Factors Influencing Smoking Among School-Going Adolescents in Dhaka City–A Mediating Effect of Point of Sales

Introduction

Bangladesh is one of the largest tobacco consuming countries in the world. Over 58% of men and 29% of women use some form of tobacco, whether smoked or smokeless. At present, adolescent school going students’ tobacco use has become a growing problem in Bangladesh. The use of smoked items among school going students age between 12-19 is increasing at a rate of 4%. Bangladesh faces considerable health and economic consequences from tobacco. Over 57,000 deaths are attributed to tobacco use each year. The economic costs of tobacco use in Bangladesh accounted for over 3% of GDP in 2004 (Table 1). With a thorough review of recent research nearly one out of five people on the planet smoke cigarettes and an estimated 800 million of these are in developing countries. It is estimated that one third of the world’s adult population, of whom 200 million are female, are smokers. Globally, 47% of men and 12% of women are smokers. Tobacco use has become one of the leading causes of preventable death in world. According to (Sultana P, et al. [1]), “Bangladesh is one of the highest tobacco consuming countries in the world, with reported 21.2% of the population as daily smokers, 24.3% as smokeless tobacco users, and 36.3% as adult passive smoker”. This numbers are increasing daily at an alarming rate. Presently, about four million people globally die yearly from tobacco related diseases. Smoking and other nonsmoking tobacco related items are being used by all classes of people.

Table 1: Response rate of the study by Age group of the students.

The use of tobacco among students are also increasing very fast. A study in Bangladesh showed that 22.1% of students have smoking habit and it is increasing terrifyingly due to avoid anxiety and tension, feeling of maturity, symbol of manliness and unhappy family environment (Haq, et al. [2]). Adolescent school going students have also started to use tobacco related items. If current trend goes on, there will be one death in every three seconds by 2030 and majority death will be in developing countries. Tobacco kills up to half of its users and causes premature mortality and morbidity, contributes to health inequalities and exacerbates poverty. The Global Adult Tobacco Survey (GATS) 2017 revealed the social gradient in tobacco use in Bangladesh in which prevalence increases with decreasing socioeconomic status. About 24% of those in the highest wealth quintile use tobacco compared to 48% of those in the lowest wealth quintile. Smoking and passive smoking are collectively the biggest preventable cause of death in Bangladesh, with major public health burden of morbidity, disability and mortality and community costs. Bangladesh is one of the top ten countries in the world with high current smoking prevalence of 44.7% among men .This country is distinguished as the first signatory of the World Health Organization (WHO).

Framework Convention on Tobacco Control (WHO FCTC) which was ratified on 10 May 2004. The ratification was made concrete with the passage of the Tobacco Control Act (TCA) on 15 March 2005. These tobacco control measures are expected to reduce smoking. Yet a robust science base exists on social, biological, and environmental factors that influence young people to use tobacco. These students are at high risk of initiating and continuing smoking as they are likely to be exposed to peers who smoke. At the same time, they face social, emotional, and educational challenges when they enter the university settings. In Bangladesh, the numbers of tobacco smokers are increasing rapidly because of the availability of cheap tobacco products, lack of strong tobacco control regulations, and weak enforcement of existing regulations (Barakat A, et al. [3]). The Global Adult Tobacco Survey conducted by WHO reported that Bangladesh is one of the top ten countries in the world with high tobacco use (both smoking and smokeless forms) with a prevalence of 43.3% among adults (41.3 million), with 44.7% of men and 1.5% of women engaging in tobacco smoking. A study based on demographic and health survey data reported that the prevalence of tobacco smoking among men in Bangladesh is 60%. Another study among male university students in 2009 stated that 36.1% students smoked tobacco. Among fourth-year dental students, the prevalence of cigarette smoking was reported to be 49.5% and 1.7% in males and females, respectively. An increasing trend of tobacco smoking is anticipated to occur among students and this could be related to perceived alleviation of stress, life problems, peer pressure, social acceptance, class history of smoking, lower educational level of parents, and the desire to attain higher societal class. Smoking among students in Bangladesh has been poorly investigated that students may be lacking knowledge on the link between smoking and adverse health effects.

School going students are smoking because of personal factors and social factors. Not only that, the tobacco point of sales has a great impact in increasing the use of smoking among school going children. If the use of smoked tobacco items among adolescent school going children at the rate of 4% continues then in near future, we will have more adult passive smokers (Siddiqi, et al. [4]). School going students mainly buying and using tobacco because of its availability, word of mouth, experimental attitude, showoff tendency and the influence of media. If corrective measure is not taken at the initial stage, then this rate will grow at a faster rate. At the present time policy makers are focusing only adult tobacco user but problem lies bottom of the pyramid which is school going children. Approximately 50% of men in developing countries are smokers and cigarette consumption is steadily rising in these countries, particularly among women and youth. Tobacco use is predicted to be one of the major causes of death and disabilityadjusted life years (DALYs) in the 21st century (Champman, et al. [5,6]).

Significance of the Study

Total number of students is 4.21 million in Dhaka city. There is very limited study has been conducted on the use of tobacco items among youth and university going students. But there is no study found focusing the school going students’ use of smoked tobacco items and its accessibility. The aim of the study was to estimate the prevalence of tobacco smoking among students and to identify factors that may be related to both initiation and prevalence of tobacco smoking. Tobacco use and other high-risk behaviors are emerging as significant problems in our society. The unhealthy behaviors acquired during adolescence are continued throughout the life cycle, resulting in adverse effects on the individual, family and society.

Methodology

In the present study, the chosen methodology is quantitative analysis, because the research involved examination of the variables to determine possible relationships among variables by using statistical analysis. Quantitative study frequently tests the assembled hypothesis with findings are predictive, explanatory and confirming. Targeted population for this study was school going adolescent students in Dhaka city.

Sampling Technique and Data Collection

Multistage cluster sampling has been used to derive the sample where two city corporation areas have been selected, first stage/ cluster, different selected area under each city corporation have been second stage/cluster, schools in the selected fields have been the third stage/cluster and adolescent students in the chosen school have been the last stage/cluster of this study. The questionnaire have been divided in 2 sections, first one was demography information and second section was related to variables. The questionnaires were distributed to the students of selected classes after explaining the purpose of the study and the instructions to fill it.

Ethical Consideration

All the information have collected for the research purpose and not disclose to anyone outside the research team. Verbal consent have taken from all participating school’s authority and students.

Results

General characteristics of the study population: The total number of completed survey questionnaire fill up by the respondents in the study was 200. The majority (45.0%) of the respondents were of the age group 16-17 years. Followed by 14-15 years old adolescent’s school going students and 12.5 % is 12-13 years old. About (56%) of the respondents were from governmental schools and (44%) were from the private school. (Fig 4). As per the survey results are showing, 45% of the total respondents are in the age group of 16-17, 42.5 % are in 14-15 years old and 12.5% are 12-13 years old. School going adolescents whose age is 16- 17 smoke more followed by 14-15 and 12-13 years respectively (Table 1). As per the result of the survey analysis, students who are studying class 9 participated in the survey more with 45%. Class ten students were 29%. Students who are studying in class eight was 16% and seven was 10% (Table 2). From the table it’s clear that class nine students are more exposed to smoking than any other class students. Adolescents from the nuclear family participated more in the survey with 76.5%, and joint family adolescents were 23.4 % (Table 3).

Table 2: Percentage of level of education of the students.

Table 3: Percentage of the students by Smoking Experience.

Respondents Smoking Experience

Among the 200 respondents, around 131 experienced smoking which is 65.5 % of the total respondent and only 34.5 % never experienced the smoking before but may get influenced by the majority of the cluster. (Tables 3 & 4) shows that, Among 200 students 49% percent are smoke less than 6 months and only 4% have experience more than 1 years. Rest of the students have smoking experience within 6 months to 1 year (Table 4).

Table 4: Percentage of students according to duration of smoking.

Respondents get influenced for Smoking

Friends influence the adolescents to experience smoking items more with 48.5% whereas family members contribute to the influencing factor by 23.5% and relative influences 28%. From the result is very clear that friends and relatives are the most influential person in smoking (Figure 1). Association between smoking habit and age of respondents : There is an association between smoking habit and age of respondents. Age 14-15 School going adolescent smoke more than any other smoking age group (Table 5).

Table 5: Association between smoking habit and age of respondents.

Figure 1: The Influences of the persons worked on students for smoking.

Association between Smoking Habit and Level of Education

As per the result, students studying class nine smoke (49.62%) and a second-highest smoking group of students are from class eight (Table 6). Class seven students smoke (13.74%) and class ten students’ (15.27%) respectively. Each subscript letter denotes a subset of Education categories whose column proportions differ significantly from each other at the .05 level (Figure 2).

Figure 2: Sampling design.

Table 6: Association between smoking habit and Education level of respondents.

Association between Smoking Habit and Age of Respondents

There is an association between smoking habit and age of respondents. Age 14-15 School going adolescent smoke more than any other smoking age group.

Association between Smoking Habit and Level of Education

As per the result, students studying class nine smoke (49.62%) and a second-highest smoking group of students are from class eight (Table 6). Class seven students smoke (13.74%) and class ten students’ (15.27%) respectively. Each subscript letter denotes a subset of Education categories whose column proportions differ significantly from each other at the .05 level.

Association Between Smoking Habit and Influential Person

Table 12 shows, 61.83% of school-going adolescent students are highly influenced by the peer group or friends. However, 14.50% of students are influenced by relatives. It is also noticeable that 15.27% of students are smoking as because their elder brother smokes not only that around 8.39% reported they started smoking as their father smoke at home. Each subscript letter denotes a subset of Influenced categories whose column proportions differ significantly from each other at the .05 level.

Data Analysis for variables

This section deals with the tobacco use pattern of adolescent students among grades7, 8, 9 and 10 from both governmental and private schools of Dhaka North and South. All the variables, Personal factors, Social factors, POS and Use of tobacco Items are analyzed descriptive statistics and checked the mediation type. The Chi Square statistic Chi-Square for testing relationships between categorical variables (Statistical Solution, 2019). It is also explained that the Chi-Square statistic is most commonly used to evaluate Tests of Independence when using a cross-tabulation (Table 7).

Table 7: Association between smoking habit and Influential Person.

Table 8: Relationship Between Personal factor ( PF) and Used of tobacco items (UTI ).

Note: a. This table has expected count less than 5. The minimum expected count is .05.

Table 9: Relationship Between Social factor (SF) and Used of tobacco items (UTI).

Note: a. 250 cells (98.0%) have expected count less than 5. The minimum expected count is .05.

Table 10: Correlation Coefficient between personal factor, Social factor and Point of sales.

Note: a. Dependent Variable: UTI (Use of tobacco items).

Association between Personal Factor and Used Smoked Tobacco Items

The result of table 13 shows that there is a significant relationship between personal factor and used smoked tobacco items. Association between Social Factor and Used Smoked Tobacco Items: As per the result of Table 14, there is a significant relationship between social factor and used smoked tobacco items. As per the result of table 14, there is a significant relationship between social factor and used smoked tobacco items. To check the correlation of variables and items, we performed Descriptive Statistics and Coefficient analysis (Tables 8-10).

Discussion

Nearly half (65.5%) of adolescent students studying in grades seven, eight, nine and ten were ever users of tobacco. Adolescents from nuclear family smoke more than joint family. In most of the cases, school-going adolescent experiences their first tobacco smoke by the influence of friends though family members and relatives have also influenced these students to smoke. Schoolgoing adolescents are exposed to tobacco items more because of the point of sale. According to GYTS (2002), 4 out of 5 students buy tobacco item from nearby shops. Among adolescents, school-going students, the experimental use of tobacco, showoff and influence of media was higher than other influential factors in the various study. Thus, the use of tobacco is increasing in Dhaka city as a higher proportion of adolescents are currently experimenting the tobacco products especially smoked tobacco products like bidi and cigarette (Pandey, et al. [7]). The average age of initiating tobacco use was 12-13 years. During this age, adolescents’ activities are less supervised by their parents than in their earlier life and also are more influenced by the activities and behavior of friends and relatives. As both younger and older students are studying in the same school, the younger ones were influenced by the behavior of older ones.

These older students could have a real influence on the younger students in terms of the younger ones wanting to follow the behavior of the older ones. A significant proportion (23.5%) of the adolescent students reported that at least one of their family members (parents, siblings and other members residing permanently) use any tobacco products. Tobacco use of family members is expected to influence adolescents. When adolescents are exposed to the tobacco use habit of family members, they are more likely to perceive tobacco use as positive and acceptable behavior. Thus, this helps to develop favorable personal beliefs and subjective norms about tobacco use, and ultimately leading youth to take up the habit (Green, et al. [8-11]). Nearly (28%) of schoolgoing adolescent students reported that at least one of their cousin use tobacco. On the other hand, around (48.5%) mentioned their friends using tobacco. During adolescence, the relationship with the relatives and peer group become stronger than family members, and thus, young people are influenced more by the habits of their friends. Imitating the behavior of friends is a common practice among adolescents who want to be like their peers and affects them to use tobacco. The recent Nepali study (Calverton, et al. [12]) also explained that youths close friends who smoke were more likely to experiment and continue smoking and the effect of peer group is stronger.

Although detailed studies on the influence of family members and friends were not available in Bangladesh, different international studies suggested results similar to this study. A positive correlation was observed with parental tobacco use from a study in Tunisia. Several other studies showed that adolescents with a parent or older siblings or a friend who smoke cigarettes are more likely to be smoker. Nearly one third (42%) of adolescent students reported that they saw tobacco promotional advertisements in the media or at social or sporting events. Although the advertisement of tobacco products in national electronic media (i.e. Radio and Television) is already banned, national newspapers and magazines with high youth readerships are still publishing the attractive advertisements of tobacco products. Besides, youths are being targeted through large billboards on city corners and through sporting events, music concerts, street festivals and other social events and gatherings that are sponsored by the tobacco companies. Around ( 31.4%) respondent claims point of sale nearby school is the most accessible to purchase smoked tobacco items another (12%) mentioned floating seller helps to buy cigarette easily in front of school gate.

Tobacco companies have long been known to design marketing strategies aimed at young potential tobacco users through different marketing approaches. After all tobacco advertisement in national television and radio was banned in Bangladesh long back, tobacco companies targeted adolescents through attractive advertisements in social media, large billboards on major city corners and by sponsoring different social activities. In addition, adolescents continue to be exposed to tobacco advertisements on foreign television channels that are accessible in Bangladesh. The results of this study showed that adolescent students exposed to tobacco promotional advertisements were 1. 11 times more likely to use tobacco than those who were not exposed. Similarly, other research has shown that youth who were regularly exposed to such advertisements were more likely to use tobacco.

Conclusion and Recommendations

Although a majority (85.4%) of adolescent students claimed to know the hazards of tobacco use, the results showed that about one fourth (14.6%) of the respondents, Personal factors have great impact on school going adolescent students to experience the smoke tobacco items but the impact of social factors is even higher among adolescent. Whereas the impact of point of sale plays another vital role to attract adolescent to smoke. A high proportion (65.5%) of adolescent students were ever-users of tobacco. These students more commonly used smoking items like cigarette and Biri. Most of the ever-users initiated tobacco use by 12-13 years of age. The majority of them are experimental users but are potential regular users in the future. Among ethnic groups, a higher proportion of adolescent students of the nuclear family were using tobacco than other groups (Choudhury, et al. [13]). For building mass awareness, School-based educational programs focusing on all forms of smoking and its effects should be planned and implemented.

Individual attention and culturally- appropriate education programs should be targeted at the adolescent students of the nuclear family. Different intervention programs should focus on various aspects like programs to discourage the uptake of tobacco among adolescent students between 12-13. Tobacco use by close relatives, friends and family members were influential factors for tobacco use of adolescent students. A substantial proportion of adolescent students are being exposed to the tobacco use behavior of family members and friends, creating an environment to develop more tobacco users in the future. Parental counseling is necessary to inform them about the influence of their tobacco use on their children. Programs to protect every adolescent from being exposed to the tobacco use of others are necessary (WHO, et al. [8,9,14]). Health education programs should be provided to adolescent students to raise the level of awareness of the threats of tobacco use and to change their perceptions. More anti-tobacco messages in the print, radio and/or television and psycho-social support to help students to develop a positive self-image to counter-act the pro-tobacco message are necessary.

This study suggests that exposure to tobacco promotional advertisements steers students towards the use of tobacco. Although tobacco-related advertisements are already banned in national electronic media, youths are being targeted through billboards, magazines and newspapers, and by the sponsorship of social and other events of youth interest by the tobacco companies (Zikmund, et al [15,16]). Efforts should be made to legislate a complete ban of all direct and indirect tobacco advertisements in the print and broadcast media [17-26]. Restrictions should be made in sponsoring targeted youth activities by tobacco companies. Besides, school authority should not allow shops allowing cigarette or bidi. This study suggests, therefore, that knowledge of health hazards is not sufficient to protect individuals from initiating tobacco use, although education is a necessary component of a comprehensive tobacco control program.

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Immunomodulatory Strategies for Spinal Cord Injury

Introduction

Traumatic Spinal Cord Injury (SCI) results in an initial primary injury, followed by secondary events including ischemia, anoxia, and excitotoxicity for the first minutes, hours, and days after injury [1-4]. As a key secondary event, inflammation is initiated in part by the rapid influx of circulating immune cells such as neutrophils, inflammatory monocytes, and macrophages [5-7]. These cells begin to release various inhibitory factors which contribute to an inhibitory microenvironment and damage adjacent intact tissue, thereby inducing permanent loss of locomotor and somatosensory functions below the level of injured spinal cord [8-10]. Although inflammatory response induces additional secondary damages, it is necessary to trigger wound healing and tissue regeneration after injury [5,11]. Additionally, there can be unwanted results such as systemic infection or disease when depleting immune cell populations. Therefore, systemic suppression of immune responses is unlikely to provide long-term therapeutic effects for SCI treatment. This review will focus on therapeutic strategies that reprogram the pro-inflammatory microenvironment towards a pro-regenerative milieu to limit inflammation-mediated secondary injury and promote functional regeneration after SCI.

Gene Therapy

Various gene therapy-based approaches have been introduced in the last twenty years and continue to be utilized as a powerful therapeutic approach to alter or correct defective genes for the treatment of multiple diseases [12,13]. While direct delivery of biologics is effective for short-term dosage, gene therapy provides the long-term sustained expression of the gene of interest. Vectors for gene therapy can be broadly classified as non-viral and viral vectors14. Non-viral vectors are cost-effective and less immunogenic, however, their ability to transfect cells is limited particularly in vivo [14]. Viral vectors can overcome these limitations by incorporating their genetic information into the host cell’s genome, inducing greater transduction efficacy and long-term expression of target genes. Choosing an appropriate vector will be the most important factor to achieve immunomodulatory therapy for SCI. The timing of onset of delivered transgene expression can be critical. The transgene expression by herpes simplex virus (HSV) or adenovirus occurs within 24 hours after delivery, which is faster than other viral vectors [15]. The transgene expression by lentivirus (LV) exhibits within 48 hours after administration and peaks the expression level of transgene after 3 days [6,16,17]. Vector size also can be an important factor since retrovirus can carry a maximum of 10 kb of single-stranded RNA and adeno-associated virus (AAV) can contain less than 5 kb of single-stranded DNA. Adenovirus and HSV can contain relatively higher transgene capacities. Viral vectors can be immunogenic compared to non-virial vectors which can result in unwanted side effects. While adenovirus can cause significant inflammatory responses, LV and AAV are considered less immunogenic and exhibit a transient immune response after administration [18].

Nanoparticles

Nanomedicine employing polymeric nanoparticle (NP) has received significant attention due to its inherent therapeutic potential to modulate immune responses to cure inflammatory responses-mediated disorders including SCI [19-22]. Previous study utilizing poly(lactide-coglycolide) (PLG)-based NPs with a negative surface charge indicated that intravenously administered NPs distracted circulating immune cells such as neutrophils and inflammatory monocytes prior to extravasation into the injured spinal cord, thereby reducing the pathological system indirectly after SCI [22]. NPs with highly negative zeta potential are taken up by targeted circulating immune cells through the scavenger receptors such as macrophage receptor with collagenous structure (MARCO) and reprogram them to affect their migration to the spleen [23]. Furthermore, some NP-positive cells accumulate at the injury, enabling upregulation of the expression of pro-regenerative factors that directly promote a more permissive environment after SCI [24,25]. Additionally, locally delivered poly (2-hydroxyethyl methacrylate) (PHEMA) and polycaprolactone (PCL) based NPs have been shown to modulate immune responses by attenuating the activation of pro-inflammatory macrophages and microglia specifically after SCI [26]. Other NPs including polystyrene [27], poly (lactic acid) [28], iron oxide [29], and gold [30] have also been employed to reprogram immune cells to modulate inflammatory responses after injury.

Stem Cell Therapy

Stem cells can proliferate and differentiate into any cell type present in the body. Considering their therapeutic potential and abilities of self-renewal and differentiation, stem cells are widely used following SCI [31,32]. In a preclinical study, the transplantation of mouse embryonic stem cells (ESC) into a rat spinal cord after SCI, demonstrated differentiation of transplanted stem cells into astrocytes, oligodendrocytes and neurons improving hindlimb weight support and coordination [33]. In a clinical study, bonemarrow derived mesenchymal stem cell transplanted patients showed improvement in American Spinal Injury Association Impairment Scale (AIS) grade from A to B in SCI patients [34]. Adult stem cells are more susceptible to immune rejection compared to ESC since they express major histocompatibility complex (MHC)- II and CD86 [35]. However, adult stem cells can reprogram the inflammatory environment by releasing multiple anti-inflammatory cytokines [36]. The neural stem cells (NSC) in the spinal cord have the ability to reprogram the immune cells after SCI [37]. They promote macrophage polarization towards a more pro-regenerative phenotype enhancing the expression of anti-inflammatory factors to improve nerve regeneration. NSC release multiple factors after an injury such as TGF-b, prostaglandin E, and nitric oxide that increase the number of regulatory T cells enhancing the expression of pro-regenerative factors. In addition, NSC can directly contact T cells and increase regulatory T cell populations, thereby enhancing the expression of anti-inflammatory factors while limiting the expression of pro-inflammatory factors [37]. Mesenchymal stem cells (MSC) can also modulate the inflammatory responses after SCI. MSC secrete interleukin 1 receptor antagonist (IL1-RA) that can induce the polarization of macrophages into pro- regenerative phenotype [38]. In addition, MSC-mediated over-expression of IL-6 and hepatocyte growth factor (HGF) can influence monocytes and induce the secretion of the high level of pro-regenerative factors including IL-10 in the injured site for immunomodulation [39]. Moreover, delivered MSC into the injured spinal cord limited the infiltration of the circulating inflammatory subset of immune cells to the injured site and promoted the phenotypical changes of macrophages and microglia into a more anti- inflammatory phenotype. MSC also restored the broken blood spinal cord barrier to prevent additional damage, thereby enhancing functional recovery after SCI [39].

Conclusion and Future Directions

Immunotherapeutic approaches for the treatment of SCI have the capacity to indirectly and/or directly enhance functional recovery. Although many preclinical studies have demonstrated therapeutic effects of immunomodulatory factors on SCI, the introduction of these strategies in the clinic faces various limitations for SCI victims. Further investigations will be required to assess how each of the immunomodulatory factors can be employed synergistically to reprogram the immune system to promote functional recovery after SCI while limiting life-threatening side effects.

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Comparative Analysis of Salivary Cellular Composition in Patients with Chronic Inflammatory Bowel Disease

Introduction

The oral cavity reflects the overall condition of the body. Signs of systemic disease are commonly manifested in the oral cavity before the systemic disease itself suspected. The various oral tissues (lips, tongue, gingiva, mucous surfaces, teeth) and fluids (saliva and cervical fluid) are involved in the presentation of disease state [1]. Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBD) with multifactorial origin including the immune system (autoimmune response and autoantibodies against organ-specific cellular antigens shared by the gastrointestinal tract and other systems), genetic sensitivity and environmental factors (diet, use of antibiotics or NSAIDs and the presence of enteral infections) [2]. Both the intestinal mucosa and the oral mucosa contain a large number of immune cells that make up the so-called mucosal immune system, functioned to preserve the body’s constancy against antigens that have penetrated the digestive system [3]. The commensal microbial flora promotes immune processes at the level of secretion of antimicrobial peptides, regulatory and effector immune cells [4]. This symbiosis with the microbiome helps maintain homeostasis, while dysbiosis induces altered immune responses with an inflammatory response [5]. CD and UC can engage not only the mucosa of the gut, but also have extra-intestinal manifestations in the oral cavity and other organs (joints, skin, eyes, bile ducts). Oral lesions as a clinical manifestation were first described in 1969 by Dudeney [6]. The incidence ranges from 0.5% to 30% most commonly seen in Crohn’s disease (cheilitis, ulcerations, fissures and glossitis) [7]. Oral manifestations in 5-10% of cases may be the first sign of bowel disease [8] or precede the appearance of intestinal lesions by a year or more [9]. Some of the oral changes are considered disease-specific and others are non-specific (aphthous stomatitis, pyostomatitis), but they can help diagnose and monitor the activity of the process [10,11].

Oral manifestations in IBD patients may be due to other causes, such as drug reactions, infections, malnutrition, adherence to specific diets, complications (inflammatory activity, dyselectrolytemia anemia, and hypovitaminosis) [1]. The relationship between changes in the oral cavity and IBD has not been sufficiently clarified. The inflammatory response, autoimmune genesis, dysbacteriosis, and infection are thought to be factors leading to specific changes in the oral cavity, most pronounced in the activity of inflammatory bowel mucosa [12]. The oral cavity and its oral fluid are an integral part of the gastro-intestinal tract (GIT), have a common phylogenetic and morpho-functional origin and structure, obey general neurohumoral regulation and, in addition to digestive function, have a protective role for the digestive system [13]. Saliva is an aqueous secretion with a slightly acidic to neutral pH of 6.0-7.0 and includes dissolved inorganic ions and various organic substances, including proteins (mucins, immunoglobulins, enzymes), epithelial cells, bacteria, leukocytes and food residues [14]. Oral fluid is a mirror of the metabolic, functional, hormonal and emotional state of the body [15]. Investigation of saliva as a non-invasive biological material may answer the question whether the pathological process and inflammatory activity in IBD can influence and alter its cellular content and functions. These cellular and functional alterations could serve us to monitor patients’ status. The aim of the present study is to investigate the salivary cell content and composition in patients with IBD and evaluate the effect of intestinal inflammatory activity on it.

Material and Methods

Subjects

The study included 54 patients (30 women and 24 men) with IBD (40 with UC and 14 with CDs), mean age 43.9±14.7 (range: 19 to 74 years) admitted to the gastroenterology wards at the University Hospital “St. Marina” and Military Hospital - Varna during September 2017 – May 2019 for the diagnosis of IBD or exacerbation of the disease, as well as for routine control colonoscopies. The diagnoses of UC and CD of the examined patients were made based on the criteria of ECCO Consensus 2019, year (European Crohn’s and Colitis Organization), including a complex of anamnestic, clinical, laboratory and instrumental studies. All participants enrolled in the study have signed informed consent. The local Ethical Committee approved the study (Protocol No 64/13.07.2017). As a control group, 80 healthy subjects (43 women and 37 men) aged between 20-65 years (mean: 43.1±10.8 years, corresponding to that of the patient group; p>0.05) undergoing routine prophylactic examinations, including dental status at the Military hospital-Varna were examined. The exclusion criteria were inflammatory changes in the mouth and dental procedures within 48-72 hours before the examination. The preliminary requirements for the collection and primary treatment of biological material have been clarified and respected. Unstimulated native saliva is collected in the morning by a passive droplet.

Collection Protocol

In all patients, saliva was collected under the same conditions: on an empty stomach (in the morning from 8-10 h) without the use of tonic drinks (coffee) and smoking. Unstimulated saliva is used. The oral fluid is collected in special sterile containers (conical bottom and graduated) by passively repeatedly removing the amount collected in the mouth within 5-10 minutes to a total amount of 2-3 ml. For reliable results, patients were instructed to comply with the following conditions: More than 30 minutes have elapsed since the last meal, drink, chewing gum or the last toothbrush and toothbrush cleaning. Mouth 5 min before the test, rinse twice with grunt for 10 sec. with saline or mineral water.

Material Processing

The saliva is hypotonic, lytic cellular processes are accelerated, so the material is quickly processed within 15-20 minutes. Until then, the samples are refrigerated at 4°C. On the graduated scale of the containers, we count the amount of saliva collected. We spin the containers at 2500 rpm for 5 minutes. The supernatant was separated. Resolve the sediment with dilute (dilution buffer for biological material at FUS 100 cell count) of the same amount to the original volume. We homogenize well. Cell counting is performed by the FUS 100 automated urinary sedimentation system (DIRUI). The method is based on flow cytometric microscopic high-speed images to enumerate and identify the formed elements in the sample. The pictures of the sample cells are compared to a database of images of various morphologies and crystals embedded in the instrument software and classified according to shape, structure and size. The number of images is calculated as count/μL. The morphological characteristics of the different cellular elements obtained by the automatic analysis are also verified visually. Routine laboratory methods were used for the assay of the inflammatory markers: WBC count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). All IBD patients were tested for FC, a specific biomarker for intestinal inflammation, by the quantitative immunochromatographic method Point-of-Care Test (Quantum Blue®fCAL, BÜHLMANN).

Statistical Analysis

Descriptive statistical analysis, non-parametric T-test for comparison of the mean values and Spearman correlation test were used for data processing. Values of p <0.05 were considered significant.

Results

The mean disease duration was 7.44±9.51 (1-31) years. All patients are on therapy (mono- or combination therapy) given in (Table 1). The pharmacological management of IBD aims to reduce inflammation and maintain remission. Therapy includes sulfasalazine, 5-aminosalicylates, glucocorticoids and immunosuppressants, and a series of biological agents, monoclonal antibodies (adalimumab, golimumab, infliximab). The classification of patients with IBD according to disease activity/severity is given in (Table 2). It is based on the indices used to evaluate activity or disease severity: for CD - CDAI (Crohn’s Disease Activity Index), and for UC - Mayo index. According to these indices, we divided the patients included in the study into two groups. The first group was patients with active disease (CDAI> 220 and Mayo index ≥2), and the second group was patients with mild or remission. The number of cellular elements (epithelial cells, erythrocytes, leukocytes and bacteria) are presented in (Table 3). (Figure 1) Leukocytes and epithelial cells, as significant components of the mucosal epithelial barrier, are most affected by changes in physiological and pathological conditions. Physiological factors such as gender and age have been found to influence the values of these parameters in both the control and patient groups. The comparative analysis showed that the number of cells in the norm had a gender variation. Women have slightly higher values than men.

Table 1: Types of drug therapy in the IBD study group.

Table 2: Demographic and clinical characteristics of patients with.

Table 3: Cellular elements in unstimulated saliva in the three study groups.

Note: *Differences in the number of cell elements in the three study groups were evaluated using One way ANOVA Bonferroni correction

**The reference limits were determined for the purposes of our study in a representative group of clinically healthy persons from the Bulgarian population. The selection of persons from the reference group (n =186) was carried out randomly - from persons passing to MMA-Varna, subject to a routine annual preventive examination or expert assessment, without subjective and objective deviations (healthy persons) and normal dental status.

Figure 1: Comparison of salivary cell elements in the three studied groups

A) Erythrocytes,

B) Leukocytes,

C) Epithelial cells and

D) Bacteria (One way ANOVA with Bonferroni correction is attached).

The data are presented in (Table 4). Similar observations are described by Rijkschroeff, et al. [16]. Many studies show that the oral mucosa is sensitive to the effect of sex hormones (estrogens and progesterone). Different periods in a woman’s life, such as puberty, pregnancy, and menopause, are associated with changes in their levels affect oral mucosa. Donald, et al. [17,18] disclose wellexpressed rhythmic changes in the oral cavity cells, coinciding with changes detected in vaginal smears, thus reflecting the hormonal state of the menstrual cycle [17,18]. We also find a moderate positive correlation (r=0.358; p=0.009) between age and increased oral leukocytes, but not with epithelial cells. The number of leukocytes in the mouth decreases in proportion to the reduction of teeth due to their inability to migrate through the gingival crevices. The mean disease duration was 7.44±9.51 (1-31) years. We found no relationship between disease duration and cellular element values. They are influenced by the activity of the inflammatory process.

Table 4: Values of leukocytes and epithelial cells in control and patient groups and their sex dependence.

Discussion

Cells found in the oral fluid have different origins. Leaving the ductus of the parotid gland, the saliva contains no cellular elements [19]. They are added by buccal mucosa, migration of leukocytes and erythrocytes from different areas of the oral mucosa. The largest is the abundance of epithelial cells that are usually excreted in chewing processes. The oral cavity is covered by squamous epithelial cells, which are a barrier against mechanical, chemical damage and pathogenic microbiological invasion [20]. The normal mucosal epithelium is a multilayer flat with high regenerative capacity. Due to its involvement in the digestive process, however, it remains un keratinized or partially corrosive in certain places (such as the intricately arranged mucous membrane of the tongue with the papillae available) and secretes mucus to facilitate the passage of food. It is already known that epithelial cells are not passive bystanders, but are metabolically active and able to respond to external stimuli by synthesizing several cytokines, adhesion molecules, growth factors, chemokines and matrix metalloproteases [21]. Morphologically, squamous cells have a small nucleus and a large polygonal cytoplasm with numerous granulations. Cell sizes are 85-125 μm, while those of the gingiva and periodontium are smaller in size. According to Watanabe, the number of epithelial cells in the saliva is 4x105/ml. [22].

Several studies have tracked the number and change in morphology of epithelial cells in screening and diagnosis of precancerous conditions and oral cancer [23]. Inflammatory lesions and periodontal disease also lead to an increase in the number of squamous cells secreted. Our study found that the control group had higher values of epithelial cells, given by some researchers as the norm. This can be explained by the presence of dental bridges and dental crown, whose finding in the oral cavity increases with age. Smoking is also an essential factor acting irritably on the oral mucosa and a significant risk factor for inflammatory and neoplastic processes. In a previous study, we found a statistically significant difference in the number of epithelial cells and leukocytes in smokers compared to non-smokers. Of the subjects in our control group, smokers were 36, representing 45%, and 23 (43%) from the patient group. We observed an increased number of epithelial cells in patients with IBD, which was statistically significant relative to the control group (p=0.0192). However, no such dependence was observed between the two patient groups. The finding is believed to be due to the chronic inflammatory process, dehydration and poor nutrition, especially during relapse. Little is known about the factors that affect the relative number of epithelial cells and leukocytes in the oral cavity. The buccal mucosa is relatively permeable and has a rich blood supply, with continuous flow and migration of leukocytes from the gingival fluid through the gingival gap in the saliva [24].

Healthy buccal mucosa contains several cells involved in immune function, including lymphocytes (T cells), polymorphonuclear cells (including neutrophils and eosinophils), and mast cells [25,26]. They protect the oral cavity and are an element of the innate immune response. Leukocyte counts in norm and pathology have been the subject of several studies. It is estimated that about 1 – 4x105 cells are normally found in 1 ml of saliva. Their number has inter-individual but also circadian intra-individual variation [19]. Leukocytes are predominantly represented by polymorphonuclear cells (PMNs) - 90% and 10% monomorphonuclear cells (MMNs). The major representatives of MMNs are B lymphocytes (60%), T cells (about 20-30%) and macrophages (~10%), which are integral parts of mucosa-associated lymphoid tissue (MALT). Its role is to induce an immune response to specific antigens from the environment and to develop local immunity. In a healthy state, approximately 30,000 oral PMNs (oPMN) per minute arrive through gingival cervical fluid (GCF), which inflows into the oral cavity from the periodontal sulcus [27]. Eighty percent of oPMNs are viable and functional in the gingival gap and play a phagocytic and antibacterial role. In the hypotonic saliva, oPMNs undergo rapid lytic changes. Functional PMNs are paramount to innate immunological processes, including maintenance of oral health.

In acute and chronic inflammatory processes in the oral cavity, the amount of oPMN increases. Rijkschroeff, et al. [16] report significantly higher expression of CD11b in oPMNs compared to circulating blood PMNs in healthy individuals, suggesting their facilitated migration through oral mucosal tissues. There are insufficient studies on the effects of systemic diseases on salivary cell composition and oral hemostasis. We found a statistically significant difference (p<0.0001) in leukocyte counts between the control group and patients with IBD, but there was no similar relationship between the two patient groups (with activity and mild/remission). The active involvement of oral mucosa in the immunological response and inflammation most likely causes leukocyte growth in the saliva. We did not find a correlation between the total number of circulating blood leukocytes and the number of salivary leukocytes. Although bPMNs reflect the general inflammatory state of an individual, the number of oPMNs varies greatly depending on local influences in the oral cavity [16,28]. A similar weak correlation was observed between oPMN and C-reactive protein as a marker of inflammation. Corticosteroid therapy probably affects the content of cellular elements in the oral fluid, causing increased permeability and migration of erythrocytes and leukocytes through the gingival crevices. Calprotectin is a cytoplasmic protein secreted by the degranulation of neutrophil leukocytes and monocytes locally at the site of inflammation.

Faecal calprotectin (FC) has in recent years become a marker reflecting the activity of the inflammatory mucosal response in IBD. Majster, et al. [28] examined calprotectin in saliva from patients with IBD, finding elevated values in both stimulated and unstimulated saliva. In our patients, we routinely examine FC for disease monitoring. By correlation analysis, we checked whether there was a relationship between oral leukocyte count and FC as a marker of inflamed intestinal mucosa. A weak correlation r=0.2043 was found. For comparability, the correlation between the number of salivary leukocytes and salivary calprotectin should be used. The normal amount of erythrocytes in the oral fluid is negligible, although there is no accurate literature data on their normal count per milliliter. Most studies look at the increase in red blood cells and the presence of salivary blood in fissures, lesions, periodontal or cancerous diseases in the oral cavity [29,30]. Our study found equally higher erythrocyte values in both patient groups, regardless of the inflammatory process activity. This is most likely due to side effects of medicines leading to increased permeability of the vessels in the oral cavity, ecchymoses and bleeding. It is more commonly observed in patients with mono or combination therapy with Infliximab (a TNFα inhibitor) [31]. The oral cavity and gastrointestinal tract are highly colonized sites [16]. Bacteria are the most numerous cells, represented mainly by normal microflora, but pathological species can also be found.

In healthy individuals, the microflora have a symbiotic relationship with the host organism and possess important and unique functions, including metabolic function. The human oral microbiome is regarded as a community of more than 300 species of microorganisms, with a total salivary total of 106-108 CFU/ml (colony-forming unit) [32]. Gram positive bacteria predominate, and anaerobic bacteria are about 10 to 100 times the amount of aerobes [33]. In the study group, especially those with inflammatory process activity, the bacterial count was statistically significantly lower. During the relapse, antibiotic therapy is included as part of the treatment and treatment of the inflammatory process. Most likely, antibiotics lead to microflora suppression by reducing their population or leading to dysbiosis. The thesis about the reducing role of AB against the oral microflora is confirmed by the fact that in the second patient group and without antibiotic therapy, the number of salivary bacteria is almost similar to that of the control group.

Conclusion

Oral health and homeostasis, maintaining oral hygiene is crucial for patients with IBD during their treatment and remission. The concept of enumeration of cellular elements in saliva with the FUS 100 automated system may be proposed as a possible screening method for the indication of inflammatory changes in the mouth. A rapid, non-invasive qualitative and quantitative assessment of cellular composition would be a new approach in laboratory or clinical diagnosis and monitoring and management of patients with IBD.

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