A quantitative assay method of Toxoplasma gondii HSP70 mRNA by quantitative competitive-reverse transcriptase-PCR

Parasitology International 53 (2004) 49–58

A quantitative assay method of Toxoplasma gondii HSP70 mRNA by quantitative competitive-reverse transcriptase-PCR Lian-Xun Piao, Fumie Aosai, Mei Chen, Hao Fang, Hye-Seong Mun, Kazumi Norose, Akihiko Yano* Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan Received 18 August 2003; accepted 13 November 2003

Abstract Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) has been identified as a virulent molecule expressing only in T. gondii tachyzoites during lethal acute infection. Therefore, it is of importance to determine the expression of T.g.HSP70 mRNA in a quantitative manner for analysis of virulence of T. gondii in tissues. We have constructed a competitor T.g.HSP70 and have successfully established a quantitative competitive-reverse transcriptase– polymerase chain reaction (QC-RT-PCR) targeting T.g.HSP70 gene. By using the established QC-RT-PCR method, we have demonstrated that the copy number of T.g.HSP70 mRNA per T. gondii tachyzoite was highest in the lung among the organs examined in interferon-g knockout (GKO) mice. 䊚 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Toxoplasma gondii; T.g.HSP70; QC-RT-PCR; Danger signal

1. Introduction Toxoplasma gondii is an opportunistic parasite with an extensive range of warm-blooded animal hosts, including humans w1x. In mice and humans two T. gondii stages are encountered, tachyzoites Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; gDNA, genomic DNA; GKO, interferon-g knockout; LI, large intestine; MLN, mesenteric lymph nodes; p.o., perorally; P.I., post infection; QC-RT-PCR, quantitative competitive-reverse transcriptase–polymerase chain reaction; SI, small intestine; T. gondii, Toxoplasma gondii; T.g.HSP70, T. gondii-derived heat shock protein 70; WT, wild type *Corresponding author. Tel.: q81-43-226-2071; fax: q8143-226-2076. E-mail address: [email protected] (A. Yano).

and bradyzoites. T.g.HSP70 molecule is specifically expressed by tachyzoites w2,3x. We have cloned the T.g.HSP70 gene and previously demonstrated the major properties of T.g.HSP70 as a downregulator of host immune responses in T. gondiiinfected mice. Firstly, the T.g.HSP70 whose expression increased 1–2 days before death of the host, deteriorating the host defense by downregulating nitric oxide (NO) release from peritoneal macrophages in T. gondii-infected mice w2x. Secondly, T.g.HSP70 induced anti-HSP70 autoantibody production by B-1 cells of T. gondii-infected mice. The VH1-JH1 B-1 cells producing antiHSP70 autoantibody and IL-10 downregulated the host defense of mice to T. gondii infection w4,5x.

1383-5769/04/$ - see front matter 䊚 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2003.11.001

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Fig. 1. Construction of competitor T.g.HSP70 DNA. T.g.HSP70 gene constructed into pME18100 plasmid was cleaved with restriction enzyme Spl I at 1115 and 1295 bps, and the fragment between 1115 and 1295 bps was deleted. The enzyme-cleaved blunt ends were autoligated. The DNA fragments of original T.g.HSP70 and the competitor T.g.HSP70 were detected as 500 bps and 320 bps, respectively, by using the primers described in Section 2.

Regarding the pathogenicity or virulence of T. gondii tachyzoites, the direct destruction of host cells by multiplying tachyzoites represents an obligate intracellular parasitic protozoan. This type of pathogenicity was recognized as tachyzoites-mediated pathogenicity, and we tentatively designated this stage of T. gondii as destructive tachyzoites w3x. Moreover, we proposed a new category of tachyzoite stage of T. gondii (virulent tachyzoites) expressing high level of T.g.HSP70 that indirectly manifests its pathogenicity by downregulating host defense responses w3x. Based on a functional analysis of T.g.HSP70 in host immune responses, we attempted to establish a method for quantitatively measuring the copy number of T.g.HSP70 mRNA in tissues of various organs in T. gondii-infected mice, and we established QC-RT-PCR targeting T.g.HSP70 gene. By using the QC-RT-PCR method, we analyzed the virulence of organ-specific T. gondii tachyzoites in GKO and wild type (WT) BALByc mice.

strain) of T. gondii were used for infection experiments as previously described w7–9x. GKO and WT BALByc mice were perorally (p.o.) infected with 100 T. gondii cysts of Fukaya strain and their survival was monitored daily. 2.2. Construction of competitor T.g.HSP70 DNA

2. Materials and methods

The cDNA fragment of T.g.HSP70 (1941 bps; GenBank accession number AB019539) inserted in pME18100 plasmid w10x was cleaved with the restriction enzyme Spl I at 1115 and 1295 bps, and the small fragment of 180 bps between 1115 and 1295 bps was deleted. The cleaved blunt ends of T.g.HSP70 in pME18100 were autoligated by ligation kit (Takara, Tokyo, Japan). The autoligated competitor T.g.HSP70 in the pME18100 plasmid was transfected to Escherichia coli, DH5a by the heat shock method. The pME18100 plasmid carrying the competitor T.g.HSP70 was purified from the transformed E. coli colony (Multiple WizardR Plus Minipreps, Promega Co., Madison, WI, USA) and was used as a competitor T.g.HSP70 (Fig. 1).

2.1. Mice and T. gondii strain

2.3. QC-PCR

Sex-matched GKO and WT BALByc mice were used at 8 weeks of age. WT BALByc mice were purchased from SLC (Hamamatsu, Japan). GKO BALByc mice were genotyped by PCR w6x. Cysts of an avirulent Fukaya strain (tissue cyst-forming

T. gondii tachyzoites were collected from the peritoneal cavity of GKO BALByc mice at day 7 after intraperitoneal infection with 200 T. gondii cysts of Fukaya strain. After deleting peritoneal exudate cells (PECs) by centrifugation, free tachy-

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zoites were counted by microscopy. Genomic DNA (gDNA) was prepared from various numbers (104, 3=103, 103, 3=102, 102, 3=101, 101, 3=100) of T. gondii tachyzoites and was coamplified with 1 pg of the competitor T.g.HSP70 using the following primers: forward (59GCGAAGTTGTGTTGGTTG-39) corresponding to nucleotides 1001–1018 bps of T.g.HSP70 gene, and reverse (59-AAGTCCACCGGAAAGAGC-39) corresponding to nucleotides 1483–1500 bps of T.g.HSP70 gene. The amplification reaction cycle was as follows: 94 8C for 3 min., 54 8C for 1 min. and 72 8C for 2 min. for the first cycle, 94 8C for 1 min., 54 8C for 1 min., and 72 8C for 2 min. for 35 cycles, and 94 8C 1 min., 54 8C for 1 min. and 72 8C for 5 min for the last cycle. The amplified products were separated by agarose gel electrophoresis and stained with ethidium bromide. The DNA fragments of targeted T.g.HSP70 and the competitor T.g.HSP70 were detected as 500 bps and 320 bps, respectively. By measuring the ratios of band densities of QC-PCR products of T.g.HSP70 gDNA and competitor T.g.HSP70, a standard curve was drawn. The GKO and WT BALByc mice were p.o. infected with 100 T. gondii cysts of Fukaya strain and were euthanized 8 days post infection (P.I.) by asphyxiation with CO2. After measuring the weight of various organs such as the brain, heart, lung, spleen, mesenteric lymph nodes (MLN), kidney, stomach, liver, small intestine (SI), caecum and large intestine (LI), gDNA was extracted from tissue of each organ. One microgram of tissue gDNA was co-amplified with 1 pg of the competitor T.g.HSP70 targeting T.g.HSP70 gene. The ratio of band density of genomic T.g.HSP70 to that of the competitor T.g.HSP70 from each sample was applied to the standard curve to measure the T. gondii number per microgram of tissue gDNA. Three mice were used for each experimental group. The experiment for the standard curve was repeated three times. 2.4. QC-RT-PCR Tissue mRNAs from the brain, heart, lung, spleen, MLN, kidney, stomach, liver, SI, caecum and LI of GKO and WT BALByc mice infected

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with T. gondii cysts were isolated using MicroFast Track娃 2.0 kit (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. Ten nanogram mRNA was reverse transcribed in 20 ml using an RNA PCR kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. The cDNA in 10 ml of RT product was co-amplified with 1 pg of the competitor T.g.HSP70 as mentioned above for QC-RT-PCR. The product was electrophoretically separated. The ratios of band densities of QC-RT-PCR products of T.g.HSP70 cDNA and competitor T.g.HSP70 (TyC ratio) were applied to the standard curve to measure the copy number of T.g.HSP70 mRNA per nanogram tissue mRNA. The expression of T.g.HSP70 mRNA copy number per T. gondii zoite in the various organs of T. gondii-infected GKO and WT BALByc mice was calculated using the following formula: copy number of T.g.HSP70 mRNA per T. gondii zoites(copy number of T.g.HSP70 mRNA measured by the standard curve with the TyC ratio of QC-RT-PCR product)=(amounts of tissue mRNA from constant tissue weight)y(T. gondii number measured by the standard curve with TyC ratio of QC-PCR product)=(amounts of tissue gDNA from constant tissue weight). The formula was constructed on the premise that one molecule of T.g.HSP70 cDNA was transcribed from one copy of T.g.HSP70 mRNA by RT. Three mice were used for each experiment. The experiments were repeated at least twice. 2.5. Assessment of liver function Serum liver enzyme levels of aspartate aminotransferase (AST, IUyl) and alanine aminotransferase (ALT, IUyl) were quantified using a commercial kit (Sigma, St. Louis, MO, USA) that was adapted for use in a 96-well plate. 3. Results 3.1. Co-amplification of T.g.HSP70 gene of T. gondii Fukaya strain and competitor T.g.HSP70 by QC-PCR Molecular genetic studies have shown that HSP70 gene is a single copy in both virulent and

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avirulent strains of T. gondii w11x. Thus, the gDNA of a determined number of T. gondii (104, 3=103, 103, 3=102, 102, 3=101, 101, 3=100) was coamplified with 1 pg of competitor T.g.HSP70 DNA using the primers mentioned in Section 2. The intensities of the PCR products of genomic T.g.HSP70 increased relative to T. gondii numbers while those of competitor T.g.HSP70 gradually decreased (Fig. 2a). The standard curve between the cell number of T. gondii (i.e. copy number of T.g.HSP70 gDNA) and the logarithm of the ratio of T.g.HSP70 gDNAycompetitor T.g.HSP70 (TyC ratio) had a linear relationship (Fig. 2b). Thus, the QC-PCR method targeting the T.g.HSP70 gene for measuring the T. gondii number quantitatively was established. 3.2. T. gondii distribution in GKO and WT BALBy c mice measured by QC-PCR All GKO BALByc mice succumbed 8–10 days after p.o. infection with T. gondii cysts, while all

Fig. 3. Survival curve of GKO and WT BALByc mice. GKO and WT BALByc mice were p.o. infected with 100 T. gondii cysts of Fukaya strain and survival of the mice was monitored daily. Five mice were used for each experimental group and the experiments were repeated three times.

WT BALByc mice survived more than 20 days P.I. (Fig. 3). By using the QC-PCR method targeting the T.g.HSP70 gene, T. gondii numbers in various

Fig. 2. Establishment of QC-PCR targeting T.g.HSP70 gene. (a) The gDNAs derived from 104 , 3=103, 103, 3=102, 102, 3=101, 101, 3=100 T. gondii tachyzoites of Fukaya strain were co-amplified with 1 pg of competitor T.g.HSP70 by using T.g.HSP70 primers. Lanes 1 and 2 represent 104 tachyzoites. Lanes 3 and 4 represent 3=103 . Lanes 5 and 6 represent 103 . Lanes 7 and 8 represent 3=102. Lanes 9 and 10 represent 102. Lanes 11 and 12 represent 3=101. Lanes 13 and 14 represent 101. Lane 15 represents 3=100. Lane 16 represents competitor T.g.HSP70 alone. Lane 17 represents gDNA of 104 tachyzoites alone. (b) The ratio of T.g.HSP70 gDNAycompetitor T.g.HSP70 (TyC ratio) was calculated as described in Section 2. The standard curve between the T. gondii number and the logarithm of TyC ratio was drawn.

