 × 108/kg. Recovery of peripheral blood count was rapid, and no regimen-related toxicity was observed. Chimerism by short tandem repeat analysis of bone marrow mononuclear cells on day 28 after UBMT was 100% recipient type, indicating graft rejection with autologous hematopoietic cell recovery. FK506 was then discontinued. From day 48 after UBMT until the current day, WBC count has been almost 10. 0 × 109/L. Despite graft rejection, the KRAS 13G>D mutation was not detected by bone marrow aspiration on day 219, and her peripheral blood counts were normalized. Four years after diagnosis, the KRAS 13G>D mutation in the peripheral blood, nails, buccal mucosa, and hair was not detected, but the KRAS13G>D mutation was not. She has been managed without treatment and remained in complete remission for over 5 years since receiving UBMT. Discussion In JMML patients with specific RAS mutations, spontaneous improvement in hematologic abnormalities has been reported. HSCT was needed in this case because the patient developed respiratory failure due to pulmonary infiltration of JMML cells. In JMML patients with gene mutation, JMML-specific gene mutations could not be detected after engraftment of HSCT. In contrast, most JMML cases relapse and need a second HSCT after rejection of the first. However, this patient's condition normalized after rejection of UBMT. Nowadays, minimal residual disease in JMML is analyzed by detection of JMML cell-specific gene mutations. The KRAS mutation can be detected in spontaneously regressed JMML following hematological improvement. We suggest that a myeloablative conditioning regimen including BU, Flu, and CY could eradicate JMML clones, and in some JMML cases, this could prevent the need for a second HSCT after rejection of the first. Disclosures: No relevant conflicts of interest to declare.

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Baculovirus-Vectored Multistage Plasmodium vivax Vaccine Induces Both Protective and Transmission-Blocking Immunities against Transgenic Rodent Malaria Parasites

Infection and Immunity ◽

10.1128/iai.02040-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4348-4357 ◽

Cited By ~ 24

Author(s):

Masanori Mizutani ◽

Mitsuhiro Iyori ◽

Andrew M. Blagborough ◽

Shinya Fukumoto ◽

Tomohiro Funatsu ◽

...

Keyword(s):

Fusion Protein ◽

Plasmodium Vivax ◽

Mammalian Cells ◽

Expression System ◽

Viral Envelope ◽

Vaccine Platform ◽

Transmission Blocking ◽

Content Type ◽

Sexual Stages

ABSTRACTA multistage malaria vaccine targeting the pre-erythrocytic and sexual stages ofPlasmodiumcould effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistagePlasmodium vivaxvaccine which simultaneously expressesP. vivaxcircumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cellsin vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenicP. bergheiparasites expressing the correspondingP. vivaxantigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen.

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Neutrophil pseudoplatelets in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156298 ◽

1989 ◽

Vol 47 ◽

pp. 862-863 ◽

Cited By ~ 1

Author(s):

J Hanker ◽

E.J. Burkes ◽

G. Greco ◽

R. Scruggs ◽

B. Giammara

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Peripheral Blood ◽

Gingival Crevicular Fluid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Staining Reaction ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Crevicular Fluid

The mature neutrophil with a segmented nucleus (usually having 3 or 4 lobes) is generally considered to be the end-stage cell of the neutrophil series. It is usually found as such in the bone marrow and peripheral blood where it normally is the most abundant leukocyte. Neutrophils, however, must frequently leave the peripheral blood and migrate into areas of infection to combat microorganisms. It is in such areas that neutrophils were first observed to fragment to form platelet-size particles some of which have a nuclear lobe. These neutrophil pseudoplatelets (NPP) can readily be distinguished from true platelets because they stain for neutrophil myeloperoxidase. True platelets are not positive in this staining reaction because their peroxidase Is inhibited by glutaraldehyde. Neutrophil pseudoplatelets, as well as neutrophils budding to form NPP, could frequently be observed in peripheral blood or bone marrow samples of leukemia patients. They are much more prominent, however, in smears of inflammatory exudates that contain gram-negative bacteria and in gingival crevicular fluid samples from periodontal disease sites. In some of these samples macrophages ingesting, or which contained, pseudoplatelets could be observed. The myeloperoxidase in the ingested pseudoplatelets was frequently active. Despite these earlier observations we did not expect to find many NPP in subgingival plaque smears from diseased sites. They were first seen by light microscopy (Figs. 1, 3-5) in smears on coverslips stained with the PATS reaction, a variation of the PAS reaction which deposits silver for light and electron microscopy. After drying replicate PATS-stained coverslips with hexamethyldisilazane, they were sputter coated with gold and then examined by the SEI and BEI modes of scanning electron microscopy (Fig. 2). Unstained replicate coverslips were fixed, and stained for the demonstration of myeloperoxidase in budding neutrophils and NPP. Neutrophils, activated macrophages and spirochetes as well as other gram-negative bacteria were also prominent in the PATS stained samples. In replicate subgingival plaque smears stained with our procedure for granulocyte peroxidases only neutrophils, budding neutrophils or NPP were readily observed (Fig. 6).

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