Introduction
Nowadays, about 1.25 billion people are known to be smokers worldwide and
approximately 5 million death save attributed to smoking each year [1]. It has
been reported that cigarette smoke
consists of about 4000 substances known to be antigenic, cytotoxic, mutagenic,
and carcinogenic
influencing various human body organs both locally and systemic ally [2]. Both
arms of immune system including innate and adaptive responses also were
affected by cigarette smoking [3].Therefore,cigarette smoking influences cell mediated
and antibody mediated immune responses [2,3]. The principle function of the
immune system is defense against infections and killing of tumor cells [4,5].
Accordingly,
cigarette smoking was considered as a significant risk factor for cancers as
well as infections [6,7]. Tetanus is an infectious disease caused by
neurotoxin secreted from the gram-positive anaerobic bacillus Clostridium (C)
tetani. Despite being preventable with an effective vaccine, tetanus remains a
significant cause of morbidity and mortality worldwide [8]. Contamination of a
wound with spores of C. tetani usually causes tetanus, due to release of
neurotoxin. Tetanus is rare in people with history of complete vaccination
course. However, inadequate tetanus toxoid vaccination and wound prophylaxis
are associated with tetanus. Risks of fatal disease is also reported to be
higher in patients 60 years of age and older [9,10]. The current recommendation
of Advisory Committee on Immunization
Practices and Centers for Disease Control and Prevention is to administrate
the diphtheria, tetanus, and acellular
pertussis vaccine (DTaP) at ages 2, 4, 6, 15-18 months with a booster at 4-6
years [8]. In Iran, immunization against diphtheria, tetanus and pertussis has
been applied since 1950 using a local vaccine manufactured by Razi Institute
(Razi-DTwP) [11,12]. In most countries, booster immunization of diphtheria and
tetanus is recommended to be performed every 10 years. Booster
tetanus-diphtheria (Td) vaccine is recommended to start at the age of 11-12
years old [13].
Cigarette smoking has local and
systemic effects on the immune system and increases the susceptibility to
infections and also alter the course of infectious diseases [6]. The results of
some studies also demonstrated that smoking
impairs the antibody response to infectious agents
such as viruses and bacteria [14]. Moreover, lower antibody responses to some
vaccines have been reported in smokers in comparison to non-smokers [15]. Furthermore, cigarette smoking
is associated with delay in wound healing such as skin wounds [16,17].
Accordingly, it is expectable that cigarette smokers may
be at a higher risk for tetanus than non-smoker subjects. This study
conducted for the first time to evaluate the serum levels of anti-TTA in cigarette smoker subjects and in a healthy
non-smoking group.
Material and Methods
This
descriptive study was carried out in Rafsanjan (a city that located in South-East
of Iran in Kerman province) From January 2012 to June 2012. One hundred cigarette smoking men (mean age: 44.29
± 11.66 years) and 100 healthy non-smoker men (mean age: 43.38 ±
8.76 years) were included in the study.
As
mentioned, the universal vaccination of infants and children
against diphtheria, tetanus and pertussis has been incorporated in the
national immunization scheme in Iran since 1950 [11]. Accordingly all subjects
born after this time (including subjects enrolled to this study) have received
DTwP vaccine. Based-Line vaccination has been done by using 3 doses of vaccine
at 2, 4, and 6 months of age with 3 booster reminder doses at age 18–24 months,
5–6 years and 18-24 years. There have been no other vaccination records
regarding tetanus for participants.
The cigarette smokers were selected from subjects referring to the
Pathobiological Laboratory of Rafsanjan University of Medical Sciences.
Cigarette smoking, defined as smoking ≥1 cigarettes per day for at least
one year. Smoking status was assessed via questionnaire and all smokers
recruited for the study were current smokers. A healthy non-smoking group (with
similar socioeconomic status to the smoker group) was recruited among blood
donors of Rafsanjan Blood Transfusion Center. The non-smokers were basically health,
with no history of cigarette smoking or
living as passive smokers. Subjects with disease such as history of recurrent infections, asthma,
allergy and atopic diseases, any suspected immunological disorders and
use of any drugs were all excluded from
the study. Other exclusion criteria included any acute or chronic illnesses, malignancy, surgery and major
trauma within 6 months prior to blood collection. Also the participants had not
received any immunomodulating treatments during the 6 months prior to sampling.
All participants had normal CBC, normal liver and normal renal function tests.
Moreover, the blood lipids profile (including cholesterol and triglyceride) of
all subjects were within the normal range.
