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Depletion of lymphocytes, but not neutrophils, via apoptosis in a murine model of Vibrio vulnificus infection

^1,2Laboratory of Veterinary Public Health¹, and Laboratory of Veterinary Anatomy², School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23-35-1, Towada Aomori 034-8628, Japan ³Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan

Correspondence Takashige Kashimoto kashimot{at}vmas.kitasato-u.ac.jp

Received August 12, 2004
Accepted October 1, 2004

Vibrio vulnificus causes severe sepsis in humans. There are several reports about the relationship between host immunity and bacterial growth in V. vulnificus infection. However, the effect on leukocytes of V. vulnificus infection in vivo has not been elucidated. A murine model of V. vulnificus infection was used to investigate its effects on leukocytes in this study. Bacteria were recovered from the blood of mice 3 h after subcutaneous injection in the right lower flank. They were detected in 87.5 % (n = 7/8) of mice at 6 h, but this value decreased to 12.5 % (n = 1/8) at 12 h. In contrast, the number of lymphocytes in peripheral blood had already started to decrease at 3 h, and reached a minimum at 6–9 h post-inoculation. Typical DNA laddering, a hallmark of apoptosis, was also detected in thymocytes and splenocytes at 6 and 9 h, and showed a tendency to disappear by 12 h. Although the number of lymphocytes decreased in the model, the numbers of neutrophils did not. These results suggested that V. vulnificus has selective cytotoxicity for lymphocytes in peripheral blood in vivo, and the lymphocyte depletion was probably associated with apoptosis in vivo.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Vibrio vulnificus, a Gram-negative estuarine bacterium, causes septicaemia in humans who suffer from liver cirrhosis, alcoholism, haemochromatosis, immunocompromised conditions or diabetes (Hlady & Klontz, 1996; Linkous & Oliver, 1999; Strom & Paranjpye, 2000). Primary septicaemia is caused by the ingestion of seafood contaminated with V. vulnificus or through wound infections involving exposure to contaminated sea water or marine products. Mortality from this infection exceeds 50 %, and increases to more than 90 % in patients who go into shock shortly after admission to hospital (Koenig et al., 1991). Infected individuals with primary septicaemia usually present with fever and chills. Within 24 h of these first signs of illness, secondary cutaneous lesions appear, including ecchymoses, bullae and progressive cellulitis (Starks et al., 2000).

Several putative virulence factors of V. vulnificus, including a capsular polysaccharide, a metalloprotease and iron-sequestering systems, have been studied in vitro and in vivo (Fan et al., 2001; Johnson et al., 1984; Miyoshi et al., 1998; Morris et al., 1987). Of these, the capsular polysaccharide confers resistance to phagocytosis by human polymorphonuclear leukocytes or murine peritoneal macrophages in vitro (Kreger et al., 1981; Tamplin et al., 1985). Indeed, encapsulated phenotypes of V. vulnificus have higher lethality towards mice than unencapsulated phenotypes (Wright et al., 1981; Yoshida et al., 1985). This is the one of the reasons why encapsulated strains are predisposed to cause sepsis in humans.

In a recent study, it was reported that extensive loss of lymphocytes via apoptosis was observed in patients who died by polymicrobial sepsis (Hotchkiss et al., 2001). In a mouse model of polymicrobial sepsis, general-caspase inhibitor and caspase-3 inhibitor prevented lymphocyte apoptosis and improved survival (Hotchkiss et al., 2000). Therefore, lymphocyte apoptosis is involved in lethality in polymicrobial sepsis. However, the relationship between lethality and lymphocyte apoptosis has yet to be clarified.

There are few reports concerning the relationship between apoptosis of host cells and growth of V. vulnificus in vivo. We reported that clinical isolates of V. vulnificus can induce apoptosis in the macrophage-like cell line J774, and in mouse peritoneal macrophages both in vitro and in vivo (Kashimoto et al., 2003), indicating that V. vulnificus might induce immunodysfunction by interacting with leukocytes. However, the effects of V. vulnificus infection on leukocytes in vivo are not fully understood. This study aimed to clarify the effect on leukocytes by using the murine model of V. vulnificus infection.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Growth conditions of V. vulnificus and Escherichia coli strains.

V. vulnificus strain K44 was isolated from the blood of a septicaemic patient at Kurashiki Central Hospital in Japan. K44 was routinely grown in Marine broth (Difco) at 37 °C for 12 h. Injected bacteria were prepared as follows. The bacterial concentration was adjusted to an OD₆₀₀ of 0.5, and then 100 µl adjusted bacterial culture was added to 5 ml fresh Marine broth. This was then grown for a further 2 h at 37 °C with agitation to obtain exponential-growth-phase bacteria. Bacteria were harvested by centrifugation at 2500 g and 4 °C for 10 min, and were suspended in PBS (pH 7.4) at a concentration of about 10⁷ c.f.u. ml^–1. The solution was adjusted to the desired bacterial concentration by measuring the OD₆₀₀. Concentrations were verified by plating serial dilutions of the samples on thiosulfate/citrate/bile salt/sucrose (TCBS) agar (Nissui Pharmaceutical) and counting the c.f.u. after incubation. E. coli DH5 was used as a control; it was prepared by the same method as above except that Luria–Bertani (LB) broth and agar were used.

Animals and treatment.

Male, 5-week-old ddY, C.B-17 +/+, C.B-17 scid/scid, C3H/HeJ and C3H/HeN mice were used in this study. Mice were housed in plastic cages in groups and were maintained on a standard laboratory diet (Rat Chow MF; Oriental Yeast) and tap water, and exposed to a 12 : 12 h light and dark cycle. The ambient temperature during the study was maintained at about 21 °C. The Kitasato University guidelines for animal treatment, which meet the principles of laboratory animal care, were followed. Injection of the bacteria into the mice was carried out as described previously (Kashimoto et al., 2003). Mice were given subcutaneous injections of 5x10⁶ V. vulnificus K44 or E. coli DH5 suspended in 0.5 ml PBS, in the right lower flank, and then killed at the indicated times by an overdose of anaesthetic. Infection of mice was verified by culture of blood obtained by heart puncture under sterile conditions. Aliquots (100 µl) of the blood were incubated for 24 h at 37 °C. The number of bacteria was determined on TCBS agar for V. vulnificus and on LB agar for E. coli.

Analysis of DNA fragmentation.

DNA fragmentation was analysed as described previously with some modification (Szondy et al., 1998). Briefly, thymus and spleen tissues from the mice were gently glass-ground to dissociate the cells, and then suspended in cold Hanks’ balanced salt solution (HBSS), which was then gently passed through a stainless steel mesh to obtain single-cell suspensions (thymocytes and splenocytes). Cells (5x10⁶) in HBSS were centrifuged, and the pellet was suspended for 10 min at 4 °C in lysis buffer (10 mM Tris/HCl, pH 7.4; 10 mM EDTA; 0.5 % Triton X-100). Samples were then centrifuged at 14 000 g for 5 min, and the supernatant containing fragmented DNA was digested with 200 µg RNase ml^–1 (Sigma Aldrich) for 1 h at 37 °C. The sample was then treated with 200 µg proteinase K ml^–1 (Merck) for 30 min at 50 °C. The supernatant was precipitated overnight using isopropyl alcohol. DNA fragments were separated on 2.0 % agarose gels, visualized with ethidium bromide, and photographed.

Histology.

Thymus and spleen were fixed in 10 % (v/v) buffered formalin (pH 7.2), dehydrated in a graded alcohol series, and embedded in paraffin wax. Three-micrometre sections were stained with haematoxylin and eosin (H&E). Terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labelling (TUNEL) staining was performed to detect apoptotic cells. Briefly, paraffin-embedded sections were dewaxed in xylene and rehydrated by passage through a graded ethanol series, ending with PBS. Sections were permeabilized using proteinase K. After washing, the 3'-OH ends of DNA fragments were stained by the method described by the manufacturer (In situ Apoptosis Detection kit POD; Roche Diagnostics). The sections were visualized with 3,3'-diaminobenzidine tetrahydrochloride solution and counterstained with methylene green.

Percentage of apoptotic lymphocytes.

The apoptotic splenocytes were detected by TUNEL staining on sections of spleen from C3H/HeJ (LPS non-responder) and C3H/HeN (LPS responder) mice 9 h after inoculation with the bacteria (n = 5). The percentages of apoptotic splenocytes were calculated from a sample of at least 300 cells in the white pulp of the spleen.

Flow cytometry.

The single-cell suspensions of thymocytes and splenocytes, prepared as described above, were washed twice in cold HBSS. After washing, 1x10⁶ cells were resuspended in 50 µl PBS with 1 % bovine serum albumin (BSA) and 0.01 % sodium azide. To detect each cell surface marker, the antibodies anti-mouse CD4-PE, anti-mouse CD8-FITC, anti-mouse CD3-FITC and anti-mouse CD45R (B220)-PE (Immunotech) were added to the cell suspension at appropriate dilutions, and incubated for 30 min at 4 °C. Cell suspensions were washed twice with PBS containing 1 % BSA and 0.01 % sodium azide, and then the numbers of stained cells were determined by counting about 10 000 cells on a fluorescence-activated cell sorter (FACS 440; Becton Dickinson).

Statistical analysis.

All results are shown as means ± SDs. For multiple comparisons of the data, we used one-way ANOVA followed by Tukey's test; P < 0.05 was considered statistically significant. Data from studies with only two groups were analysed by the Student's t-test for equal variance or Welch's t-test for unequal variance after Bartlett's test.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Depletion of lymphocytes, but not neutrophils, in V. vulnificus-infected mice

Chronic liver disease, alcoholism, haemochromatosis or other iron-overload disorders increase susceptibility to V. vulnificus infection in humans (Linkous & Oliver, 1999; Strom & Paranjpye, 2000). In particular, chronic liver diseases such as cirrhosis or viral hepatitis are associated with the highest risk of fatal outcomes for V. vulnificus infections because of the decreased synthesis of iron-binding proteins in the liver: 80 % of people suffering from chronic liver diseases die when infected with V. vulnificus (Strom & Paranjpye, 2000). In fact, it had been experimentally demonstrated that iron overload in mice enhanced susceptibility to V. vulnificus administered by intraperitoneal or intravenous injection (Stelma et al., 1992; Wright et al., 1981). We also confirmed that the 50 % lethal dose (LD₅₀) of the K44 strain used in this study by intraperitoneal challenge was decreased from 5.0x10⁷ to less than 1.0 through iron-overloading (data not shown). However, lymphocyte apoptosis was induced in iron dextran injected control mice without the injection with bacteria, indicating that the iron-treated model was not appropriate for our purpose. Therefore, we observed the effects of V. vulnificus on leukocytes in the mice model without iron overload in this study.

The number of mice in which V. vulnificus could be detected from blood samples started to increase by 3 h (2/8 mice), and reached a maximum at 6 h (7/8) after injection (Fig. 1a). The number of bacteria isolated from the blood increased from 10² to 10⁴ c.f.u. ml^–1 (data not shown) in a time-dependent manner until 9 h after injection. However, the number of leukocytes showed a tendency to decrease at 3 h, and reached a minimum value 6–9 h after injection (Fig. 1a). The number of leukocytes was still depressed at 12 h (Fig. 1a). No bacteria were detected throughout the experimental period in the mice that were injected with E. coli DH5 and served as controls (Fig. 1a). The types of depleted leukocytes in peripheral blood were determined. As shown in Fig. 1(b), the number of lymphocytes tended to decrease at 3 h, and decreased to about 25 % of the control value by 6 h post-inoculation. The numbers of lymphocytes were significantly decreased in the K44-injected mice in comparison with those of the control groups injected with E. coli strain DH5 (P < 0.01) (Fig. 1b). Despite the severe decrease in lymphocytes in the K44-injected mice, the numbers of neutrophils showed no significant differences from the control groups at any time points (Fig. 1b). It was clear that lymphocyte numbers declined in the peripheral blood of K44-injected mice; therefore, we examined the number of lymphocytes in the thymus and spleen of these mice. The numbers of both thymocytes and splenocytes were markedly decreased at 3 h after injection, reaching a minimum at 6 h (Fig. 1c). The numbers of thymocytes and splenocytes tended to recover at 12 h (Fig. 1c). It is interesting to note that the decreases in thymocytes and splenocytes were caused earlier than the loss of peripheral blood lymphocytes (Fig. 1b, c), suggesting that the loss of peripheral lymphocytes was a result of the decrease in lymphocytes in the lymphoid tissues. Thus the experimental time points at which the depletion or recovery of lymphocytes was observed corresponded with bacterial growth in vivo.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Lymphocyte depletion is associated with bacterial growth. (a) Leukocyte depletion and numbers of mice in which bacteria were detected in the blood at various time intervals (n = 8). ‘Detection of the bacteria’ indicates the number of V. vulnificus-positive mice detected from 100 µl blood (detected/total). ND, Not detected. (b) Depletion of lymphocytes in mice injected with V. vulnificus. Numbers of lymphocytes and neutrophils were determined from a total leukocyte count and differential cell count. (c) Depletion of thymocytes and splenocytes in V. vulnificus-injected mice. The thymocytes and splenocytes were isolated as described in Methods. •, K44-injected mice; , control mice injected with E. coli DH5. Data represent means ± SDs. Data were analysed by ANOVA. **Significant decrease compared with the mice before treatment (P < 0.01). Significant decrease compared with the mice injected with E. coli DH5 at the same time point (P < 0.01).

Depletion of lymphocytes was associated with apoptosis

Histological analysis of the thymus and spleen at 9 h after injection of K44 showed phagocytosis of dead cells by macrophages as evidenced by a ‘starry sky’ appearance in H&E-stained sections (Fig. 2d, f; arrowheads). Atrophy of lymphoid follicles (white pulp) in the spleen was observed in the K44-injected mice (Fig. 2b). These results suggested that the rapid depletion of lymphocytes occurred by cell death. In the thymus and spleen sections stained using the TUNEL method, the dead cells were phagocytosed by macrophages (identical to the ‘starry sky’ appearance; arrowheads) and several lymphocytes showed positive reactions (Fig. 2h, j; arrows). These changes were not observed in the control groups injected with E. coli DH5 (Fig. 2a, c, e, g and i). In a recent study, it was reported that extensive loss of lymphocytes via apoptosis was observed in patients who died by polymicrobial sepsis (Hotchkiss et al., 2001). A decrease in the area of lymphoid follicles could also be demonstrated by microscopic examination (Hotchkiss et al., 2001). In our experiment with V. vulnificus-infected mice, we detected atrophy of lymphoid follicles in the spleen (Fig. 2b). These results suggested that a similar effect on lymphocyte apoptosis occurred in our V. vulnificus-infected mouse model as well as polymicrobial sepsis in human. Hotchkiss et al. (2001) reported that general-caspase inhibitor and caspase-3 inhibitor prevented lymphocyte apoptosis and improved survival in polymicrobial sepsis in a mouse model. To investigate the role of lymphocyte apoptosis in lethality, we injected V. vulnificus into the mice which had been administered the pan-caspase inhibitor (z-VAD-fmk; 6 mg kg^–1) 2 h before injection of the bacteria. However, the survival rates and time were not significantly affected by the treatment with z-VAD-fmk compared with z-FA-fmk administered mice (data not shown). Because we cannot exclude the possibility that the various different points (bacterial species, mouse strains, challenge dose and concentration of inhibitor) between our mouse model and the model of polymicrobial sepsis have an influence on this result, we could not decide whether the prevention of lymphocyte apoptosis will become an effective therapy for V. vulnificus infection at the present time.

View larger version (142K): [in this window] [in a new window]   Fig. 2. Histology of the thymus and spleen in mice inoculated with V. vulnificus or E. coli. The sections of thymus and spleen were stained with H&E (a–f) and TUNEL (g–j). Atrophy of the white pulp appeared in the V. vulnificus-injected mice (b). Dead cells were observed in the macrophages as a ‘starry sky’ appearance in both thymus and spleen in the V. vulnificus-injected mice (d and f; arrowheads). Some lymphocytes (arrows) and dead cells phagocytosed by macrophages (arrowheads) appeared positive for TUNEL staining (h and j). Little pathological change could be observed in mice injected with E. coli DH5 (a, c, e, g and i). Bars, 100 µm (a and b) and 40 µm (c–j).

Lymphocyte apoptosis was associated with bacterial growth in vivo

To confirm that positive reactions of TUNEL staining on histological analysis were associated with lymphocyte apoptosis and that the degree of apoptosis was related to bacterial growth in vivo, we measured DNA laddering, a hallmark of apoptosis, in the thymocytes and splenocytes obtained from the V. vulnificus-injected mice at all experimental time points. As shown in Fig. 3, K44 induced DNA fragmentation in the thymus and spleen at 3, 6 and 9 h. Only slight fragmentation of DNA was detected at 12 h (Fig. 3). However, no fragmentation of DNA in the thymus or spleen of control mice was observed (Fig. 3). In addition, we showed that the number of lymphocytes in peripheral blood reached a minimum 6–9 h post-inoculation, and that the number of thymocytes and splenocytes clearly decreased by 3 h and then recovered by 12 h after injection (Fig. 1a, c). Typical DNA laddering observed at 6 and 9 h tended to disappear by 12 h in these tissues (Fig. 3). Thus the experimental time points at which the depletion or recovery of lymphocytes was observed corresponded to a distinct presence or absence of DNA fragmentation in the lymphocytes. These results indicate that lymphocyte apoptosis was associated with bacterial growth in vivo.

View larger version (71K): [in this window] [in a new window]   Fig. 3. V. vulnificus infection induced DNA fragmentation in thymocytes and splenocytes. Low-molecular-mass DNA was extracted from the cells of mice inoculated with V. vulnificus or E. coli, and electrophoresed on 2.0 % agarose gels, as described in Methods. Fragmentation of DNA was detected in both thymocytes and splenocytes of V. vulnificus-injected mice as early as 3 h post-inoculation (lower panels). However, only slight fragmentation could be detected at 12 h post-inoculation. No such changes were seen in mice injected with E. coli DH5. One representative experiment of three replicates is shown. M, 100 bp DNA ladder marker.

Phenotypic characterization of lymphocyte subsets following V. vulnificus infection

We determined the subsets of thymocytes and splenocytes that were affected by apoptosis. The cell surface phenotypes of lymphocytes were detected using flow cytometry at 6 h after inoculation of bacteria. In both the thymocytes and splenocytes, there were no differences in the percentages of analysed subsets of the K44-injected group compared with the group injected with E. coli DH5 (Fig. 4). We calculated the absolute numbers of all analysed subsets of lymphocytes from the total numbers of lymphocytes (Fig. 1b) and the percentage of each subset (Fig. 4a). The absolute numbers of all subsets in the K44-injected group were decreased in comparison with the DH5-injected group (Fig. 4b).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Subtyping and absolute numbers of lymphocytes. Thymus and spleen were dissociated from V. vulnificus- or E. coli-injected mice at 6 h post-inoculation. Thymocytes and splenocytes were isolated as described in Methods. There were no changes in lymphocyte subsets in mice injected with either V. vulnificus or E. coli DH5 (a). The absolute numbers of thymocytes and splenocytes were calculated from the total numbers of lymphocytes and the percentage of each subset (b). Open bars, non-injected mice; filled bars, mice injected with E. coli DH5; hatched bars, K44-injected mice. **Significant decrease compared with the mice injected with E. coli DH5 (P < 0.01). 4+, CD4+ T-subset; 8+, CD8+ T-subset; DP, CD4+ CD8+ lymphocytes; DN, CD4– CD8– lymphocytes; 3+, CD3+ T cells; B220+, CD45R (B220)+ B cells.

Participation of LPS in lymphocyte apoptosis

It is well known that LPS, a cell-wall component of Gram-negative bacteria, is an important mediator of the inflammatory response and is an important factor of lymphocyte apoptosis in infection with Gram-negative bacteria. LPS activates the cells through Toll-like receptors (TLRs). Among TLR family members, both TLR2 and TLR4 have been shown to recognize LPSs (Erridge et al., 2004). C3H/HeJ mice have selectively impeded LPS signal transduction, caused by the mutation in the third exon of the TLR4 gene (Poltorak et al., 1998). However, C3H/HeN mice maintain normal LPS signal transduction by TLR4. To investigate the role of TLR4 in lymphocyte apoptosis in our experimental model, we compared the number of lymphocytes and percentage of apoptotic splenocytes between C3H/HeJ and C3H/HeN mice 9 h after inoculation with V. vulnificus. As shown in Fig. 5, there was no significant difference in the number of lymphocytes or percentage of apoptotic lymphocytes between these mice groups, indicating that TLR4 does not participate in V. vulnificus-induced lymphocyte apoptosis. Recent studies have demonstrated that TLR2 is largely required for signalling by LPSs from many Gram-negative bacteria (Erridge et al., 2004). Therefore, it is possible that the lymphocyte apoptosis in our model was induced through TLR2 signalling. The other possibility involves the cytolysin of V. vulnificus, which is one of the major secreted toxins and induces apoptosis in various types of cells in vitro (Fan et al., 2001; Kim et al., 1998; Kwon et al., 2001). Since the effect of cytolysin on lymphocytes has not been clearly demonstrated in vivo at the present time, further investigation is necessary to determine the contribution of this toxin to lymphocyte apoptosis in our mouse model.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Influence of V. vulnificus infection on the number of leukocytes and percentage of apoptotic splenocytes in C3H/HeJ mice and C3H/HeN mice (n = 5). The numbers of total leukocytes in these mice before inoculation were compared with the values at 9 h post-inoculation of K44 strain (a). The apoptotic splenocytes were detected by TUNEL staining of a section of the spleen, and the percentages of apoptotic splenocytes were calculated from at least 300 cells in the white pulp of the spleen (b). Data represent means ± SDs. Data were analysed using Student's t-test. **Significant decrease compared with the mice before the treatment (P < 0.01). Significant increase compared with the mice injected with E. coli DH5 (P < 0.01).

Effect on the number of neutrophils in V. vulnificus-injected severe combined immunodeficient (SCID) mice

The total number of leukocytes reached a minimum 6–9 h after injection of K44 into ddY mice (Fig. 1a). However, the number of neutrophils in these mice was not decreased by V. vulnificus injection (Fig. 1b). To gain further clarity on the question of whether V. vulnificus induces neutrophil apoptosis in vivo, we injected the K44 strain into the SCID mouse, which has a high percentage of neutrophils (about 70 %) and very few lymphocytes in peripheral blood. The total number of leukocytes and neutrophils in the K44-injected SCID mice increased more than threefold compared with the pre- injection value (Fig. 6). However, in the K44-injected control mice (+/+), the total number of leukocytes decreased to 70 % of pre-injection numbers within 6 h (Fig. 6). These results indicated that growth of V. vulnificus did not clearly induce apoptosis in neutrophils in vivo. From the results shown in Figs 1(c) and 6, we concluded that V. vulnificus appeared to induce apoptosis specifically on lymphocytes in peripheral blood in vivo, which led to a loss in overall leukocyte numbers in the peripheral blood, and the lymphocyte apoptosis must be closely related to the growth of V. vulnificus in vivo.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Influence of V. vulnificus infection on the numbers of neutrophils in control and SCID mice (n = 5). Control strain, C.B17 +/+; SCID strain, C.B17 scid/scid. Data represent means ± SDs. Data were analysed using Student's t-test. *,**Significant difference compared with the mice before treatment (P < 0.05 or 0.01, respectively).

In conclusion, we report here that V. vulnificus induces lymphocyte, but not neutrophil, depletion in association with apoptosis in vivo.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study was supported by Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the School of Veterinary Medicine and Animal Sciences, Kitasato University.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES