*Article* **First-degree Relatives of Celiac Disease Patients Have Increased Seroreactivity to Serum Microbial Markers**

**Liisa Viitasalo 1,2, Sari Iltanen 1,3, Heini Huhtala 4, Päivi Saavalainen 5, Katri Kaukinen 6,7, Katri Lindfors 6 and Kalle Kurppa 1,8,\***

Received: 23 March 2020; Accepted: 8 April 2020; Published: 13 April 2020

**Abstract:** Risk of celiac disease (CD) is increased in relatives of CD patients due to genetic and possible environmental factors. We recently reported increased seropositivity to anti-*Saccharomyces cerevisiae* (ASCA), *Pseudomonas fluorescens*-associated sequence (anti-I2) and *Bacteroides caccae* TonB-linked outer membrane protein (anti-OmpW) antibodies in CD. We hypothesized these markers also to be overrepresented in relatives. Seropositivity and levels of ASCA, anti-I2 and anti-OmpW were compared between 463 first-degree relatives, 58 untreated and 55 treated CD patients, and 80 controls. CD-associated human leukocyte antigen (HLA)-haplotypes and transglutaminase (tTGab) and endomysium (EmA) antibodies were determined. One or more of the microbial antibodies was present in 75% of relatives, 97% of untreated and 87% of treated CD patients and 44% of the controls. The relatives had higher median ASCA IgA (9.13 vs. 4.50 U/mL, *p* < 0.001), ASCA IgG (8.91 vs. 5.75 U/mL, *p* < 0.001) and anti-I2 (absorbance 0.74 vs. 0.32, *p* < 0.001) levels than controls. There was a weak, positive correlation between tTGab and ASCA (r = 0.31, *p* < 0.001). Seropositivity was not significantly associated with HLA. To conclude, seropositivity to microbial markers was more common and ASCA and anti-I2 levels higher in relatives of CD patients than controls. These findings were not associated with HLA, suggesting the role of other genetic and environmental factors.

**Keywords:** celiac disease; relatives; microbiota; *Saccharomyces cerevisiae*; *Pseudomonas fluorescens*; *Bacteroides caccae*

#### **1. Introduction**

Celiac disease (CD) is an immune-mediated condition characterized by gluten-induced small-bowel enteropathy. Almost all patients carry human leukocyte antigen (HLA) alleles encoding DQ2 or DQ8 heterodimers [1]. These alleles are nevertheless also present in up to 35% of the general population and do not fully explain the genetic risk [2]. Recent genome-wide association studies and immunogenetic studies have identified numerous non-HLA loci and single nucleotide polymorphisms that may modify CD risk [3,4]. Partly due to shared genetic predisposition, the relatives of patients have an increased susceptibility to CD, the average prevalence among first-degree relatives being approximately 8% [5] compared with 1%–2% in the general population [6,7].

However, only a minority of at-risk individuals develop CD, and the concordance even varies between identical twins [8,9], which implicates environmental factors. The prevalence may also vary between adjacent countries with similar genetic backgrounds and gluten consumption [10], and retrospective measurements of stored samples indicate a rise in the true incidence [6,11,12]. As one potentially associated factor, the role of intestinal microbiota in the development of CD has aroused particular interest [13–15]. Previously, we and others observed elevated levels of antibodies to microbial markers *Saccharomyces cerevisiae* (ASCA), *Pseudomonas fluorescens*-associated sequence (anti-I2) and *Bacteroides caccae* TonB-linked outer membrane protein (anti-OmpW) in inflammatory bowel disease [16–18]. We have shown increased seroreactivity to these markers also in overt CD [19] and a decrease of the antibody levels during gluten-free diet (GFD) [20]. Further, these microbial markers are detectable in early stages of the disease even before the presence of villous atrophy and serum CD-specific autoantibodies [21].

We hypothesized that close relatives of CD patients, with partially shared living environments and genetic factors, could have increased seroreactivity to microbial markers. This was investigated by comparing their frequency of seropositivity and levels of microbial antibodies with those in untreated and treated CD patients and in healthy controls.

#### **2. Materials and Methods**

#### *2.1. Study Participants*

The study was carried out at Tampere University and Tampere University Hospital. Previously diagnosed CD patients were recruited in a nationwide search through newspaper advertisements and via patient societies. Their medical records were obtained with permission, and only subjects with a biopsy-proven diagnosis were included. Relatives of these patients were invited to a screening study comprising personal interviews and measurement of CD serology. Additional blood samples were drawn for research purposes. Exclusion criteria for the relatives were previously diagnosed CD or dermatitis herpetiformis, or otherwise initiated gluten-free diet (GFD). Altogether, 3031 relatives met the inclusion criteria and entered the original screening study. Duodenal biopsy was offered for all relatives with positive CD serology. For the present study, serum samples from 463 first-degree relatives were randomly selected for the measurement of ASCA, anti-I2 and anti-OmpW. The CD control group comprised 58 biopsy-proven patients who underwent measurements of the CD serology and microbial markers at diagnosis and after one year on GFD (*n* = 55). In addition, 80 adult blood donors with negative CD serology served as non-CD controls.

#### *2.2. CD Autoantibodies and Genotyping*

Serum immunoglobulin A (IgA) class endomysium autoantibodies (EmA) were tested by an indirect immunofluorescence method using human umbilical cord as substrate [22]. Titers 1: ≥ 5 were deemed positive and diluted up to 1:4000 or until negative. Serum IgA class tissue transglutaminase autoantibodies (tTGab) were measured by an enzyme-linked immunosorbent assay (ELISA, INOVA diagnostics, San Diego, CA) according to the manufacturer's instructions. A cutoff ≥ 30 U/mL was applied for seropositivity. Some of the CD autoantibody-positive relatives declined the biopsy, but, due to the high specificity of EmA/tTGab [23], the vast majority of them are also likely to have CD. They were therefore analyzed as a separate group.

The CD-associated HLA DQ haplotypes (DQ2.5, DQ2.2, DQ8) were determined from the relatives and CD patients with the tagging single nucleotide polymorphism method or with the Olerup SSP DQ low-resolution kit (Olerup SSP AB, Stockholm, Sweden) as described elsewhere [24,25].

#### *2.3. Microbial Antibodies*

Serum IgA and IgG class ASCA were measured by a commercial ELISA (Quanta Lite ASCA, INOVA Diagnostics Inc., San Diego, CA) considering levels ≥ 25 U/mL positive. *E. coli* XL-1 blue and *E. coli* BL-21 (Stratagene, La Jolla, CA) strains and previously reported antigen purification techniques [26,27] were used to produce I2-GST and OmpW antigens. The serum samples were diluted 1:50, and IgA anti-I2 and anti-OmpW antibodies were measured with an in-house ELISA. For anti-I2, the cutoff level for positivity was set at absorbance 0.5. For anti-OmpW, it was set at 0.6 in children and 1.0 in adults based on our previous studies showing age differences in the normal range [16,19].

#### *2.4. Statistical Analysis*

Quantitative data are shown in tables as percentages or as medians with lower and upper quartiles. The data were cross-tabulated in order to ascertain the overlap of seropositivity for microbial antibodies in different study groups. The Kruskal–Wallis test was used to compare the differences in microbial antibody levels between the groups. Correlations between autoantibodies and microbial markers were tested with Spearman's rank correlation coefficient. Associations in the seropositivity to microbial antibodies within and between the families were also tested. The chi-square statistic for the change in the -2 log-likelihood from the constant only model to the model with "family" was used to determine whether the inclusion of "family" contributed significantly to model fit. A *p* value < 0.05 was considered significant. Statistical analyses were carried out with SPSS Statistics for Windows (IBM Corp., Armonk, NY, USA).

#### *2.5. Ethical Aspects*

The study protocol was approved by the Ethics Committee of the Pirkanmaa Hospital District, study identification code ETL R05183. All participants or, in the case of children, their legal guardians gave written informed consent. The paper follows the rules of the Declaration of Helsinki.

#### **3. Results**

The gender distribution was fairly equal among the relatives, whereas a majority of CD patients were women, and there were more men in the non-CD control group (Table 1). There were no major differences in the median ages between the groups (Table 1), but 49 (10.6%) of the relatives were <18 years of age, while the other groups comprised only adults.

**Table 1.** Demographic data on relatives of celiac disease (CD) patients, CD patients and non-celiac controls.

\* Negative serum endomysium (titer 1: <5) and tissue transglutaminase (<30 U/mL) antibodies. GFD, gluten-free diet.

The relatives were divided into CD autoantibody-negative (*n* = 414) and autoantibody-positive (*n* = 49) groups and were analyzed separately (Table 1). Among the autoantibody-negative relatives, seropositivity for at least one of the microbial markers was more common than in the non-CD controls but less frequent than in the CD patients (Figure 1). The most notable difference was seen in ASCA, as 19% of the relatives without CD-autoantibodies and none of the controls were seropositive for ASCA IgA, ASCA IgG, or both. In addition, anti-I2 and anti-OmpW positivity was more common among the autoantibody-negative relatives than controls (61% and 40% vs. 31% and 24%, respectively; Figure 1).

**Figure 1.** Distribution of seropositivity to antibodies against *Saccharomyces cerevisiae* (ASCA), *Pseudomonas fluorescens*-associated sequence (anti-I2 antibodies) and *Bacteroides caccae* TonB-linked outer membrane protein (anti-OmpW antibodies) among autoantibody-negative relatives of celiac disease (CD) patients, CD patients (at diagnosis and on a GFD) and controls.

The median levels of ASCA IgA, ASCA IgG and anti-I2 were also significantly higher in the autoantibody-negative relatives than those in the control group (Figure 2a–c), whereas anti-OmpW was higher only in untreated and treated CD patients (Figure 2d). ASCA IgG was higher in both untreated and treated CD patients and anti-I2/OmpW in untreated patients when compared with autoantibody-negative relatives (Figure 2b–d).

Altogether, 46 out of the 49 autoantibody-positive relatives had HLA-DQ2 haplotype, DQ8 haplotype, or both. As many as 86% of them showed seroreactivity to at least one microbial marker compared to 73% of the CD antibody-negative relatives, and the median levels of the microbial antibodies were also higher (ASCA IgA 11.1 vs. 8.90 U/mL, *p* = 0.019; ASCA IgG 12.8 vs. 8.37 U/mL, *p* = 0.001; absorbance for anti-I2 0.93 vs. 0.71, *p* = 0.320 and for anti-OmpW 1.00 vs. 0.81, *p* = 0.022, respectively). In contrast to the autoantibody-negative group, anti-OmpW levels were also significantly higher than in the controls (absorbance 0.79, *p* = 0.043).

Adjusting for age and gender or exclusion of children from the comparisons did not affect the results of the prevalence of seropositivity nor median levels of the microbial markers, although the medians were significantly lower in children than in adults (ASCA IgA 6.30 vs. 9.64 U/mL, *p* < 0.001; ASCA IgG 7.13 vs. 9.18 U/mL, *p* = 0.070; absorbance for anti-I2 0.34 vs. 0.79, *p* < 0.001 and for anti-OmpW 0.54 vs. 0.87, *p* < 0.001, respectively).

Seropositivity to anti-I2 and anti-OmpW was significantly more frequent between relatives in the same family than between different families (*p* < 0.001 for anti-I2 and *p* = 0.001 for anti-OmpW, respectively). In ASCA, this was observed only when autoantibody-positive relatives were also included in the analysis (*p* = 0.007).

There were no significant differences in the distribution of seropositivity across microbial markers when the relatives were categorized according to their HLA haplotypes (Table 2).

There was a weak, positive correlation between the values of tTGab and ASCA IgA (r = 0.31, *p* < 0.001), whereas correlation coefficients between the other microbial markers and tTGab or EmA were <0.3.

**Figure 2.** Serum levels of antibodies to *Saccharomyces cerevisiae* (ASCA) in IgA (**a**) and IgG (**b**) classes, *Pseudomonas fluorescens*-associated sequence (anti-I2) (**c**) and *Bacteroides caccae* TonB-linked outer membrane protein (anti-OmpW) (**d**) in autoantibody-negative relatives. Horizontal lines indicate the cutoff level for seropositivity of each antibody.

**Table 2.** Frequency of seropositivity to microbial markers in autoantibody-negative relatives of celiac disease patients with different human leukocyte antigen (HLA) haplotypes.

ASCA, Anti-*Saccharomyces cerevisiae* antibodies; anti-I2, antibodies to *Pseudomonas fluorescens*-associated sequence; anti-OmpW, antibodies to *Bacteroides Caccae* TonB-linked outer membrane protein; DQ2, HLA-DQA1\*05-DQB1\*02 (DQ2.5) or HLA-DQA1\*02-DQB1\*02 (DQ2.2); DQ8, HLA-DQA1\*03-DQB1\*0302. There were no statistically significant differences between the groups in the distribution of seropositivity.

#### **4. Discussion**

The main finding of the present study was increased seroreactivity to microbial markers in the relatives of CD patients compared with controls even after the exclusion of CD autoantibody-positive individuals. This was observed particularly with ASCA and anti-I2, the median levels of which were also significantly higher than levels in the controls, although they were lower than in CD patients. To the best of our knowledge, the only study to report on this issue so far was a conducted by Da Silva et al., who investigated seropositivity to ASCA in relatives of CD patients [28]. They divided 76 relatives into EmA/tTGab negative and positive groups, while 57 individuals with negative CD autoantibodies and no family risk served as controls. Partly in contrast to us, there was a significantly higher frequency of positivity to ASCA IgA/G only in autoantibody-positive relatives compared with the controls [28]. This discrepancy may, at least in part, be explained by the smaller number of participants since there was a trend toward overrepresentation of ASCA, also among the CD autoantibody-negative relatives. There may also have been methodological differences, as the authors did not report the kits used for the ASCA measurements.

Owing to the high specificity of tTGab and EmA [23], most of the autoantibody-positive relatives were likely CD patients. Therefore, their increased seroreactivity to microbial markers is logically in line with that observed in already-diagnosed CD. By contrast, the increased frequency of seroreactivity to a part of the microbial markers in the autoantibody-negative relatives is not as easily explained. It is to be noted that Setty and colleagues [29] previously reported that tTGab-negative relatives of CD patients had signs of intestinal epithelial stress, demonstrated by ultrastructural alterations of microvilli, and increased expression of heat shock proteins and interleukin-15 along with elevated expression of activating NK receptors on intraepithelial cytotoxic T cells. Thus, even in the absence of CD autoantibodies or characteristic histological damage to the intestine, at least some of the relatives appeared to display proinflammatory responses reminiscent of CD. This raises the question of whether the observed abnormal microbial antibody production could also be implicated in this process.

Setty et al. also speculated about a possible genetic predisposition to epithelial stress [29] and suggested a possible HLA and other as yet-unidentified genetic associations. We observed no significant association between the distribution of ASCA, anti-I2 and anti-OmpW positivity and the CD-related HLA haplotypes, suggesting that at least HLA genetics does not markedly affect the serological response. In line with this, HLA DQ2/8 are not overexpressed in inflammatory bowel disease (IBD) patients [30] who also may have increased seropositivity to microbial markers [16,17]. Genetics may still play a role in microbial antibody production in intestinal diseases, as demonstrated by two studies comparing levels of microbial antibodies between monozygous and dizygous twin pairs with IBD. Amcoff et al. reported that the differences in the anti-I2 antibody levels were smaller within than between monozygous twin pairs, even if only one of them had IBD [31]. However, this was not seen in dizygous twins with one suffering from IBD and the other being healthy and having partly discordant genetics, supporting the role of genetic factors [31]. By contrast, similar ASCA levels were observed only in a subgroup of monozygous twins both having IBD [31,32]. Bearing this in mind, it is interesting that we found stronger associations of anti-I2 positivity between the relatives from the same family than between the families, whereas with ASCA this was seen only when autoantibody-positive relatives were included in the analysis. Taken together, it seems that both genetic and environmental factors have a role in the antibody production, with this varying depending on the microbial marker, but further studies are needed.

Environmental factors including gluten intake [33,34] and infections in early life [35–37] have also been associated with increased CD risk. Other suggested, although controversial [38,39], risk factors include bacterial infections and frequent use of antibiotics [40,41]. Interestingly, the incidence has been reported to vary depending on socioeconomic circumstances [10], leading to the hypothesis that slight microbial exposure increases CD risk by driving immune reactions toward autoantigens and dietary components [42]. Close relatives usually share the living milieu and may, thus, experience similar environmental modulatory effects on the microbiota and immune system that, in addition to genetics, could give rise to parallel responses to microbial antigens. It remains unclear, however, which external factors drive these responses and whether the microbial markers have a causal role [43]. It is likely that a complex interaction between multiple factors, such as dysregulation of the immune system, changes in the epithelial barrier, and dysbiosis causes the loss of tolerance to microbial antigens [13,44–46]. In addition, a very recent study showed that *Pseudomonas fluorescens* peptides mimic gluten epitopes and activate gliadin-reactive T cells, with this cross-reactivity possibly contributing to the onset of CD [47].

We previously found most of the potential CD patients to already exhibit the microbial markers before the development of villous damage or autoantibodies [21], reflecting the situation in the relatives in the present study. Interestingly, Torres and colleagues recently showed that ASCA also predicts forthcoming Crohn's disease up to five years before the diagnosis [48]. More studies are needed to determine the role of these markers in early development of CD and whether they could be utilized to predict the disease in at-risk groups.

The main strengths of our study include the large and well-defined cohort of relatives of CD patients who underwent systematic screening for CD-associated HLA and autoantibodies and the representative control groups. As a weakness, however, large differences between the group sizes could have influenced the results. Furthermore, only the groups with relatives contained pediatric subjects, although the results remained unchanged after excluding children from the analyses. Genetic data of the non-HLA alleles were also lacking, which could be an even more significant limitation among relatives with a less marked HLA predisposition to CD. Since we did not have detailed information on the health condition of the relatives, and the histological status of their intestines remains unknown, it is possible that some of them had unreported CD or another disease affecting the results. Furthermore, dietary data of the relatives was lacking, and it is possible that cross-reactions between food antigens influenced the microbial antibody levels. ASCA is known to cross-react with other yeast strains [49], and the lack of correlation between ASCA antibodies and *Saccharomyces cerevisiae* DNA on intestinal mucosa [50] indicates the possibility of some yet-unidentified cross-reactive antigens. In accord with our previous study [51], for currently unclear reasons, ASCA levels were generally higher in the IgG class than the IgA class. By contrast, IgA class ASCA seems to be more consistently elevated in IBD [48,52]. Which of these two antibody classes is the more useful marker in CD would be an interesting subject for further research. The median duration of GFD in the CD group was only one year, which may have biased the serological results, as histological and serological recovery often take longer despite a strict diet [53]. Finally, a few adults here had surprisingly high anti-OmpW values compared with our previous studies. Although we still believe that the used cutoff was valid, we recommend that it be confirmed in other populations.

In conclusion, we found increased seroreactivity to serum microbial markers, particularly ASCA and anti-I2, in relatives of CD patients even in the absence of the disease-specific autoantibodies or other signs of active CD. This observation was not explained by the presence or absence of predisposing HLA haplotypes, thereby suggesting the role of other genetic and environmental factors.

**Author Contributions:** L.V.: Conceptualization, writing the original draft; S.I.: Conceptualization, supervision, writing—review and editing; H.H.: Conceptualization, methodology, writing—review and editing; P.S.: Conceptualization, methodology, writing—review and editing; K.K. (Katri Kaukinen): Conceptualization, funding acquisition, writing—review and editing; K.L.: Conceptualization, methodology, funding acquisition, writing—review and editing; K.K. (Kalle Kurppa): Conceptualization, funding acquisition, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Academy of Finland, the Finnish Medical Foundation, the Sohlberg Foundation, the Paulo Foundation, the Sigrid Juselius Foundation, the Foundation for Pediatric Research, the Competitive State Research Financing of the Expert Area of Tampere University Hospital, Helsinki University Hospital Research Fund, the Finnish Medical Foundation and the Finnish Coeliac Society.

**Acknowledgments:** We would like to acknowledge Jonathan Braun, Pathology and Laboratory Medicine, UCLA David Geffen School of Medicine, UCLA Health System for his support.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**5. Applications of solution-processed functional oxides for TFTs and display**

#### **5.1. Low-temperature all solution-derived amorphous oxide TFTs**

TFTs, in which all the layers were fabricated using simple chemical solution-processed, vacuum-free routes, followed by thermal annealing at 400°C, were demonstrated [34]. A ruthenium oxide (RuO2 ) was used for both gate and source/drain electrodes. Amorphous LZO and zirconium-indium-zinc oxide (ZIZO) films were used as the gate insulator and channel layer, respectively, which enabled the fabrication of a TFT with the desired performance at a sufficiently low temperature. Transfer characteristics of the low-temperature all solutionprocessed TFT are shown in **Figure 10**. The obtained "on/off" ratio, *SS*-factor, and *μ* were ∼6 × 105 , 250 mV/decade, and 5.80 cm2 V−1 s−1, respectively.

Low-Temperature Solution-Processable Functional Oxide Materials for Printed Electronics http://dx.doi.org/10.5772/intechopen.75610 93

**Figure 10.** Transfer curves of low-temperature all solution-processed oxide TFT.

**Material Method T (°C) ρ/σ μ (cm2 V−1 s−1) Eg**

CuCrO2

92 Green Electronics

CuCrO2

Ca3 Co4

Y:CuAlO2 Li:ZnO

talline bulk Bi3

given in **Table 5**.

nium oxide (RuO2

**display**

∼6 × 105

Ru3

Electrohydrodynamic

Ln-M-O (M = Ru, Ir, and Ln is a lanthanide)

A-B-O (A = Bi, Pb; B = Ru, Ir)

CuAlO2 So-gel 900 250 Ω cm — — 2002 [92]

CuFeO2 So-gel 700 0.36 S cm−1 0.7 3.05 2012 [97]

, CuAl0.5Cr0.5O2 Sol-gel 600 11–16 Ω cm — — 2009 [96]

:Mg CSD 600 0.31 Ω cm 0.7 2012 [98]

O9 CSD 650 57 mΩ cm — — 2014 [92] CuCrO2 Combustion 180 0.14 S cm−1 0.23 3.0 2018 [99] CuCrO2 Spray pyrolysis 345 12 S cm−1 6.4 × 10−3 2.4 2015 [100]

temperatures [95]. The lowest necessary temperatures for a-BiRuO, a-PbRuO, and a-BiIrO were 240, 290, and 350°C, respectively, resulting in RT DC resistivities of 3.8, 1.7, and 3.8 mΩ cm, respectively. The resistivity of a-BiRuO film has nearly reached the value of crys-

p-type oxides, the low-resistivity values of these amorphous oxides are matched only by epitaxial LaCuOSe:Mg annealed at a high temperature of 1000°C (1.1 mΩ cm). A summary of electro-optical characteristics of some solution-processed p-type conductive oxides is

**5. Applications of solution-processed functional oxides for TFTs and**

TFTs, in which all the layers were fabricated using simple chemical solution-processed, vacuum-free routes, followed by thermal annealing at 400°C, were demonstrated [34]. A ruthe-

and zirconium-indium-zinc oxide (ZIZO) films were used as the gate insulator and channel layer, respectively, which enabled the fabrication of a TFT with the desired performance at a sufficiently low temperature. Transfer characteristics of the low-temperature all solutionprocessed TFT are shown in **Figure 10**. The obtained "on/off" ratio, *SS*-factor, and *μ* were

**5.1. Low-temperature all solution-derived amorphous oxide TFTs**

, 250 mV/decade, and 5.80 cm2 V−1 s−1, respectively.

580 Ω cm 15.06 Ω cm

1100 500

**Table 5.** Electro-optical characteristics of some solution-processed p-type conductive oxides.

CSD <400 10−3–10−2 Ω cm — — 2012 [93]

CSD 240 1.3–3.8 mΩ cm — 0.2 2012 [95]

O11 (1.4 mΩ cm), which suggests that the films are of high quality. In

) was used for both gate and source/drain electrodes. Amorphous LZO

– 0.918 **(eV)**

– – 2015 [101]

**Ref.**


*Article* **Urbanization and Floods in Sub-Saharan Africa: Spatiotemporal Study and Analysis of Vulnerability Factors—Case of Antananarivo Agglomeration (Madagascar)**

**Fenosoa Nantenaina Ramiaramanana \* and Jacques Teller**

Local Environment Management and Analysis (LEMA), Urban & Environmental Engineering Department, University of Liège, 4000 Liège, Belgium<EMAIL_ADDRESS>**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Flooding is currently one of the major threats to cities in Sub-Saharan Africa (SSA). The demographic change caused by the high rate of natural increase, combined with the migration toward cities, leads to a strong demand for housing and promotes urbanization. Given the insufficiency or absence of adequate planning, many constructions are installed in flood-prone zones, often without adequate infrastructure, especially drainage systems. This makes them very vulnerable. Our research consists of carrying out a spatiotemporal analysis of the agglomeration of Antananarivo (Madagascar). It shows that urbanization leads to increased exposure of populations and constructions to floods. There is a pressure on land in flood-prone zones due to the exponential growth of the population at the agglomeration level. Some 32% of the population of the Antananarivo agglomeration lived in flood-prone zones in 2018. An analysis of the evolution of built spaces from 1953 to 2017 highlights that urban expansion was intense over those years (6.1% yearly increase of built areas). This expansion triggered the construction of built areas in flood-prone zones, which evolved from 399 ha in 1953 to 3675 ha in 2017. In 2017, 23% of the buildings in the agglomeration, i.e., almost one out of every four buildings, were in flood-prone zones. A share of the urban expansion in flood-prone zones is related to informal developments that gather highly vulnerable groups with very little in terms of economic resources. Better integration of flood risk management in spatial planning policies thus appears to be an essential step to guide decisions so as to coordinate the development of urban areas and drainage networks in a sustainable way, considering the vulnerability of the population living in the most exposed areas.

**Keywords:** demographic change; urbanization; flooding; drainage system; vulnerability; Sub-Saharan Africa; Antananarivo


*Article* **Life Cycle Environmental Costs of Buildings**

**Yuanfeng Wang 1,\*, Bo Pang 1, Xiangjie Zhang 1, Jingjing Wang 2, Yinshan Liu 1, Chengcheng Shi 1 and Shuowen Zhou 3**

Received: 21 February 2020; Accepted: 12 March 2020; Published: 14 March 2020

**Abstract:** Energy consumption and pollutant emissions from buildings have caused serious impacts on the environment. Currently, research on building environmental costs is quite insufficient. Based on life cycle inventory of building materials, fossil fuel and electricity power, a calculating model for environmental costs during different stages is presented. A single-objective optimization model is generated by converting environmental impact into environmental cost, with the same unit with direct cost. Two residential buildings, one located in Beijing and another in Xiamen, China, are taken as the case studies and analyzed to test the proposed model. Moreover, data uncertainty and sensitivity analysis of key parameters, including the discount rate and the unit virtual abatement costs of pollutants, are also conducted. The analysis results show that the environmental cost accounts for about 16% of direct cost. The environmental degradation cost accounts for about 70% of the total environmental cost. According to the probabilistic uncertainty analysis results, the coefficient of variation of material production stage is the largest. The sensitivity analysis results indicate that the unit virtual abatement cost of CO2 has the largest influence on the final environmental cost.

**Keywords:** building; environmental costs; green GDP, China; uncertainty analysis; sensitivity analysis


**Acknowledgements**

Financial support of this work by the National Nature Science Foundation of China under Grants 51505031, as well as BIT National Key Laboratory for VRC Basic Research Foundation is highly acknowledged. Acknowledgments go also to the colleagues at the Department of Mechanical Engineering of Beijing Institute of Technology and all Postdoctoral Fellows/ Research Associates, Ph.D., MASc/MEng students who contributed to this work.


**NUCLEAR REACTORS**

Edited by **Amir Zacarias Mesquita**

**INTECHOPEN.COM**

http://dx.doi.org/10.5772/1919 Edited by Amir Zacarias Mesquita

#### **Contributors**

Amir Zacarias Mesquita, Antonella Lombardi Costa, Claubia Pereira, Maria Auxiliadora Fortini, Patricia A. L. Reis, Daniel Artur Palma, Chaitanya Deo, Mihály Makai, Yuri Orechwa, Kazuyuki Takase, Takeharu Misawa, Hiroyuki Yoshida, Mehdi Hashemi-Tilehnoee, Farshid Javidkia, Sergey Pelykh, Maksim Maksimov, Seyed Alireza Mousavi Shirazi, Motoo Fumizawa, Salah El-Din Belal El-Morshedy, Isao Kataoka, Kenji Yoshida, Masanori Naitoh, Hidetoshi Okada, Tadashi Morii, Georgy Levanovich Khorasanov, Anton Valter, Anatoly Blokhin, Osman Gencel, Turgay Korkut, Fuat Köksal, Yoshitomo Inaba, Alejandro Algora, Wargha Peiman, Igor Leonardovich Pioro, Kamiel Gabriel

#### **© The Editor(s) and the Author(s) 2012**

The moral rights of the and the author(s) have been asserted.

All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH's written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department (permissions@intechopen.com).

Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html.

#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in Croatia, 2012 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

Legal deposit, Croatia: National and University Library in Zagreb

Additional hard and PDF copies can be obtained from<EMAIL_ADDRESS>
Nuclear Reactors Edited by Amir Zacarias Mesquita p. cm. ISBN 978-953-51-0018-8 eBook (PDF) ISBN 978-953-51-6117-2


*3.2. Structure Analysis*

Figure 3 are FTIR spectra and XPS survey spectra of the linear WPU-SS samples. In Figure 3a, all curves have no absorption peak at 2270 cm−1, which indicates that −NCO groups in the system are all involved in the reaction [16,17]. Compared with the three curves, the distribution of all characteristic peaks was essentially the same, indicating that −OH and −NCO on HEDS reacted to form carbamate, and no new chemical bond was formed. The three curves are in line with the characteristic peaks of waterborne polyurethane. The band located at 3321 cm−1 corresponds to the N−H stretching vibration and the band at 1531 cm−1 is assigned to the N–H in-plane bending vibration [18,19]. The absorption bands at 1460 and 1360 cm−1 are assigned to the –CH2– bending vibrations. The bands at 2940 and 2856 cm−1 are associated with the C–H asymmetry and symmetric stretching vibration of methylene in polyurethane molecular chain [20,21]. The band at 1110 cm−1 corresponds to bending vibration of C−O (aliphatic ether) in polyether polyol [22]. The characteristic band about 1707 and 1631 cm−1 is related with nonhydrogen bond C=O hydrogen bond C=O in urea, respectively [23–25]. HEDS in polymer chain has a characteristic absorption peak of SS bond at 637 [26]. With the increase of disulfide bond content, the absorption peak of the corresponding SS bond at 637 cm−1 became more obvious, which proved that SS bond was successfully introduced into waterborne polyurethane to form WPU-SS.

**Figure 3.** (**a**) FTIR spectra of WPU-SS samples, (**b**) XPS survey spectra of WPU2, (**c**) C 1s of WPU2, and (**d**) O 1s of WPU2.

The XPS measurement was used to further confirm the construction by analyzing the chemical compositions in the WPU-SS system. The peaks in XPS survey spectrum indicated the present of S element (163.5 eV) in the samples due to disulfide bond should be successfully chemically linked to the matrix of waterborne polyurethane. In addition, the C 1s spectrum was further measured to explore the types of carbon bonds to analyze the existence of interfacial interactions, as shown in Figure 3c. The C 1s peak was curvedfitted into four main components including C-C/C-H, C-N, C-O, C-S and C=O bonds corresponding to the peaks at 284.8, 285.7, 286.5, 287.7 and 288.8 eV, respectively. The O 1s peak was curved-fitted into four main components including C-O-C and C=O bonds. The peak of C-S bond appeared implying that disulfide bond was successfully grafted onto polyurethane chains, as supported by FTIR results.

#### *3.3. Dynamic Thermodynamic Analysis*

The DMA results are shown in Figure 4. During the heating process of WPU-SS at −100~120 ◦C, the polymer with microphase separation structure showed two glass transition temperatures. The storage modulus and loss modulus of WPU-SS films are shown in Figure 4a,b, respectively. It could be found that the storage modulus (E ) decreased significantly at about −70 to −60 ◦C, while the loss modulus (E") increased significantly at the same temperature range, which corresponding to the glass transition of the hard segments, moreover, with the increase of SS bonds, the E' did not change much while the peak value of E" changed obviously, which meant the move of the polymer chains took more energy. The decrease of disulfide bond content leads to the increase of soft segment content, which reduced the E and E" of the films at room temperature. This is mainly due to the less restriction of the slip of the soft segment molecular chain, which leads to the easier entanglement of the molecular chain and the easier interaction with the main chain [27]. At the same time, the degree of microphase separation is reduced but the damping performance is improved.

**Figure 4.** (**a**) Storage modulus, (**b**) loss modulus and (**c**) tan delta of WPU-SS samples with different contents of SS as a function of temperature.

The link between tan delta and temperature is determined in Figure 4c. With the loss of SS bond loading, the soft segment rises, and the microphase separation diminishes gradually. When the microphase separation is cut down to a certain extent, the glass transition temperature of the hard segment cannot be shown in the experiment. Since the glass transition temperature of the hard segment is much lower than room temperature, the film is in a highly elastic state and exhibits the properties of an elastomer at room temperature.


**4.1 Hardware architecture**

The hardware requirements are exemplified in Figure 11, where there are four PC interconnected through a fast Ethernet local area network. Each PC must have the processor and memory required to execute the simulator smoothly, and to support high processing demand functions like execution faster than real-time. The monitors of the operator consoles must be of a similar size of the ones in the actual power plant. In the case of the Figure 11, the configuration depicts monitors of 20" and 50". During the training session, the trainee

Fig. 11. Hardware architecture.

can use any one of his consoles to supervise and control any process of the power plant. The instructor console is provided with two monitors, hence, besides of using the instructor functions described previously, he can display any screen of the operator consoles with the purpose of watching any operative action carried out by the trainee. In many architectures of simulators is common to find an additional PC, the maintenance station (not shown in Figure 11), which serves as a backup if the instructor console fails or as a test station, this means that any software modification is tested and validated in this station before any change is carried out in the simulator.