This study was evaluated
and approved by the Ethical Committee of Rafsanjan University of Medical
Sciences and all
of the
participants gave written informed consent to take part in the study.
Peripheral blood (2-4 milliliter) was
collected from the subjects of 2 groups and the serum was separated and stored
at –20 C.
Serum
levels of anti-TTA was measured by an enzyme-linked immunosorbentassay (ELISA)
method by using commercial kits (IBL-Hamburg GmbH, Hamburg, Germany).
Principally, the wells of ELISA plate are coated with tetanus toxin as antigen.
Specific antibodies of the sample binding to the antigen coated wells are
detected by a secondary enzyme conjugated antibody specific for human IgG.
After the addition of substrate the intensity of the color is proportional to
the amount of IgG-specific antibodies. Results of samples can be directly determined
using the standard curve.
In
the test procedure, after pipetting 100 μL of each standard and diluted
serum (1/100) into the respective wells, plates were incubated for 60 min at 25
C. At the end of the incubation period, plate strips were washed four times
with 300 μL washing buffer per well. Then, 100 μL conjugate was added
into each well. After the incubation period (for 30 min at 25 C), plate strips
were washed as previously described. Thereafter, 100 μL of
Tetramethylbenzidine (TMB) substrate solution was added into each well and
incubated for 20 min at 25 C in dark. The substrate reaction was stopped by an addition
of 50 μL of sulfuric acid into each well. Optical density of each well was
measured with a photometer at 450 nm within 60 min after pipetting of the stop
solution.
Anti-TTA
was measured by using standard samples with known concentrations of anti-TTA
expressed as IU/mL, provided by the manufacturer. According to manufacturer
guideline, an antitoxin concentration ≥0.1 IU/mL was considered as
protective level of antibody. Tetanus antitoxin levels less than 0.1 IU/ml were
considered to denote susceptibility [11,12]. The sensitivity of ELISA kit was
0.004 IU/ml.
Differences in antibody levels were
analyzed using t-test and Chi-square test where appropriate and P-values of less
than 0.05 were considered significant. All data were analyzed by SPSS software
(version 15, Chicago, IL).
Results
The seroprotective rate of anti-TTA
in healthy non-smoking group (99%) was significantly higher than that observed
in smoker group (78%, P<0.0001). The mean titer of anti-TTA in non-smoking
group (5.32 ± 0.26
IU/ml) was also significantly higher than that in smoker subjects (1.03
± 0.16 IU/ml;
P<0.0001).
Discussion
The results of the present study showed that the seroprotective
rate and the mean titer
of anti-TTA were lower in cigarette smoking group as compared with non-smoker
subjects. These findings represent that cigarette smoker subjects have a
greater susceptibility to tetanus infection. There is no studies regarding the
tetanus immunity in cigarette smoker subjects. In one study it was reported
that in children with smoker parents the mean titer of anti-TTA was
significantly lower in comparison with those who had non-smoker parents [18].
Moreover, it has been reported that the persistence of immunity to hepatitis B
in cigarette smokers was lower than nonsmokers [19]. It has been recently
reported that the immune response (within the nasal mucosa) to attenuated
influenza virus vaccine were suppressed in smokers as compared to nonsmokers [20]. Furthermore, it has
been reported that cigarette
smokers are susceptible to infection and sepsis [21]. Some immunologic disorders have been
shown in cigarette smokers. Several investigators have shown that long-term
smoking significantly reduces serum levels of immunoglobulins (including IgG,
IgA and IgM) in humans [22,23]. The suppressive effects of smoking on cell
mediated immune responses have been also reported [24]. Moreover, cigarette
smoking has been associated with depression of macrophages functions, decrease
of CD4+/CD8+ ratio, reduction of the
numbers of dendritic cells, impairment of intracellular signaling pathways, and
diminished production cytokines and chemokines [25,26]. In our previous study, it has been
observed that cigarette smoke-exposed saliva samples have profound suppressive effects on both
cellular and humoral immune responses in a mouse animal model [27].
Furthermore, we observed that cigarette smoke-exposed saliva
have lethal effects on human lymphocytes [28].
It
should be noted that vaccination is one of the most significant tools for inducing
the prophylaxis against infection diseases. This prophylaxis is mediated via
inducing of immunologic memory [29]. It has been
demonstrated that cigarette smoke reduces the numbers of circulating memory T
cells [30]. Accordingly, the lower levels of anti-TTA in cigarette
smokers may also be attributable in part to impairment of
immunological memory in this subjects.
The results of the present
study also demonstrated that both the titer of anti-TTA and the seroprotection
rate were significantly lower in individuals with smoking duration of >10
years in comparison to smokers with smoking duration of ≤10 years.
Moreover, the titer of anti-TTA in individuals with cigarettes smoking of
>10 daily was also significantly lower in comparison with smokers with
cigarettes smoking of ≤10 daily. The association of cigarette smoking
intensity with several pathological outcomes such as increase in the risk of
bladder cancer, higher dental caries, male infertility, increase in the risk of
type 2 diabetes mellitus has also been reported in different studies [31,32].
Moreover, the association of cigarette smoking intensity with some inflammatory
parameters such as higher levels of plasma complement C3, higher expression of inducible nitric oxide
synthase has also been reported [33,34]. An association between
cigarette smoking intensity and more susceptibility
to human papillomavirus has been demonstrated too [35]. There is no
study regarding the association of immune responses and cigarette smoking
intensity. However, the results of the present study represent that the
influence of cigarette smoking on the titer of anti-TTA mediated in a time- and
dose-dependent manner. Thus, there is a negative association between the
smoking burden and the titer of anti-TTA.
According
to serum levels of anti-TTA we have arbitrary classified the subjects to
several subgroups. In non-smoking group 75% of subjects have the anti-TTA >3
IU/mL whereas only 8% of smoker subjects have the anti-TTA >3 IU/mL. On
other hand, the proportion of subjects with low anti-TTA (<1 IU/mL) was
higher in smoker groups in comparison to the non-smoking group. This
classification can be use for designing of the re-vaccination program. According
to manufacturer guideline, for individuals with antitoxin level <0.1 IU/ml
(no protection), vaccine should be administered. Individuals with antitoxin
level 0.1-1 IU/ml should be controlled after 1-2 years. Individuals with
antitoxin level 1.1-5.0 IU/ml should be controlled after 2-4 years. Individuals
with antitoxin level >5.0 IU/ml should be controlled after 8 years. Similar
guideline has been presented for re-vaccination by Centers for Disease Control
and Prevention [36, 37]. The CDC's Advisory Committee on Immunization Practices
recommended that all smokers aged 19-64 years be vaccinated with the
pneumococcal polysaccharide vaccine [38, 39]. The incorporation of other
vaccines such as tetanus vaccine in a smokers vaccination program need more
consideration in future studies.
Conclusion
The results of the present study
showed lower levels of anti-TTA in cigarette smokers. Moreover, a reverse
association was also observed between the titer of anti-TTA and smoking burden.
Acknowledgements
This
work was financially supported by a grant from Rafsanjan University of Medical
Sciences, Rafsanjan, Iran.
Conflict of
interest: Non declared
References
1.
Lenney W, Enderby B. "Blowing in the wind": a
review of teenage smoking. Arch Dis Child 2008; 93(1):72-5.
2.
Domagala-Kulawik J. Effects of cigarette smoke on the
lung and systemic immunity. J Physiol Pharmacol 2008; 59 Suppl 6:19-34.
3.
Arnson Y, Shoenfeld Y, Amital H. Effects of tobacco
smoke on immunity, inflammation and autoimmunity. J Autoimmun 2010; 34(3):J258-65.
4.
Maryono S, Guntur A, Suharti C. Serum complement 3 (C3)
and complement 4 (C4) level in febrile neutropenia patients at Dr. Kariadi
Hospital, Semarang and Dr. Moewardi Hospital, Surakarta. Acta Med Indones 2008;
40(1):14-8.
5.
Wraith DC, Nicholson LB. The adaptive immune system in
diseases of the central nervous system. J Clin Invest 2012; 122(4):1172-9.
6.
Huttunen R, Heikkinen T, Syrjanen J. Smoking and the
outcome of infection. J Intern Med 2011; 269(3):258-69.
7.
Hymowitz N. Smoking and cancer: a review of public
health and clinical implications. J Natl Med Assoc 2011; 103(8):695-700.
8.
Ataro P, Mushatt D, Ahsan S. Tetanus: a review. South
Med J 2011; 104(8):613-7.
9.
Koromath FS, Sumarmi, Hermawan AG. Infectious disease
pattern and serum albumin levels in elderly people hospitalized at Dr. Moewardi
Hospital Surakarta during 2004. Acta Med Indones 2008; 40(3):114-6.
10.
Reed DB, Westneat SC. Exposure risks and tetanus
immunization status in farmers ages 50 and over. South Med J 2009; 102(3):251-5.
11.
Zarei S, Jeddi-Tehrani M, Akhondi MM, Zeraati H,
Pourheidari F, Ostadkarampour M, et al. Primary immunization with a triple
diphtheria-tetanus-whole cell pertussis vaccine in Iranian infants: an analysis
of antibody response. Iran J Allergy Asthma Immunol 2009; 8(2):85-93.
12.
Zarei S, Jeddi-Tehrani M, Akhondi MM, Zeraati H,
Kheirkhah T, Ghazanfari M, et al. Immunogenicity of a triple
diphtheria-tetanus-whole cell pertussis vaccine in Iranian preschool children.
Iran J Immunol 2007; 4(2):101-9.
13.
Choi JH, Choo EJ, Huh A, Choi SM, Eom JS, Lee JS, et
al. Immunogenicity and safety of diphtheria-tetanus vaccine in adults. J Korean
Med Sci 2010; 25(12):1727-32.
14.
Simen-Kapeu A,
Kataja V,
Yliskoski M,
Syrjänen
K, Dillner J,
Koskela P,
et al. Smoking impairs human
papillomavirus (HPV) type 16 and 18 capsids antibody response following natural
HPV infection. Scand J Infect Dis 2008; 40(9):745-51.
15.
Koff RS.
Immunogenicity of hepatitis B vaccines:
implications of immune memory. Vaccine
2002; 20(31-32):3695-701.
16.
Kean J. The effects of smoking on the wound healing
process. J Wound Care 2010; 19(1):5-8.
17.
Thomsen SF, Sorensen LT. Smoking and skin disease. Skin
Therapy Lett 2010; 15(6):4-7.
18.
Baynam G, Khoo SK, Rowe J, Zhang G, Laing I, Hayden C,
et al. Parental smoking impairs vaccine responses in children with atopic
genotypes. J Allergy Clin Immunol 2007; 119(2):366-74.
19.
Horowitz MM, Ershler WB, McKinney WP, Battiola RJ.
Duration of immunity after hepatitis B vaccination: efficacy of low-dose
booster vaccine. Ann Intern Med 1988; 108(2):185-9.
20.
Horvath KM, Brighton LE, Herbst M, Noah TL, Jaspers I.
Live attenuated influenza virus (LAIV) induces different mucosal T cell
function in nonsmokers and smokers. Clin Immunol 2012; 142(3):232-6.
21.
Koh GC, Peacock SJ, van der Poll T, Wiersinga WJ. The
impact of diabetes on the pathogenesis of sepsis. Eur J Clin Microbiol Infect
Dis 2012; 31(4):379-88.
22.
Aral M, Ekerbicer HC, Celik M, Ciragil P, Gul M.
Comparison of effects of smoking and smokeless tobacco "Maras powder"
use on humoral immune system parameters. Mediators Inflamm 2006; 2006(3):85019.
23.
Sopori M. Effects of cigarette smoke on the immune
system. Nat Rev Immunol 2002; 2(5):372-7.
24.
Edwards D. Immunological effects of tobacco smoking in
"healthy" smokers. COPD 2009; 6(1):48-58.
25.
Tymkiw KD, Thunell DH, Johnson GK, Joly S, Burnell KK,
Cavanaugh JE, et al. Influence of smoking on gingival crevicular fluid
cytokines in severe chronic periodontitis. J Clin Periodontol 2011; 38(3):219-28.
26.
Birrell MA, Wong S, Catley MC, Belvisi MG. Impact of
tobacco-smoke on key signaling pathways in the innate immune response in lung
macrophages. J Cell Physiol 2008; 214(1):27-37.
27.
Jafarzadeh A, Bakhshi H, Rezayati MT, Nemati M.
Cigarette smoke-exposed saliva suppresses cellular and humoral immune responses
in an animal model. J Pak Med Assoc 2009; 59(11):760-3.
28.
Nozad-Mojaver Y, Mirzaee M, Jafarzadeh A. Synergistic
effects of cigarette smoke and saliva. Med Oral Patol Oral Cir Bucal 2009; 14(5):E217-21.
29.
Castellino F, Galli G, Del Giudice G, Rappuoli R.
Generating memory with vaccination. Eur J Immunol 2009; 39(8):2100-5.
30.
Vardavas CI, Plada M, Tzatzarakis M,
Marcos A, Warnberg J, Gomez-Martinez S, et al. Passive smoking alters
circulating naive/memory lymphocyte T-cell subpopulations in children. