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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 8
| Issue : 2 | Page : 115-122 |
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Genotyping of Blastocystis hominis symptomatic isolates and kinetics of associated local CD3 and CD20 cell infiltrate
Nora I Abu El-Fetouh1, Eman S Abdelmegeed2, Raifa A Attia1, Ibrahim El-Dosoky3, Manar S Azab1
1 Department of Medical Parasitology, Mansoura University, Mansoura, Egypt
2 Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
| Date of Submission | 12-May-2015 |
| Date of Acceptance | 10-Aug-2015 |
| Date of Web Publication | 27-Jan-2016 |
Correspondence Address:
Manar S Azab
PhD, Department of Medical Parasitology, Faculty of Medicine, Mansoura University, 2 El-Gomhouria Street, Mansoura 35516
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7942.175009
Background
Pathogenicity of the protozoan parasite Blastocystis hominis is a subject of debate. It has been suggested that the pathogenic outcome may be linked to specific subtypes of Blastocystis spp. Studies on experimental infection in animals have reported varying degrees of illness depending on the used genotype.
Objective
This study was designed to identify a possible link between Blastocystis genotypes and gastrointestinal illness. In addition, the CD3 and CD20 local immune response to infection using symptomatic isolates was experimentally evaluated.
Patients and methods
Blastocystis- infected symptomatic (n = 51) and asymptomatic (n = 19) patients and irritable bowel syndrome patients (n = 32) were enrolled in the study, after exclusion of individuals who had other possible fecal pathogens. Restriction fragment length polymorphism was used for genotyping of isolates. Isolates from symptomatic cases were used for experimental infection, and immunohistochemical characterization of local CD3 and CD20 response was evaluated at two time intervals (groups A and B) after infection.
Results
Genotype 3 was the most common, being detected in 55.9% of all studied participants, and genotype 4 was the least common (9.8%). Symptomatic cases constituted 90% of genotype 1, 45.6% of genotype 3, 40% of genotype 4, and 20% of genotype 2. Genotype 2 was detected in 14.7% of all studied patients, with asymptomatic patients accounting for 60% of this isolate. Twenty-four isolates of genotype 3 occurred in 42.1% of irritable bowel syndrome patients. Rats euthanized after 7 (group A) and 14 days (group B) had higher CD3 and CD20 mean cell counts compared with control rats. The mean cell count of CD3 cells was statistically significantly higher in group A compared with group B, whereas CD20 cells in group B showed statistically significantly higher mean count compared with group A.
Conclusion
We suggest that both host and pathogen factors cooperate to express the pathogenic behavior of the parasite. Keywords: Blastocystis hominis , CD20, CD3, experimental studies, genotyping, irritable bowel syndrome, immunohistochemistry, pathogenicity
How to cite this article:
Abu El-Fetouh NI, Abdelmegeed ES, Attia RA, El-Dosoky I, Azab MS. Genotyping of Blastocystis hominis symptomatic isolates and kinetics of associated local CD3 and CD20 cell infiltrate. Parasitol United J 2015;8:115-22 |
How to cite this URL:
Abu El-Fetouh NI, Abdelmegeed ES, Attia RA, El-Dosoky I, Azab MS. Genotyping of Blastocystis hominis symptomatic isolates and kinetics of associated local CD3 and CD20 cell infiltrate. Parasitol United J [serial online] 2015 [cited 2023 Nov 29];8:115-22. Available from: http://www.new.puj.eg.net/text.asp?2015/8/2/115/175009 |
| Introduction | |  |
Blastocystis spp. parasites are prevalent worldwide and are identified as the most common eukaryotic organisms in human fecal samples, with a marked increase in prevalence in recent years. Various epidemiological surveys recorded Blastocystis hominis prevalence in up to 10% of the population in developed countries and in as much as 50-60% in developing countries [1] . Poor hygiene practices and consumption of contaminated food or water are responsible for the differences in prevalence, as the feco-oral route is considered the main mode of transmission of this parasite [2] .
