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Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay
组织中克氏锥虫的定量及克氏锥虫杀灭试验   

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Abstract

Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.

Keywords: Trypanosoma cruzi(克氏锥虫), Quantitative PCR(定量PCR), Killing assay(杀灭试验), Mouse(小鼠), Heart(心脏), Liver(肝脏), Blood(血液), Bone marrow-derived macrophage(源自骨髓的巨噬细胞)

Background

Chagas disease, characterized by chronic cardiomyopathy, is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi (Bonney et al., 2015). Approximately 20 million people in Latin America suffer from Chagas disease (Ribeiro et al., 2012; Flavia Nardy et al., 2015), and it has become a global health issue owing to the migration of infected individuals (Andrade et al., 2014; Garcia et al., 2015; Requena-Mendez et al., 2015). Several drugs, such as nifurtimox and benznidazole, have been developed for treating Chagas disease. However, these drugs need to be taken for several months and severe side effects have been reported (Viotti et al., 2009). A major aim of treatment is to inhibit T. cruzi transmission via blood as well as prevent the development of heart failure. Thus, the protocol for quantification of T. cruzi and the T. cruzi killing assay presented here might aid the development of novel diagnostic methods and therapeutic strategies for Chagas disease.


Part I: Quantification of T. cruzi in tissue


The following protocol (Kitada et al., 2017) was partially modified from the previously described methods (Cencig et al., 2011; Caldas et al., 2012).

Materials and Reagents

  1. Pipette tips (Labcon, catalog numbers: 1093-260-000 , 1045-260-000 , 1036-260-000 )
  2. 15 cm culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150468 )
  3. Blood collection tubes CAPIJECT (Terumo, catalog number: CJ-NA )
  4. 10 cm Petri dishes (Sansyo, catalog number: 36-3406 )
  5. 1.5 ml microtubes (FUKAEKASEI and WATSON, catalog number: 131-415C )
  6. Disposal hemocytometer (All-Biz, catalog number: 4109:37650 )
  7. 8-week-old male and female C57BL/6 mice (Japan SLC)
  8. LLC-MK2 cells (Rhesus monkey kidney epithelial cells) gifted by Professor S. Hamano (Institute of Tropical Medicine, Nagasaki University), which are also available at ATCC. Information regarding the LLC-MK2 cells is available at https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Kidney/CCL-7.aspx#documentation
  9. Trypanosoma cruzi Tulahuen strain (gifted by professor S. Hamano (Institute of Tropical medicine, Nagasaki University))
  10. 10 mg/ml Proteinase K (Merck, catalog number: 124568 )
  11. Phenol:chloroform:isoamyl alcohol (25:24:1) (NACALAI TESQUE, catalog number: 25970-56 )
  12. Chloroform (JUNSEI CHEMICAL, catalog number: 28560-0330 )
  13. 2-Propanol (JUNSEI CHEMICAL, catalog number: 64605-0330 )
  14. 70% ethanol (JUNSEI CHEMICAL, catalog number: 17065-0382 )
  15. TE buffer solution (pH 8.0) (NACALAI TESQUE, catalog number: 32739-31 )
  16. Go Taq qPCR Master Mix (Promega, catalog number: A6002 )
  17. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
  18. Primers (Invitrogen custom DNA primers)
    T. cruzi specific primers (Tc1), 5’-cgagctgttgcccacacgggtgct-3’ and 5’-cctccaagcagcggatagttcagg-3’ (Cencig et al., 2011); and TNF-α DNA primers, 5’-tccctctcatcagttctatggccca-3’ and 5’-cagcaagcatctatgcacttagacccc-3’ (Caldas et al., 2012)
  19. Lysis buffer (see Recipes)
    1. Tris-HCl (NACALAI TESQUE, catalog number: 35434-21 )
    2. Ethylenediaminetetraacetate acid (EDTA) (NACALAI TESQUE, catalog number: 06894-14 )
    3. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 311-90271 )
    4. Potassium chloride (NaCl) (JUNSEI CHEMICAL, catalog number: 19015-0350 )
  20. RPMI 1640 culture medium (see Recipes)
    1. Fetal bovine serum (CCB, catalog number: 171012 )
    2. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    3. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023)

Equipment

  1. Pipettes (Nichiryo, catalog numbers: 00-NPX2-1000 , 00-NPX2-200 , 00-NPX2-20 , 00-NPX2-2 )
  2. Forceps (Hammacher, catalog number: HSC_553-11 )
  3. Microbalance (Chyo Balance, model: JPN-200W )
  4. Scissor (Fine Science Tools, catalog number: 91460-11 )
  5. Shaking incubator (TAITEC, model: BR-43FM MR )
  6. Centrifuge (TOMY SEIKO, model: MX-200 )
  7. Vortex
  8. Real-Time PCR thermal cycler: Step One PlusTM system Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM, catalog number: 4376600 )
  9. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )

Procedure

  1. T. cruzi preparation
    1. 5 x 105-1 x 106 LLC-MK2 cells are seeded into a 15 cm culture dish on day 1 before infection.
    2. The LLC-MK2 cells seeded in step A1 are infected with 2 x 106 T. cruzi strain Tulahuen (MOI = 2).
    3. Change the culture medium (20 ml) every 3 days. In Figure 1A, amastigotes are shown in T. cruzi-infected cells (Panel A), which differentiate into the infective form trypomastigotes (Panel B) that are released into the culture medium.
    4. After 7 days of infection, trypomastigotes in the culture supernatants are counted microscopically (Figure 1). Approximately 2 x 107 trypomastigotes are obtained.


      Figure 1. Schematic of T. cruzi in a hemocytometer. A. Amastigotes; B. Trypomastigotes.

  2. Preparation of tissue standards and quantitative PCR
    1. Collect the liver and heart from a non-infected mouse into a 15 cm Petri dish and collect the blood (more than 200 μl) in a CAPIJECT tube.
    2. Cut the heart and liver into small pieces by scissor and add 1 x 106 T. cruzi trypomastigotes to 30 mg heart, 30 mg liver, or 200 μl blood from a non-infected C57BL/6 mouse in a 1.5 ml microtube.
    3. Add 500 μl lysis buffer (see Recipes) and 5 μl of 10 mg/ml Proteinase K and incubate at 55 °C in a shaking incubator (shaking speed: 110 times/min) for 18 h.
    4. Centrifuge at 13,000 x g for 10 min at 4 °C.
    5. Transfer the supernatant to a fresh 1.5 ml microtube.
    6. Add 500 μl phenol:chloroform:isoamyl alcohol (25:24:1) and mix the contents of the microtube using a vortex.
    7. Centrifuge at 13,000 x g for 10 min at 4 °C.
    8. Transfer the upper layer to a fresh 1.5 ml microtube.
    9. Add 500 μl chloroform and mix the contents of the microtube using a vortex.
    10. Centrifuge at 13,000 x g for 10 min at 4 °C.
    11. Transfer the upper layer to a fresh 1.5 ml microtube.
    12. Add 500 μl 2-propanol and mix the contents of the microtube using a vortex.
    13. Centrifuge at 13,000 x g for 10 min at 4 °C.
    14. Aspirate the supernatant and wash the pellet in 500 μl 70% ethanol gently.
    15. Centrifuge at 5,500 x g for 5 min at 4 °C.
    16. Aspirate the supernatant and air dry for 10 min.
    17. Dissolve the DNA pellet in TE buffer solution (pH 8.0) and measure the concentration of DNA to generate 25 μg/ml tissue DNA.
    18. DNA from the tissue spiked with T. cruzi is sequentially 10-fold diluted with 25 μg/ml DNA from the tissues without T. cruzi (Figure 2). The prepared standards contain DNA from 103-10-2 parasites per 50 ng of total DNA. A standard curve to determine the amounts of T. cruzi DNA in the tissues from mice infected with T. cruzi is generated using these standards. The pipet tips should be changed in between each dilution.


      Figure 2. Scheme of the protocol to generate the standards to quantify T. cruzi in tissue

    19. For a standard curve to evaluate the amount of TNF-α DNA, 25 mg/ml tissue DNA containing 1 x 103 T. cruzi (STD1), as prepared above, is subsequently 10-fold diluted with distilled water or TE buffer (Figure 3). Internal control ‘TNF-α DNA’ is used to normalize the amount of tissue being analyzed in each PCR reaction (Cummings and Tarleton, 2003; Caldas et al., 2012). The pipet tips should be changed in between each dilution.


      Figure 3. Scheme of the protocol to prepare the standards to quantify tissue TNF-α DNA

    20. Generation of T. cruzi and TNF-α standard calibration curve by qPCR (Figures 4A and 4B)
      1. Mix following components
        10 μl of Go Taq qPCR Master Mix
        1 μl of 10 μM T. cruzi specific primers (Tc1) or TNF-α DNA primers
        2 μl of DNA standards for T. cruzi (Figure 2) or TNF-α DNA (Figure 3)
        7 μl of double distilled water
      2. Amplification protocol
        95 °C for 10 min, and 40 cycles of 94 °C for 15 sec and 64.3 °C for 1 min.
        Fluorescent emission (520 nm) is measured at the end of the elongation step. A melting curve phase program is applied with a continuous fluorescent measurement between 65 °C and 95 °C. Each DNA sample is quantified in duplicate from two independent qPCR runs.


        Figure 4. Standard curve generated with DNA from the livers of non-infected mice spiked with or without T. cruzi trypomastigotes. A and B. A 10-fold dilution series prepared as described above was amplified with primers specific to T. cruzi DNA (A) or murine TNF-α DNA (B). The standard curves were generated from the linear region of each sample application curve.

  3. Quantification of T. cruzi DNA in the tissues from T. cruzi-infected mice
    1. The mice (at least 5 mice) are injected intraperitoneally with 250 μl PBS including 5 x 102 T. cruzi trypomastigotes prepared as in steps A1-A4.
    2. At 20 and 30 days after infection, the heart, liver, and blood are collected from mice.
    3. 30 mg heart or liver, or 200 μl blood, are incubated with 500 μl lysis buffer/5 μl 10 mg/ml Proteinase K at 55 °C in a shaking incubator (110 min-1) for 18 h.
    4. DNA from the tissues isolated from T. cruzi-infected mice is extracted as described above (steps B4-B17).
    5. Normalize the T. cruzi DNA loads in the tissues.
      To normalize the amount of tissue analyzed in each PCR reaction, the murine TNF-α DNA is used to correct for intra-sample variation in the initiation sample amount, sample loading, and DNA recovery (Cummings and Tarleton, 2003). The T. cruzi DNA value and TNF-α DNA value are calculated automatically by plotting the Ct values against each standard of known concentration shown in Figure 4. Normalization of the T. cruzi DNA value = (T. cruzi DNA value/TNF-α DNA value) x 10, where 10 corresponds to the expected value for TNF-α from 30 mg heart/liver or 200 μl blood (Caldas et al., 2012).

Part II: T. cruzi killing assay

Materials and Reagents

  1. Pipette tips (Labcon, catalog numbers: 1093-260-000 , 1045-260-000 , 1036-260-000 )
  2. 10 ml syringe (Terumo, catalog number: SS-10SZP )
  3. 26 G needle (Terumo, catalog number: NN2613S )
  4. Cell strainer 40 μm nylon (Corning, Falcon®, catalog number: 352340 )
  5. 50 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  6. 10 cm culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150466 )
  7. Scraper (Corning, catalog number: 3008 )
  8. 24-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  9. Glass coverslip (13 mm) (Matsunami Glass, catalog number: C013001 )
  10. Slide glass (MUTO PURE CHEMICALS, catalog number: 110510 )
  11. Disposal hemocytometer (All-Biz, catalog number: 4109:37650 )
  12. 6 cm Petri dishes (AS ONE, catalog number: 1-8549-02 )
  13. 8-week-old male and female C57BL/6 mice (Japan SLC)
  14. Trypanosoma cruzi Tulahuen strain (gifted by professor S. Hamano, Institute of Tropical Medicine, Nagasaki University)
  15. ISOFLURANE Inhalation Solution (Pfizer)
  16. 70% ethanol (JUNSEI CHEMICAL, catalog number: 17065-0382 )
  17. HBSS (NACALAI TESQUE, catalog number: 17460-15 )
  18. 1x phosphate-buffered saline (PBS) (NACALAI TESQUE, catalog number: 14249-24 )
  19. GM-CSF (Wako Pure Chemical Industries, catalog number: 077-04674 )
  20. 0.25% trypsin (NACALAI TESQUE, catalog number: 35555-54 ) containing 0.02% EDTA (NACALAI TESQUE, catalog number: 06894-14 )
  21. IFN-γ (PeproTech, catalog number: 315-05 )
  22. 4% paraformaldehyde (NACALAI TESQUE, catalog number: 09154-85 )
  23. Diff-Quik (Sysmex, catalog number: 16920 )
  24. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
  25. ACK lysing buffer (pH 7.2–7.4) (see Recipes)
    1. Ammonium chloride (NH4Cl) (NAKALAI TESQUE, catalog number: 02423-65 )
    2. Potassium hydrogen carbonate (KHCO3) (Wako Pure Chemical Industries, catalog number: 163-03285 )
    3. EDTA (NAKALAI TESQUE, catalog number: 06894-14 )
  26. RPMI 1640 culture medium (see Recipes)
    1. Fetal bovine serum (Oregon Construction Contractors Board, catalog number: 171012 )
    2. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    3. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985-023 )

Equipment

  1. Pipettes (Nichiryo, catalog numbers: 00-NPX2-1000 , 00-NPX2-200 , 00-NPX2-20 , 00-NPX2-2 )
  2. Scissors (MIZUHO, catalog number: 09-207-00 )
  3. Forceps (Hammacher, catalog number: HSC_553-11 )
  4. Microscope (Olympus, model: CX31 )
  5. Handheld tally counter (Line Seiki, catalog number: H-102 )
  6. Centrifuge (TOMY SEIKO, model: AX-310 )

Procedure

  1. Preparation of bone marrow-derived macrophages
    Note: The following protocol referred to a previous report (Weischenfeldt and Porse, 2008).
    1. Euthanize mice with isoflurane inhalation solution.
    2. Sterilize the hind legs and abdomen with 70% ethanol.
    3. Make an incision in the midline of the abdomen.
    4. Clip outward to expose the hind legs.
    5. Remove all muscle from the bone using scissors and cut off the bones at the root of the femurs.
    6. After cutting the bone at both lower extremities, separate the femur and tibia by cutting at the knee joint in a Petri dish.
    7. Flush the bones with HBSS using a 10 ml syringe and a 26 G needle.
    8. Pass the bone marrow cells through a cell strainer in 50 ml tubes.
    9. Centrifuge at 240 x g for 5 min at 4 °C.
    10. Aspirate the supernatant and add 1 ml ACK lysing buffer (see Recipes) for the lysis of red blood cells at room temperature.
    11. After 3 min, add 9 ml cold PBS.
    12. Centrifuge at 240 x g for 5 min at 4 °C.
    13. Aspirate the supernatant.
    14. Culture 2 x 107 bone marrow cells isolated from the lower extremities of a mouse in 10 ml RPMI 1640 culture medium (see Recipes) supplemented with 10 ng/ml GM-CSF in a 10 cm culture dish.
    15. Wash the cells with PBS twice every 2 days and add fresh RPMI 1640 culture medium with 10 ng/ml GM-CSF.
    16. After 6 days, aspirate the supernatant and add 10 ml PBS.
    17. Aspirate the PBS and add 1 ml 0.25% trypsin containing 0.02% EDTA. Incubate at 37 °C for 5 min.
    18. Add 10 ml RPMI 1640 culture medium and scrape the cells with a scraper.
    19. Collect the cells in RPMI 1640 culture medium into 50 ml tubes.
    20. Centrifuge at 240 x g for 5 min at 4 °C.
    21. Aspirate the supernatant.
    22. Place the glass coverslips, sterilized by autoclave treatment, into the wells of a 24-well plate.
    23. Plate 5 x 104 bone marrow-derived macrophages on glass coverslips with 500 μl RPMI 1640 culture medium containing 10 ng/ml GM-CSF.
    24. After 18 h, bone marrow-derived macrophages are used in the T. cruzi killing assay.

  2. T. cruzi killing assay
    1. Bone marrow-derived macrophages prepared as in steps A23-A25 are cultured with 500 μl RPMI 1640 culture medium for 18 h in the presence of 0, 1, 10, or 100 ng/ml IFN-γ, which enhances parasite-killing activity in phagocytic cells.
    2. Aspirate the supernatant and wash with 500 μl PBS five times.
    3. Add 500 μl RPMI 1640 culture medium with 10 ng/ml GM-CSF.
    4. Infect 5 x 104 T. cruzi trypomastigotes with macrophages for 6 h (MOI = 1).
    5. After washing with 500 μl PBS three times, add 500 μl RPMI 1640 culture medium with 10 ng/ml GM-CSF and culture for 72 h.
    6. Aspirate the culture medium and wash with 500 μl PBS.
    7. Fix cells with 500 μl 4% paraformaldehyde and incubate for 10 min at room temperature.
    8. To visualize intracellular amastigotes, stain the cells using Diff-Quik kit.
    9. Apply a coverslip onto a slide glass using forceps.
    10. Count the number of intracellular amastigotes (Figure 5) under a microscope.


      Figure 5. The intracellular parasites. Bone marrow-derived macrophages infected with T. cruzi for 6 h in the presence or absence of IFN-γ were rigorously washed and cultured for 72 h. The cells were fixed and stained with Diff-Quik. Original magnification, 400x. * Indicates nucleus. An arrowhead indicates amastigotes.

    11. The intracellular parasite numbers in 200 macrophages are counted under a light microscope (Figure 6).


      Figure 6. The number of intracellular amastigotes. The number of parasites per 200 macrophages. Graph represents the mean ± SD from three independent experiments. *P < 0.05. Differences between the control and experimental groups were evaluated using Student’s t-test. Differences of P < 0.05 were considered significant.

Recipes

  1. Lysis buffer
    100 mM Tris-HCl (pH 8.5)
    5 mM EDTA
    0.2% SDS
    200 mM NaCl
  2. RPMI 1640 culture medium
    10% fetal bovine serum
    100 μg/ml streptomycin, 100 U/ml penicillin
    100 μM 2-mercaptoethanol
  3. ACK lysing buffer (pH 7.2-7.4)
    0.15 M NH4Cl
    1 mM KHCO3
    0.1 mM EDTA

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Agency for Medical Research and Development (to K.T. and H. K.), and by the Ichiro Kanehara Foundation and the Kurata Memorial Hitachi Science and Technology Foundation (to H.K.). The protocols provided here were partially modified from previously described methods by S. Cencing et al. (2011) and S. Caldas et al. (2012) (Quantification of T. cruzi) and by J. Weischenfeldt and B. Porse (2008) (Preparation of bone marrow-derived macrophages). We thank Kate Fox, DPhil, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The authors declare that they have no competing financial interests.

References

  1. Andrade, D. V., Gollob, K. J. and Dutra, W. O. (2014). Acute chagas disease: new global challenges for an old neglected disease. PLoS Negl Trop Dis 8(7): e3010.
  2. Bonney, K. M., Taylor, J. M., Thorp, E. B., Epting, C. L. and Engman, D. M. (2015). Depletion of regulatory T cells decreases cardiac parasitosis and inflammation in experimental Chagas disease. Parasitol Res 114(3): 1167-1178.
  3. Caldas, S., Caldas, I. S., Diniz Lde, F., Lima, W. G., Oliveira Rde, P., Cecilio, A. B., Ribeiro, I., Talvani, A. and Bahia, M. T. (2012). Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection. Acta Trop 123(3): 170-177.
  4. Cencig, S., Coltel, N., Truyens, C. and Carlier, Y. (2011). Parasitic loads in tissues of mice infected with Trypanosoma cruzi and treated with AmBisome. PLoS Negl Trop Dis 5(6): e1216.
  5. Cummings, K. L. and Tarleton, R. L. (2003). Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol Biochem Parasitol 129(1): 53-59.
  6. Flavia Nardy, A., Freire-de-Lima, C. G. and Morrot, A. (2015). Immune evasion strategies of Trypanosoma cruzi. J Immunol Res 2015: 178947.
  7. Garcia, M. N., Woc-Colburn, L., Aguilar, D., Hotez, P. J. and Murray, K. O. (2015). Historical perspectives on the epidemiology of human Chagas disease in Texas and recommendations for enhanced understanding of clinical Chagas disease in the Southern United States. PLoS Negl Trop Dis 9(11): e0003981.
  8. Kitada, S., Kayama, H., Okuzaki, D., Koga, R., Kobayashi, M., Arima, Y., Kumanogoh, A., Murakami, M., Ikawa, M. and Takeda, K. (2017). BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Trypanosoma cruzi infection. J Exp Med 214(5): 1313-1331.
  9. Requena-Mendez, A., Aldasoro, E., de Lazzari, E., Sicuri, E., Brown, M., Moore, D. A., Gascon, J. and Munoz, J. (2015). Prevalence of Chagas disease in Latin-American migrants living in Europe: a systematic review and meta-analysis. PLoS Negl Trop Dis 9(2): e0003540.
  10. Ribeiro, A. L., Nunes, M. P., Teixeira, M. M. and Rocha, M. O. (2012). Diagnosis and management of Chagas disease and cardiomyopathy. Nat Rev Cardiol 9(10): 576-589.
  11. Viotti, R., Vigliano, C., Lococo, B., Alvarez, M. G., Petti, M., Bertocchi, G. and Armenti, A. (2009). Side effects of benznidazole as treatment in chronic Chagas disease: fears and realities. Expert Rev Anti Infect Ther 7(2): 157-163.
  12. Weischenfeldt, J. and Porse, B. (2008). Bone marrow-derived macrophages (BMM): Isolation and applications. CSH Protoc 2008: pdb prot5080.

简介

感染克氏锥虫(Trypanosoma cruzi)引起恰加斯病(Chagas disease)。 这里提供的方法允许量化T。 克氏锥虫在感染腹腔的小鼠的肝脏,心脏和血液中分析,并分析克氏锥虫在体外感染的细胞的杀伤活性。
【背景】以慢性心肌病为特征的恰加斯病是由细胞内原生动物寄生虫克氏锥虫感染引起的(Bonney等人,2015年)。拉丁美洲大约有2000万人患有南美锥虫病(里贝罗等人,2012; Flavia Nardy等人,2015),已成为全球性的健康问题由于感染者的迁移(Andrade等人,2014; Garcia等人,2015; Requena-Mendez等人), 2015年)。已经开发了几种药物,如nifurtimox和benznidazole,用于治疗恰加斯病。然而,这些药物需要服用数月,并有严重的副作用(Viotti et al。,2009)。治疗的主要目的是抑制T。克鲁兹通过血液传播以及防止心力衰竭的发展。因此,定量T的协议。 cruzi 和 T。这里介绍的克鲁兹杀螨试验可能有助于发展南美锥虫病的新型诊断方法和治疗策略。

关键字:克氏锥虫, 定量PCR, 杀灭试验, 小鼠, 心脏, 肝脏, 血液, 源自骨髓的巨噬细胞


第一部分:量化T。 cruzi 在组织中


下面的方案(Kitada等人,2017)从前述方法(Cencig等人,2011; Caldas等人, em>,2012)。

材料和试剂

  1. 移液器吸头(Labcon,目录号:1093-260-000,1045-260-000,1036-260-000)
  2. 15cm培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150468)
  3. 血液采集管CAPIJECT(Terumo,目录号:CJ-NA)
  4. 10厘米培养皿(Sansyo,目录号:36-3406)
  5. 1.5ml微管(FUKAEKASEI和WATSON,目录号:131-415C)
  6. 处理血细胞计数器(All-Biz,目录号:4109:37650)
  7. 8周龄的雄性和雌性C57BL / 6小鼠(日本SLC)
  8. LLC-MK2细胞(恒河猴肾上皮细胞),由S.Hamao教授(长崎大学热带医学研究所)提供,也可在ATCC获得。有关LLC-MK 2 细胞的信息可在 https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Kidney/CCL-7.aspx#documentation
  9. 克氏锥虫图拉胡恩菌株(S.Hanmano教授(长崎大学热带医学研究所))
  10. 10毫克/毫升蛋白酶K(Merck,目录号:124568)
  11. 苯酚:氯仿:异戊醇(25:24:1)(NACALAI TESQUE,目录号:25970-56)
  12. 氯仿(JUNSEI CHEMICAL,目录号:28560-0330)
  13. 2-丙醇(JUNSEI CHEMICAL,目录号:64605-0330)
  14. 70%乙醇(JUNSEI CHEMICAL,目录号:17065-0382)
  15. TE缓冲溶液(pH 8.0)(NACALAI TESQUE,目录号:32739-31)
  16. Go Taq qPCR Master Mix(Promega,目录号:A6002)
  17. RPMI 1640(NACALAI TESQUE,目录号:30264-56)
  18. 引物(Invitrogen定制DNA引物)
    吨。 (Tc1),5'-cgagctgttgcccacacgggtgct-3'和5'-cctccaagcagcggatagttcagg-3'(Cencig et al。,2011)。和TNF-αDNA引物5'-tccctctcatcagttctatggccca-3'和5'-cagcaagcatctatgcacttagacccc-3'(Caldas等人,2012)。
  19. 裂解缓冲液(见食谱)
    1. Tris-HCl(NACALAI TESQUE,目录号:35434-21)
    2. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:06894-14)
    3. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:311-90271)
    4. 氯化钾(NaCl)(JUNSEI CHEMICAL,目录编号:19015-0350)
  20. RPMI 1640培养基(见食谱)
    1. 胎牛血清(CCB,目录号:171012)
    2. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    3. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:21985023)

设备

  1. 移液器(Nichiryo,产品目录号:00-NPX2-1000,00-NPX2-200,00-NPX2-20,00-NPX2-2)
  2. 镊子(Hammacher,目录号:HSC_553-11)
  3. 微量天平(Chyo Balance,型号:JPN-200W)
  4. 剪刀(精细科学工具,目录号:91460-11)
  5. 摇摇培养箱(TAITEC,型号:BR-43FM MR)
  6. 离心机(TOMY SEIKO,型号:MX-200)
  7. 涡流
  8. 实时PCR热循环仪:Step One Plus TM系统实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:StepOnePlus TM TM >,目录号:4376600)
  9. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)

程序

  1. 吨。 cruzi 准备
    1. 将5×10 5 -1×10 6 LLC-MK 2细胞在感染前的第1天接种到15cm培养皿中。 br />
    2. 在步骤A1中接种的LLC-MK 2细胞被2×10 6 T细胞感染。 cruzi Tulahuen(MOI = 2)。
    3. 每3天更换培养基(20毫升)。在图1A中,无花果被显示在 T中。克拉霉素感染的细胞(图A),其区分为释放到培养基中的感染形式trypomastigotes(图B)。
    4. 感染7天后,显微镜下计数培养上清液中的trypomastigotes(图1)。大约2×10 7个trypomastigotes获得。

      “”src
      图1. T的示意图。 cruzi 在血细胞计数器。 A. Amastigotes; B. Trypomastigotes。

  2. 制备组织标准品和定量PCR
    1. 将未受感染的小鼠的肝脏和心脏收集到15厘米培养皿中,并在CAPIJECT试管中收集血液(超过200μl)。
    2. 用剪刀将心脏和肝脏切成小块,加1×10-6T。在1.5ml微型管中,将来自未感染的C57BL / 6小鼠的克氏锥虫放入30mg心脏,30mg肝脏或200μl血液中。
    3. 加入500μL裂解缓冲液(见食谱)和5μL10mg/ ml蛋白酶K,并在摇动培养箱(振荡速度:110次/分钟)中于55℃孵育18小时。

    4. 13000×g离心10分钟,4℃
    5. 将上清转移到新鲜的1.5毫升微型管中。
    6. 加入500μl酚:氯仿:异戊醇(25:24:1),并用涡旋混合微管的内容物。

    7. 13000×g离心10分钟,4℃
    8. 将上层转移到新鲜的1.5毫升微型管。
    9. 加入500μl氯仿,并用涡旋混合微管的内容物。

    10. 13000×g离心10分钟,4℃
    11. 将上层转移到新鲜的1.5毫升微型管。
    12. 加入500μl2-丙醇,并使用涡旋混合微管的内容物。
    13. 在4℃下在13,000×gg下离心10分钟。
    14. 吸取上清液,并轻轻地在500微升70%乙醇洗颗粒。

    15. 5,500×g离心5分钟,4℃
    16. 吸出上清液,风干10分钟。
    17. 将DNA沉淀溶解在TE缓冲溶液(pH8.0)中并测量DNA的浓度以产生25μg/ ml组织DNA。
    18. 来自添加有T的组织的DNA。将来自不含T的组织的25μg/ ml DNA依次稀释10倍。 cruzi (图2)。制备的标准品含有每50ng总DNA含有10 -3 -10 -10 -2个寄生虫的DNA。标准曲线来确定T的量。克氏锥虫感染的小鼠的组织中的DNA是使用这些标准产生的。移液器吸头应在每次稀释之间更换。

      “”src
      图2.生成组织标准物的方案方案在组织中定量克氏锥虫

    19. 为了评估TNF-αDNA的量的标准曲线,含有1×10 3个T的25mg / ml组织DNA。 (STD1),随后用蒸馏水或TE缓冲液稀释10倍(图3)。使用内部对照'TNF-αDNA'来标准化在每个PCR反应中被分析的组织的量(Cummings和Tarleton,2003; Caldas等人,2012)。移液器吸头应在每次稀释之间更换。

      “”src
      图3.制备标准品的方案,以量化组织TNF-αDNA

    20. 通过qPCR产生克氏锥虫和TNF-α标准校准曲线(图4A和4B)
      1. 混合以下组件
        10μlGo Taq qPCR Master Mix
        1μl的10μMT。 cruzi 特异性引物(Tc1)或TNF-αDNA引物
        2微升的DNA标准。 cruzi(图2)或TNF-αDNA(图3)
        7μl双蒸水
      2. 扩增协议
        95℃10分钟,以及94℃15秒和64.3℃1分钟的40个循环。
        在伸长步骤结束时测量荧光发射(520nm)。熔解曲线阶段程序应用在65°C和95°C之间的连续荧光测量。每个DNA样本都从两个独立的qPCR运行重复进行定量。


        图4.使用或不使用T的未感染小鼠的肝脏的DNA产生的标准曲线。 cruzi痘病毒A和B.用如上所述制备的10倍稀释系列用特异于T的引物扩增。 cruzi DNA(A)或鼠TNF-αDNA(B)。标准曲线是从每个样品应用曲线的线性区域产生的。

  3. 量化 T。克氏锥虫感染小鼠的组织中的DNA
    1. 给小鼠(至少5只小鼠)腹膜内注射250μl包括5×10 -2 T的PBS。如步骤A1-A4中制备的克氏锥虫鞭毛虫。
    2. 感染后第20天和第30天,从小鼠收集心脏,肝脏和血液。
    3. 将30毫克心脏或肝脏或200微升血液与500微升裂解缓冲液/ 5微升10毫克/毫升蛋白酶K于55℃在摇动培养箱(110分钟-1)中温育18小时h。
    4. 来自从T分离的组织的DNA。如上所述提取克鲁兹感染的小鼠(步骤B4-B17)。
    5. 标准化 T。 cruzi 在组织中加载DNA。
      为了使在每个PCR反应中分析的组织量正常化,使用鼠TNF-αDNA来校正起始样品量,样品加载量和DNA回收率的样品内变化(Cummings and Tarleton,2003)。 T。 cruzi根据图4所示的已知浓度的每个标准品绘制Ct值,自动计算DNA值和TNF-αDNA值。 cruzi DNA值=(cruze cruzi DNA值/ TNF-αDNA值)×10,其中10对应于来自30mg心脏/肝脏或200μgTNF-α的预期值μl血液(Caldas等人,2012)。

第二部分:T。 cruzi 杀死测定

材料和试剂

  1. 移液器吸头(Labcon,目录号:1093-260-000,1045-260-000,1036-260-000)
  2. 10毫升注射器(Terumo,目录号:SS-10SZP)
  3. 26克针(Terumo,目录号:NN2613S)
  4. 细胞过滤器40微米尼龙(Corning,Falcon ,目录号:352340)
  5. 50ml试管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339652)
  6. 10cm培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:150466)
  7. 刮刀(康宁,目录编号:3008)
  8. 24孔板(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:142475)
  9. 玻璃盖玻片(13毫米)(Matsunami玻璃,目录号:C013001)
  10. 幻灯片(MUTO PURE CHEMICALS,目录号:110510)
  11. 处理血细胞计数器(All-Biz,目录号:4109:37650)
  12. 6厘米培养皿(AS ONE,目录号:1-8549-02)
  13. 8周龄的雄性和雌性C57BL / 6小鼠(日本SLC)
  14. 克氏锥虫图拉胡恩菌株(由长崎大学热带医学研究所滨田教授赠送)
  15. ISOFLURANE吸入溶液(辉瑞)
  16. 70%乙醇(JUNSEI CHEMICAL,目录号:17065-0382)
  17. HBSS(NACALAI TESQUE,目录号:17460-15)
  18. 1x磷酸盐缓冲盐水(PBS)(NACALAI TESQUE,目录号:14249-24)
  19. GM-CSF(Wako Pure Chemical Industries,目录号:077-04674)
  20. 含0.02%EDTA(NACALAI TESQUE,目录号:06894-14)的0.25%胰蛋白酶(NACALAI TESQUE,目录号:35555-54)
  21. IFN-γ(PeproTech,目录号:315-05)
  22. 4%多聚甲醛(NACALAI TESQUE,目录号:09154-85)
  23. Diff-Quik(Sysmex,目录号:16920)
  24. RPMI 1640(NACALAI TESQUE,目录号:30264-56)
  25. ACK裂解缓冲液(pH 7.2-7.4)(见食谱)
    1. 氯化铵(NH 4 Cl)(NAKALAI TESQUE,目录号:02423-65)
    2. 碳酸氢钾(KHCO 3)(Wako Pure Chemical Industries,目录号:163-03285)
    3. EDTA(NAKALAI TESQUE,目录号:06894-14)
  26. RPMI 1640培养基(见食谱)
    1. 胎牛血清(俄勒冈州建筑承包商委员会,目录号:171012)
    2. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    3. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:21985-023)

设备

  1. 移液器(Nichiryo,产品目录号:00-NPX2-1000,00-NPX2-200,00-NPX2-20,00-NPX2-2)
  2. 剪刀(MIZUHO,目录号:09-207-00)
  3. 镊子(Hammacher,目录号:HSC_553-11)
  4. 显微镜(奥林巴斯,型号:CX31)
  5. 手持计数器(精机线,目录号:H-102)
  6. 离心机(TOMY SEIKO,型号:AX-310)

程序

  1. 骨髓来源的巨噬细胞的制备
    注:以下协议涉及以前的报告(Weischenfeldt和Porse,2008)。
    1. 用异氟醚吸入溶液安乐死小鼠。

    2. 用70%乙醇消毒后腿和腹部
    3. 在腹部中线做一个切口。

    4. 向外夹,露出后腿。

    5. 使用剪刀去除骨骼中的所有肌肉,并切断股骨根部的骨骼
    6. 在两个下肢切骨后,在培养皿的膝关节切开股骨和胫骨。
    7. 使用10毫升注射器和26克针头用HBSS冲洗骨骼。
    8. 将骨髓细胞穿过50ml管中的细胞过滤器。

    9. 在240℃下离心5分钟
    10. 吸出上清液,并加入1毫升ACK裂解缓冲液(见食谱)在室温下裂解红细胞。
    11. 3分钟后,加入9毫升冷PBS。

    12. 在240℃下离心5分钟
    13. 吸出上清液。
    14. 在10cm培养皿中,在10ml RPMI 1640培养基(见配方)中培养2×10 7个从小鼠下肢分离的骨髓细胞(参见配方),所述RPMI 1640培养基补充有10ng / ml GM-CSF。
    15. 每2天用PBS洗涤细胞两次,加入含有10ng / ml GM-CSF的新鲜RPMI 1640培养基。
    16. 6天后,吸出上清液,并添加10毫升PBS。
    17. 吸出PBS并加入1ml含有0.02%EDTA的0.25%胰蛋白酶。
      在37°C孵育5分钟
    18. 加入10毫升RPMI 1640培养基,并用刮刀刮细胞。
    19. 收集在RPMI 1640培养基中的细胞到50毫升管。

    20. 在240℃下离心5分钟
    21. 吸出上清液。
    22. 将玻璃盖玻片经灭菌处理灭菌,放入24孔板的孔中。
    23. 在含有10ng / ml GM-CSF的500μlRPMI 1640培养基的玻璃盖玻片上板5×10 4个骨髓来源的巨噬细胞。
    24. 18小时后,在克氏锥虫杀伤试验中使用骨髓来源的巨噬细胞。

  2. 吨。 cruzi 杀死测定法
    1. 将在步骤A23-A25中制备的骨髓来源的巨噬细胞与500μlRPMI 1640培养基一起在0,1,10或100ng / ml IFN-γ存在下培养18小时,吞噬细胞。
    2. 吸出上清液,用500μlPBS洗5次。
    3. 加入500μl含10ng / ml GM-CSF的RPMI 1640培养基。
    4. 感染5×10 -4 Tm。 cruzi trypomastigotes与巨噬细胞6小时(MOI = 1)。
    5. 用500μlPBS洗涤3次后,加入500μl含有10ng / ml GM-CSF的RPMI 1640培养基并培养72小时。
    6. 吸出的培养基,并用500μLPBS洗。
    7. 用500μl4%多聚甲醛固定细胞,并在室温下孵育10分钟。
    8. 为了可视化细胞内的无鞭毛体,使用Diff-Quik试剂盒染色细胞。

    9. 使用镊子将盖玻片盖在载玻片上
    10. 在显微镜下计算细胞内无鞭毛体的数量(图5)。

      “”src
      图5.细胞内寄生虫感染T细胞的骨髓来源的巨噬细胞。 cruzi 在存在或不存在IFN-γ的情况下持续6小时,剧烈洗涤并培养72小时。将细胞固定并用Diff-Quik染色。原始放大倍率,400倍。 *表示核。箭头表示无鞭打。


    11. 在光镜下计数200个巨噬细胞内的细胞内寄生虫数量(图6)
      “”src
      图6.细胞内无鞭毛体的数目每200个巨噬细胞的寄生虫数量。图表表示来自三个独立实验的平均值±SD。 * P &lt; 0.05。对照组和实验组之间的差异使用Student's检验进行评估。 P 的差异< 0.05被认为是显着的。

食谱

  1. 裂解缓冲液
    100mM Tris-HCl(pH8.5)
    5 mM EDTA
    0.2%SDS
    200 mM NaCl
  2. RPMI 1640培养基
    10%胎牛血清
    100μg/ ml链霉素,100U / ml青霉素
    100μM2-巯基乙醇
  3. ACK裂解缓冲液(pH 7.2-7.4)
    0.15M NH 4 Cl Cl / 1mM KHCO 3
    0.1mM EDTA

致谢

这项工作得到日本文部科学省和日本医学研究开发机构(KT和HK)以及金原一郎基金会和仓田纪念日立科学的资助和技术基金会(香港)。这里提供的协议是由S.Cencing等人(2011)和S.Caldas等人的先前描述的方法部分修改的。 (2012)(克拉霉素的量化)和J.Weischenfeldt和B.Porse(2008)(骨髓衍生的巨噬细胞的制备)。我们感谢Edanz集团(www.edanzediting.com/ac)的Kate Fox,DPhil编辑本稿的草稿。作者声明,他们没有竞争的财务利益。

参考

  1. Andrade,D.V.,Gollob,K.J。和Dutra,W.O。(2014)。 急性恰加斯病:一种老的被忽视疾病的新的全球性挑战 PLoS Negl Trop Dis 8(7):e3010。
  2. Bonney,K.M.,Taylor,J.M。,Thorp,E.B.,Epting,C.L。和Engman,D.M。(2015)。 调节性T细胞的消耗减少了实验性恰加斯病的心脏寄生虫病和炎症。 > Parasitol Res 114(3):1167-1178。
  3. Caldas,S.,Caldas,I. S.,Diniz Lde,F.,Lima,W. G.,Oliveira Rde,P.,Cecilio,A. B.,Ribeiro,I.,Talvani,A.and Bahia,M.T。(2012)。 实时PCR策略用于实验性克氏锥虫的血液和组织样品中的寄生虫定量< / em>感染。 Acta Trop 123(3):170-177。
  4. Cencig,S.,Coltel,N.,Truyens,C.和Carlier,Y。(2011)。 感染克氏锥虫的小鼠的组织中的寄生虫载荷并用AmBisome 。 PLoS Negl Trop Dis 5(6):e1216。
  5. Cummings,K.L。和Tarleton,R.L。(2003)。 通过实时PCR快速定量检测宿主组织中的克氏锥虫(Trypanosoma cruzi)。 Mol Biochem Parasitol 129(1):53-59。
  6. Flavia Nardy,A.,Freire-de-Lima,C.G。和Morrot,A。(2015)。 克氏锥虫的免疫逃避策略。 J Immunol Res 2015:178947.
  7. Garcia,M.N.,Woc-Colburn,L.,Aguilar,D.,Hotez,P.J。和Murray,K.O.(2015)。 关于德克萨斯州人类南美锥虫病流行病学的历史观点以及关于增强对南美锥虫病美国南部。 PLoS Negl Trop Dis 9(11):e0003981。
  8. Kitada,S.,Kayama,H.,Okuzaki,D.,Koga,R.,Kobayashi,M.,Arima,Y.,Kumanogoh,A.,Murakami,M.,Ikawa,M。和Takeda,K。 2017年)。 BATF2通过在克氏锥虫感染期间抑制Il23a表达而抑制免疫病理学Th17应答。 J Exp Med 214(5):1313-1331。
  9. Requena-Mendez,A.,Aldasoro,E.,de Lazzari,E.,Sicuri,E.,Brown,M.,Moore,D.A.,Gascon,J.and Munoz,J。(2015)。 生活在欧洲的拉丁美洲移民中恰加斯病的发病率:一项系统评价和荟萃分析。 PLoS Negl Trop Di 9(2):e0003540。
  10. Ribeiro,A.L.,Nunes,M.P.,Teixeira,M.M.和Rocha,M.O。(2012)。 南美锥虫病和心肌病的诊断和治疗 Nat Rev Cardiol 9(10):576-589。
  11. Viotti,R.,Vigliano,C.,Lococo,B.,Alvarez,M.G.,Petti,M.,Bertocchi,G。和Armenti,A。(2009)。苯扎硝唑治疗慢性南美锥虫病的副作用:恐惧和现实。 > Expert Rev Anti Infect Ther 7(2):157-163。
  12. Weischenfeldt,J。和Porse,B。(2008)。 骨髓衍生巨噬细胞(BMM):分离和应用 CSH Protoc 2008:pdb prot5080。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kayama, H., Kitada, S. and Takeda, K. (2017). Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay. Bio-protocol 7(22): e2613. DOI: 10.21769/BioProtoc.2613.
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