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Fig. 4. Measurement of T. gondii number in tissue of T. gondii-infected mice by QC-PCR. (a) T. gondii amounts in brain (lane 1), heart (lane 2), lung (lane 3), spleen (lane 4), MLN (lane 5), kidney (lane 6), stomach (lane 7), liver (lane 8), SI (lane 9), caecum (lane 10) and LI (lane 11) of GKO and WT BALByc mice were examined by QC-PCR targeting T.g.HSP70 gene at 8 days P.I. Lane 12 represents QC-PCR of competitor T.g.HSP70 alone. Lane 13 represents QC-PCR of gDNA of 104 tachyzoites alone. (b) The TyC ratio of QC-PCR product was measured as described in Section 2 and T. gondii number per microgram of tissue gDNA from the various organs was shown. Standard deviation of Fig. 4b was less than 20% of mean.

organs of T. gondii-infected GKO and WT BALBy c were comparatively measured. In GKO BALBy c mice, T. gondii amounts in the MLN, SI, spleen, liver, heart, caecum and lung were high (9.02=107, 8.06=106, 5.02=106, 1.04=106, 6.50=105, 2.10=105 and 2.01=105 per mg gDNA, respectively), while those in the LI, kidney, stomach and brain were low (5.25=103, 4.25=102, 3.08=102 and 2.20=102 per mg gDNA, respectively). However, in WT BALByc mice, T. gondii amounts in the SI, heart, lung, spleen, caecum, MLN, LI, brain, liver, stomach and kidney were low (2.90=103, 3.16=102, 3.05=102, 3.03=102, 2.95=102, 1.81=102, 1.62=102, 1.50=102, 2.20=101, 1.52=101 and 1.33=101 per mg gDNA, respectively) (Fig. 4a

and b). Thus, T. gondii abundances were significantly different between GKO and WT BALByc mice, and they also differed according to organ. 3.3. Quantitative measurement of T.g.HSP70 mRNA expression in GKO and WT mice Messenger RNAs were isolated from tissue RNAs of various organs of T. gondii-infected GKO and WT BALByc mice. The cDNA of RT products of 10 ng mRNA was co-amplified with 1 pg of the competitor T.g.HSP70 DNA targeting T.g.HSP70 gene to measure the expression of T.g.HSP70 mRNA quantitatively (Fig. 5a). No product was obtained in PCR of the mRNA samples without RT (Fig. 5b). Copy numbers of

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Fig. 5. Expression of T.g.HSP70 mRNA in GKO and WT BALByc mice measured by RT-QC-PCR targeting T.g.HSP70 gene. (a) cDNA in RT product of tissue mRNA from the various organs of GKO and WT BLAByc mice obtained at 8 days P.I. was coamplified with 1 pg of competitor T.g.HSP70. The QC-RT-PCR data of brain (lane 1), heart (lane 2), lung (lane 3), spleen (lane 4), MLN (lane 5), kidney (lane 6), stomach (lane 7), liver (lane 8), SI (lane 9), caecum (lane 10) and LI (lane 11) were shown. Lane 12 represents competitor T.g.HSP70 alone. Lane 13 represents gDNA of 104 tachyzoites alone. (b) To examine contamination of gDNA in the templates, tissue mRNAs from various organs of T. gondii-infected GKO and WT BALByc mice were co-amplified with 1 pg of the competitor T.g.HSP70 DNA targeting T.g.HSP70 gene without RT. (c) The copy number of T.g.HSP70 mRNA per ng of tissue mRNA from the various organs was shown. Standard deviation of Fig. 5c was less than 20% of mean. (d) The copy number of T.g.HSP70 mRNA per T. gondii zoite from the various organs of T. gondii-infected GKO and WT BALByc mice was calculated by the following formula: copy number of T.g.HSP70 mRNA per T. gondii zoites(copy number of T.g.HSP70 mRNA measured by standard curve with TyC ratio of QC-RT-PCR product)=(amounts of tissue mRNA from constant tissue weight)y(T. gondii number measured by standard curve with TyC ratio of QC-PCR product)=(amounts of tissue gDNA from constant tissue weight). Bars represent mean"S.D. of the copy number of T.g.HSP70 mRNA per T. gondii zoite of three individual miceygroup.

Fig. 6. Liver function of T. gondii-infected GKO and WT BALBy c mice. Serum liver enzyme levels of AST and ALT in uninfected and T. gondii-infected GKO and WT BALByc mice were examined as described in Section 2. Statistical analysis was performed by using student t-test: *1 P-0.001, *2 P-0.05.

T.g.HSP70 mRNA in 1 ng mRNA from various organs were calculated from the standard curve (Fig. 2b) and the TyC ratio of the QC-RT-PCR product (Fig. 5a). The copy number of T.g.HSP70 mRNA in 1 ng mRNA was high in the MLN, spleen, SI, heart, liver and lung (4.38=106, 1.60=106, 6.80=105, 2.40=105, 1.66=105 and 7.02=104, respectively), while it was low in the caecum, LI, stomach, kidney and brain (5.72=103, 4.06=102, 3.11=101, 2.02=101 and 1.52=101, respectively) in GKO BALByc mice. In contrast, the copy number of T.g.HSP70 mRNA in 1 ng mRNA from various organs of WT BALBy c mice was much lower than that of GKO BALBy c mice (Fig. 5c). Then, the copy number of

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T.g.HSP70 mRNA per T. gondii zoite was quantitatively calculated from Fig. 4b and Fig. 5c, and it was revealed to differ depending on the organ. In GKO BALByc mice, the copy number of T.g.HSP70 mRNA per T. gondii zoite in the lung was 4.66, the highest among the organs examined. T. gondii in the heart and liver of GKO mice also expressed many T.g.HSP70 mRNA copies (2.22 and 2.13, respectively) compared with T. gondii zoites in other organs. The copy number of T.g.HSP70 mRNA per T. gondii zoite in the various organs of WT BALByc mice was low (Fig. 5d). 3.4. Liver function of T. gondii-infected GKO and WT mice To evaluate the liver function, serum enzyme levels of AST and ALT were quantified in uninfected and infected GKO and WT BALByc mice. Both of AST and ALT levels of T. gondii-infected mice were significantly higher than those of uninfected mice in both GKO and WT mice. AST and ALT levels of T. gondii-infected GKO mice were higher than those of infected WT mice (Fig. 6). Thus, high level expression of T.g.HSP70 mRNA in the liver of T. gondii-infected GKO mice correlated to tissue damage of the liver measured by AST and ALT. 4. Discussion The pathogenicity of T. gondii infection is apparently attributable to the host cell destruction by the intracellular proliferation of tachyzoites w12– 14x. Furthermore, previous studies have revealed that the tissue damage by T. gondii infection could not be merely attributed to the replication of intracellular parasites or apoptosis of host cells w1,15x, but that tissue damage was mediated through a soluble parasite-derived factor(s) that reaches toxic levels during lethal infections w15x. The virulence of tachyzoites is a key factor in the severity of toxoplasmosis w16x. T.g.HSP70, which is expressed by tachyzoites but not by bradyzoites of T. gondii, has been revealed to be a virulent molecule and danger signal during lethal, acute T. gondii infection in GKO mice w2,3x. T.

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gondii infection induced anti-T.g.HSP70 antibody and anti-mHSP70 autoantibody production in T. gondii-infected mice w4x, and the anti-HSP70 autoantibody downregulated the host defense response to T. gondii infection w5x. T.g.HSP70 induced B cell activation through TLR4 w17x. Furthermore, T.g.HSP70 also downregulated NO synthesis w2x. Thus, we named T.g.HSP70 expressing tachyzoites as ‘virulent tachyzoites’, and virulent tachyzoites indirectly manifest the pathogenicity by downregulating host defense responses (2–5). ‘Destructive tachyzoites’ are defined to T. gondii tachyzoites which express low level of T.g.HSP70 and destruct host cells by multiplying, representing an obligate intracellular parasitic protozoa (3). Virulent tachyzoites expressed a much higher level of T.g.HSP70 than destructive tachyzoites w2,3x. Thus, the quantitative measurement of T.g.HSP70 mRNA expression is essential for performing definitive analysis of the pathogenicity of T. gondii in tissues. In the present study, we successfully established a quantitative measurement system to assay the T.g.HSP70 mRNA copy number per T. gondii zoite by using a truncated competitor T.g.HSP70 in RTPCR. Ubiquitously expressed genes such as glyceraldehyde-3-phosphate dehydrogenase have usually been used as an internal control for analyzing the mRNA expression in conventional RTPCR, but they are not adequate for a quantitative calculation. The application of competitive RTPCR methods has been reported by several groups in virus infection w18–20x, tumor w21–24x and other specimens w25–36x in human w18,19,21– 23,25–29x, animals w20,24,26,30–33x, as well as in culture cells w34–36x. This is the first report to describe the quantitative estimation of mRNA copy numbers in the tissue of T. gondii-infected mice by the use of QCRT-PCR targeting cDNA of T.g.HSP70 mRNA. Using the QC-RT-PCR method, we quantitatively measured the copy number of T.g.HSP70 mRNA per T. gondii zoite in the tissue of various organs of GKO and WT BALByc mice p.o. infected with T. gondii cysts of Fukaya strain. The copy number of T.g.HSP70 mRNA per T. gondii zoite in lung tissue was the highest among the organs examined in GKO BALByc mice,

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followed by that in heart and liver (Fig. 5d). These results correlated with previous pathological observations of tissue damage in lung w37–42x, heart w41–46x and liver w15,37,43,44,47–49x. Derouin and Garin w13x reported an early involvement of lung in T. gondii infection in which parasite burdens were analyzed quantitatively by tissue culture method in blood, brain and lung of mice after intraperitoneal acute lethal infection with T. gondii tachyzoites of virulent RH and C56 strains w13x. In the infection with either strain, tachyzoites were first detected in lung 2–4 days P.I., and parasitic loads remained constantly at a higher level in lung than in brain until death on day 6–8 P.I. These results indicate that the infection with a virulent T. gondii strain is characterized by an early involvement of lung, with pneumonia as the principal cause of death w13x. In the present study, we have clearly demonstrated that the highest expression of T.g.HSP70 molecule, a danger signal, caused the host to suffer from pneumonia leading to mortality, by using our newly established QC-RT-PCR system. Areas of inflammation associated with tachyzoites were also observed in the heart and liver of T. gondii-infected mice w44x. Clinically, autopsy examination of patients of toxoplasmosis with acquired immunodeficiency syndrome revealed frequent pathological involvement of the heart w45,46x. Mordne et al. w15x reported the pathological involvement of the liver during lethal toxoplasmosis in mice, where widespread alterations in hepatocytes, including enlargement, cytoplasmic vacuolization, and release of liver enzymes were induced w15x. Actually, high expression of T.g.HSP70 mRNA in the liver of T. gondii-infected GKO mice was revealed to correlate with the tissue damage of the liver measured by AST and ALT (Fig. 6). In GKO BALByc mice, the intestine, spleen, MLN, brain and kidney were also target organs of T. gondii infection w9,50x. The present study indicated that a significant level of T.g.HSP70 mRNA expression was correlated with the pathological changes in these organs. Development of intestinal damage, and even fatal outcome, has been described in acute infection with T. gondii w51,52x. However, the copy numbers of T.g.HSP70 mRNA

per T. gondii zoite in the spleen and MLN were not high (Fig. 5d). Lethal T. gondii infections led to a marked acellularity in lymphoid compartments and a loss of tissue architecture in the spleen and MLN, and also induced extensive necrosisyapoptosis of non-infected cells within lymphoid tissues w15x. We suggest that the damage of the spleen and MLN was caused by another pathogenicity independent of the T.g.HSP70 molecule. Regarding the brain, Suzuki et al. w44x reported that encephalitis was the main damage by T. gondii infection in GKO mice. The present study showed a low level of T.g.HSP70 mRNA expression in the brain (Fig. 5d), and no severe encephalitis (data not shown). These discrepant observations may have resulted from the differences in infection route and T. gondii strain. In T. gondii-infected WT BALByc mice, the expression of T.g.HSP70 mRNA in the various organs was shown to be weak (Fig. 5d), reflecting the pathological observations of organ damage not being obvious under the present experimental conditions (data not shown). We previously reported that interferon-g indirectly downregulated the expression of T.g.HSP70 in T. gondii w2,3x, but the precise mechanisms regulating the expression of T.g.HSP70 remain to be unraveled. Taken together, the present study described the establishment of a quantitative assay system for the T.g.HSP70 mRNA copy number per T. gondii zoite, and it was revealed that the T.g.HSP70 expression was one of the factors that caused organ damage by the virulent stage of T. gondii tachyzoites. The measurement of T.g.HSP70 mRNA expression in tissues may be applicable for clinical diagnosis in terms of understanding the inflammatory level caused by T. gondii infection. Acknowledgments This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture, Japan. References w1x Cristina-Gavrilescu L, Denkers EY. IFN-g overproduction and high level apoptosis are associated with high

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