B. hominis is a protozoan parasite with many genotypes in nature [3] . Such complexity associated with diverse pathogenicity was explained as being due to the presence of pathogenic and nonpathogenic genotypes [4] . Tan [1] reported that, in the absence of any enteric pathogen, Blastocystis spp. was detected without significant differences between asymptomatic and symptomatic groups. Nevertheless, other reports [5],[6] noted that Blastocystis spp. may play a significant role in several chronic gastrointestinal illnesses such as irritable bowel syndrome (IBS). It is worth noting that, although B. hominis pathogenicity is debatable, stool samples should be screened for it, and if no other pathogens are detected symptomatic patients harboring B. hominis should be treated accordingly [3] .
The study of host immunity to Blastocystis spp. is undercited in the literature and offers many prospects for future study. It has been suggested that 'the protozoan does not attack the host, but rather that the host attacks the protozoan and that the disease is a result of the protozoan's defense mechanisms' [7] . The major resident cell component of the gut mucosal epithelium are intraepithelial lymphocytes, which interact with epithelial cells to maintain normal homeostasis [8] . The principal functions of B cells are to make antibodies in response to antigens, to act as antigen-presenting cells, and to develop into memory B cells after antigenic activation. B cells also release cytokines for signaling immune regulatory functions [9] . T lymphocytes in turn play a central role in cell-mediated immunity. Mucosal-associated invariant T cells are a special type of cells that appear to play a regulatory role in immunity. They are dependent on gut microbiota, being absent in germ-free mice [10] .
On the basis of these data, the present study aims to explore the genotyping of Blastocystis spp. in patients with different clinical presentations. Another objective is to find out the pattern of expression of CD3 and CD20 cells as markers of T [11] and B [12] lymphocytes, respectively, in the intestinal mucosa of rats orally infected with isolates from symptomatic patients. To understand the underlying local immune response toward the parasite, CD3 and CD20 cell expression was studied at two time intervals following infection, arbitrarily chosen to represent the sequence of immunocellular reaction.
| Patients and methods | | |
Type of study
This is a descriptive analytical and experimental study.
Participants
Patients with B. hominis identified as the only possible pathogen in stool were included in this work. They were selected from outpatient clinics of Mansoura University Hospitals during the period from June 2011 to January 2013, and ranged in age from 4 to 50 years. All patients or their guardians were acquainted with the study details and agreed to participate in it. The 102 cases were classified as symptomatic patients (n = 51) suffering from chronic nonwatery diarrhea and other symptoms related to gastrointestinal disorders, such as abdominal pain and distension; asymptomatic patients (n = 19) had manifestations of other system involvements such as fever, cough, arthritis, hypertension, etc.; IBS patients (n = 32) had gastrointestinal disorders identified on the basis of clinical data, according to Rome III diagnostic criteria [13] .
Stool samples
Bacterial enteric pathogens were excluded by standard culture methods. Saline and iodine wet mount preparation as well as formalin ethyl acetate concentration [14] were performed to exclude the presence of other parasites. Modified acid-fast stain was used to exclude cryptosporidiosis. Viral enteric pathogens were excluded by nonwatery nature of the stool samples. The annotated samples were kept frozen at −20°C until used for genotyping.
In vitro stool culture
To detect B. hominis, in vitro cultivation was performed for each stool sample by inoculating 50 mg of fresh stool in Jones medium [15] without starch [16] , under anaerobic conditions (BD GasPak EZ Gas Generating Container Systems, Becton Dickinson, Sparks, USA). The cultures were incubated at 37°C and examination of 3-4-day-old cultures was done under ×10 and ×40 magnifications. On observing the typical vacuolar or granular forms of Blastocystis organisms, they were subcultured in a new medium for several subcultures before being used for animal inoculation.
Genomic DNA extraction
Extraction of genomic DNA of B. hominis was performed using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.
Genomic DNA amplification
Following the method of Yoshikawa et al. [17] , the small subunit (SSU) rDNAs were amplified by using the forward primer SR1F: 5Ͳ-GCTTATCTGGTTGATCCTGCC AGTAGT-3Ͳ and the reverse primer SR1R: 5Ͳ-(TGATCCTTCCGCAGGTTCACCTA-3Ͳ, which were designed to prime the conserved region of the SSU rRNA sequence obtained from three B. hominis strains.
These primer pairs produced ~1780-bp product. The PCR amplification was performed with 35 cycles of 94°C for 40 s, 57°C for 60 s, and 72°C for 2 min, after an initial denaturation at 94°C for 3 min.
Restriction fragment length polymorphism of small subunit rDNA
The purified DNA was digested with HinfI or RsaI [18] in a reaction mixture of 2 μl of 10× FastDigest buffer, 1 μl of FastDigest enzyme, 10 μl of DNA solution, and 17 μl of nuclease-free water to a final volume of 30 μl at 37°C in a heat block for 5 min. The reaction was stopped by heating for 20 min at 65°C for enzyme inactivation. The digested products were electrophoresed with a 100-bp ladder (New England BioLabs Inc., Ipswich, UK) in ethidium bromide-stained 2% agarose gels and Tris-borate EDTA buffer. Gels were placed on the UV transilluminator and examined for digest product.
Animals and infection
We used Wistar male rats as a model for Blastocystis spp. infection in an attempt to explore the pathological effect at different stages of infection. Thirty immune-competent 3-week-old Wistar male rats were obtained from the experimental animal house of Theodor-Bilharz Research Institute. Stool samples of rats were subjected to direct parasitological examination and culture to confirm the Blastocystis spp.-free status. The animals were separately maintained in polycarbonate cages at 25°C, with a relative humidity of 40-60%, under a 12 h light/dark cycle and were fed a normal diet of commercial pellets and given potable water. The procedures employed in the present experiments complied with the current ethics guidelines set out by our university.
The organisms from 4-day-old cultures of symptomatic isolates, irrespective of their genotype, were pooled after being purified using the subtraction method by centrifuging samples more than five times [19] . Numbers of B. hominis cells were adjusted using a hemocytometer; 20 rats were inoculated with 1 × 10 5 cells [20] by oral gavage, and 10 were kept at the same nutritional and environmental conditions as the control group. Animals were screened for Blastocystis spp. infection by daily stool analysis using direct wet mount and culture. When the typical vacuolar or granular forms of Blastocystis spp. were detected by light microscopy, infection was judged to be positive.
Histopathology
Infected rats were divided into two groups, comprising 10 rats each. Group A rats were euthanized 7 days after infection, whereas group B rats were euthanized after 14 days. Uninfected control rats were euthanized on the same day as the corresponding infected animal groups (five rats at a time). Tissue samples of colon were fixed in 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined under ×40 magnification.
Immunohistochemistry
The procedure was performed on two separate sections from each group of rats using either CD3 mouse monoclonal antibody (dilution 1/50, code M7254, clone F7.2.38) or CD20 mouse monoclonal antibody (dilution 1/200, code M0755, clone L26), followed by incubation with the labeled polymer, using two sequential 30-min incubations (EnVision+System-HRP, code K4006; Dako Denmark A/S). The final reaction was obtained by 10 min incubation with 3,3Ͳ-diaminobenzidine+substrate chromogen, which results in a brown-colored precipitate at the antigen site according to the manufacturer's instructions (Dako Denmark A/S). Slides were digitized using an Olympus digital camera installed on an Olympus microscope with a 1/2× photo adaptor, using ×100 objective. The resultant images were analyzed on an Intel Core I3-based computer using Video Test Morphology software (Russian Federation, Saint-Petersburg, Russia) with a specific built-in automated object counting routine for immunohistostaining analysis and stain density.
Statistical analysis
Statistical analysis was performed using statistical package for the social sciences (SPSS, version 17.0; SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed as mean ± SD, whereas qualitative data were expressed as number and percentage. For parametric data, comparisons were made with analysis of variance followed by Tukey's test. For comparison between frequencies, Pearson's χ2 -test was used. A P value of less than 0.05 was considered statistically significant.
| Results | | |
The study included 102 B. hominis-infected cases. The highest frequency of infection was among those below 18 years (48%), followed by those older than 40 (39.2%). The least was among patients aged 18-40 years (12.7%).
Genotypes of B. hominis
The genotyping of B. hominis in 102 stool samples was interpreted according to the banding pattern based on the difference of the restriction fragment length polymorphism profiles of SSU rDNA when digested with HinfI and RsaI restriction enzymes ([Figure 1] and [Figure 2]). This revealed four B. hominis genotypes ([Table 1]). Genotype 3 was the most common, being detected in 55.9% (57/102) of all studied patients, followed by genotype 1 (19.6%, 20/102) and genotype 2 (14.7%, 15/102); the least was genotype 4 (9.8%, 10/102). Ninety percent of genotype 1, 45.6% of genotype 3, 40% of genotype 4, and 20% of genotype 2 were symptomatic. Statistically significant differences were found between genotype 1 and other genotypes in the symptomatic group (P < 0.05 for each comparison). Sixty percent of patients with genotype 2 were asymptomatic, with statistically significant differences compared with genotype 1 and genotype 3 in the asymptomatic group (P < 0.05 for each comparison). In the IBS group, a statistically significant difference (P = 0.04) was noted between genotype 3 (24 isolates) and genotype 1 (only two isolates). | Figure 1: Restriction enzyme profi le of B. hominis small subunit rDNA digested with HinfI. M is the ladder DNA at 100 bp. Lanes 2, 3, and 7 are genotype 3 and lanes 4 and 1 0 are genotype 4.
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 | Figure 2: Restriction enzyme profi les of B. hominis small subunit rDNA digested with HinfI. M is the ladder DNA at 100 bp. Lanes 3 and 10 are genotype 1 and lanes 2, 4, and 7 are genotype 3.
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 | Table 1: Frequency of different genotypes of B. hominis in the study groups (total = 102)
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Histopathology
All infected rats showed positive infection 3-4 days after oral inoculation of cultured parasites. Colonic mucosa of rats euthanized 7 days after infection showed focal atrophy of mucosa with scarce inflammatory cells, including aggregates of eosinophils ([Figure 3]a1), and the parasite was seen at the luminal aspect of the colon ([Figure 3]a2). In comparison, the pathological changes 14 days after infection showed dense inflammatory infiltrate ([Figure 3]b1) including eosinophils in a diffuse and aggregate manner with focal atrophy of mucosa. In addition, intraepithelial lymphocytes and mucosal sloughing appeared in two sections ([Figure 3]b2). | Figure 3: Colonic sections of B. hominis-infected rats euthanized at 7 days after infection showing infl ammatory cells, mainly eosinophils (a1; black arrows), parasite inside the intestinal crypts (a2; black arrow); and euthanized at 14 days after infection showing mixed infl ammatory cells (b1; black arrows; a, eosinophils; b, lymphocytes; c, macrophages) and mucosal sloughing (b2). Hematoxylin– e osin stain, ×400.
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Immunohistochemistry
CD3 ([Figure 4]a) and CD20 ([Figure 4]b) were chosen as markers of T and B lymphocytes, respectively. [Table 2] illustrates that both rat groups, A and B, showed a statistically significant increase in the mean count of CD3 and CD20 cells compared with controls (P < 0.05 for each comparison). The mean count of CD3 cells was statistically significantly higher in group A (30 ± 3.4) compared with group B (20 ± 2.3) (P<0.05), whereas CD20 cells in group B recorded statistically significantly higher mean count (30 ± 4.1) compared with group A (20 ± 3.1) (P < 0.05). | Figure 4: Immunohistochemical staining of (a) CD3 T lymphocytes and (b) CD20 B lymphocytes in colonic sections of euthanized B. hominis-infected rats (bl ack arrows). ×400.
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| Discussion | | |
Recent molecular studies of Blastocystis spp. have directed much attention toward elucidating which particular genotype of the organism may be the potentially pathogenic one [7],[21],[22] . In our study, the genotyping of B. hominis in stools of 51 symptomatic, 32 IBS, and 19 asymptomatic patients, based on the difference of the restriction fragment length polymorphism profiles of SSU rDNA when digested with HinfI and RsaI restriction enzymes, identified only four B. hominis genotypes ([Table 1]). According to the banding pattern, genotype 3 was the most common, having been detected in 55.9% of all studied patients, followed by genotype 1 (19.6%) and genotype 2 (14.7%); the least was genotype 4 (9.8%). This is in agreement with a study conducted by Souppart et al. [21] who reported that genotype 3 was the most common (61.90%) in a sample of symptomatic Egyptian patients, followed by genotype 1 (19.05%) and genotype 2 (19.05%). Interestingly, Abaza et al. [23] supported the predominance of genotype 3 (56.1%) in Egyptian patients followed by genotype 1 (35.1%) and genotype 2 (3.5%) and recorded mixed infection with genotypes 1 and 3 in 5.3% of patients. In a Turkish study [7] , genotype 3 similarly predominated and genotype 2 came second. It is worth mentioning that genotype 3 was the only genotype identified in a recently conducted research among both symptomatic and asymptomatic Egyptian patients [22] .
The variability of symptoms in Blastocystis spp.-infected patients could be attributed to the presence of different genotypes with different pathogenic potential. Understanding the underlying mechanism(s) is important for deciphering the parasite pathobiology. We recorded statistically significant relations between genotypes and the presence/absence of symptoms ([Table 1]). Ninety percent of genotype 1, 45.6% of genotype 3, 40% of genotype 4, and 20% of genotype 2 were symptomatic. Genotype 1 was not detected in the asymptomatic infected group. Statistically significant differences were found between genotype 1 and other genotypes in the symptomatic group. This coincides with another study by Hussein et al. [24] , which verified that genotype 1 was clinically and statistically related to the pathogenicity of B. hominis, whereas genotype 2 was irrelevant. This also agrees with our results. In that same study it was suggested that genotypes 3 and 4 include pathogenic and nonpathogenic strains.
Study of isolates from China revealed an association between genotype 1 and disease, whereas genotype 3 was isolated predominantly from asymptomatic individuals [25] . However, we detected genotype 3 in 51% of symptomatic patients and in 75% of IBS patients, with statistically significant difference compared with genotype 1. These findings are in accordance with several reports on genotype 3 predominance in patients with chronic gastrointestinal illness in Singapore [26] , USA [5] , and Malaysia [27] . In Egypt [28] , the most prevalent genotype in IBS patients was genotype 3 (49%), followed by genotype 1 (26%) and genotype 4 (24%). In a study conducted by Alfellani et al. [29] , there was a higher record of genotype 4 in samples from IBS clinics in the UK, although it did not reach statistical significance. In contrast, in a study from Pakistan [30] , genotype 1 was the most common in IBS samples. Thus, it is clear that no consistent pattern exists between IBS-linked samples and the genotype of Blastocystis spp. detected, as the observed distribution is very different in each country [29] . Clark et al. [31] reported that differences in methodology (genotyping vs. sequencing) as well as many other variables, not the least of which is geography, are responsible for such differences, and indicated that the potential relationship needs further investigation, concluding that no single genotype is found in IBS patients.
Several studies [32],[33],[34],[35] have concluded no definite correlation between Blastocystis spp. exact genotypes and their pathogenic potential, where genetically highly polymorphic populations of human Blastocystis spp. may differ in their ability to produce human disease [36] . Recently, Abaza et al. [23] found no significant difference in the distribution of genotypes 1 or 3 among the study groups. However, genotype 3 was the most dominant in the IBS group (68.4%), followed by genotype 1 (21.1%).
The parasite despite being noninvasive is capable of causing pathology. Inflammatory aggregates of eosinophils and focal atrophy of colonic mucosa were seen in colonic sections of rats euthanized 7 days after infection ([Figure 3]a1) and the parasite was found at the luminal aspect of the colon ([Figure 3]a2). The pathological changes were more intense 14 days after infection ([Figure 3]b1) and mucosal sloughing appeared in two sections ([Figure 3]b2). This is in concordance with a study in which a murine model of B. hominis infection showed dense inflammatory cell infiltration, edematous lamina propria, and mucosal sloughing of the cecum and colon [37] . An Egyptian study described mild inflammatory reaction in colonic and cecal mucosa of rats in response to infection with isolates from IBS and asymptomatic patients, compared with severe inflammatory reaction in sections from rats infected with isolates from acute symptomatic patients. The parasite vacuolar forms were seen at the edge and invading the mucosa [23] . Nevertheless, some studies reported no pathological lesions associating B. hominis infection in humans [38] or experimentally in rats [39] apart from an increase in the number of goblet cells and in neutral-mucin, but not in acid-mucin-containing goblet cells. This controversy in pathogenicity may result from the fact that various subtypes modulate host responses differently, and also because of the existence of virulent and avirulent strains of B. hominis that proved to be antigenically and genetically heterogeneous [40] .
To our knowledge, this is the first trial to explore the pattern of host immune response toward B. hominis infection immunohistochemically using CD3 ([Figure 4]a) and CD20 ([Figure 4]b) as markers of T [11] and B [12] lymphocytes, respectively. Groups A and B showed a statistically significant increase in the mean count of CD3 and CD20 cells compared with controls. The mean count of CD3 cells was statistically significantly higher in group A compared with group B, whereas CD20 cells in group B showed a statistically significantly higher mean count compared with group A ([Table 2]). As reported with other intestinal pathogens, we noted that actual invasion of the intestinal epithelium by Blastocystis spp. is not necessary for the induction of an inflammatory response [41] . Although little is known about cytokine responses against noninvasive microorganisms in the colon, it was hypothesized that Blastocystis spp.-derived molecules (or antigens) could be absorbed through the paracellular or transcellular pathway of the epithelium and stimulate intraepithelial or mucosal immune cells [39] . In addition, parasite secretory components, such as cysteine proteases, may exert a variety of detrimental effects on host cells, resulting in cytopathic effects, barrier compromise [42] , and the production of proinflammatory cytokines [1] . Previous studies showed that Blastocystis strains NandII and WR1 stimulated IL-8 release from a human colonic epithelial cell line in vitro, mediated possibly by the organism-derived cysteine protease [43],[44] . Iguchi et al. [39] reported upregulation of the gene transcription of IFN-g, IL-12, and TNF-α in cecal mucosa of rats experimentally infected with the Blastocystis strain RN94-9, and suggested that the colonization of Blastocystis parasites in the cecum provokes the activation (or influx) of T cells, monocytes/macrophages, and/or natural killer cells in local tissues.
Predominance of CD3 cells in group A rats might address the crucial regulation within the mucosal immune system, and several T-cell subsets appear to serve this purpose [45] . The observed higher CD20 mean cell count in group B rats is possibly due to the shift of immune response toward antibody secretion. This agrees with the described kinetics of antibody production in intestinal secretions against B. hominis [46] , in which three isotypes, IgM, IgG, and IgA, showed an increasing trend from week 1 after immunization. Local antibodies at the mucosal surfaces play a central role in the primary defense against pathogens by preventing the binding of the microbes and their produced toxins to the epithelium [47] . Secretory IgA is a central component of noninflammatory gut defenses that mediate quiescent host-microbe relationships. It was proposed that secretory IgA hinders contact of pathogens with mucosal surfaces by facilitating entrapment in mucus followed by peristaltic or ciliary clearance. Other proposed mechanisms are steric hindrance of the microbial epitopes that mediate epithelial attachment, interception of incoming pathogens within epithelial cell vesicular compartments, or mediation of pathogen export back into the lumen [48] . IgG also appeared to be involved in secretory immunity being produced locally from plasma cells in the lamina propria [47] , or from circulating molecules that enter the intestinal tissue in response to immunogen detection [49] .
In conclusion, this work adds to previous research noting a higher genotype-symptom relationship, with genotype 1 and genotype 3 of the Blastocystis parasite possibly linked to potential pathogenicity. To our knowledge, this is the first study that highlights the kinetics of local host CD3 and CD20 responses at two time intervals following infection with symptomatic isolates. Comparative studies are needed to validate our results as animal models can help in understanding the pathobiology and role of Blastocystis spp. in chronic unexplained diarrhea and gut dysfunction.
Acknowledgements
The authors highly acknowledge the valuable help and discussion of the molecular work by Dr Hossam Zaglool. They also thank Dr Sameh Abdel-Ghany for statistical analysis of data and Dr Shady Shariff for image analysis of the immunohistochemical analysis.
Author Contribution: NI Abu El-Fetouh performed the experiment; ES Abdel Megeed assessed samples bacteriologically; RA Attia, I. El-Dosoky, and MS Azab designed the research and supervised the practical work; MS Azab interpreted the results, and wrote and revised the manuscript for submission.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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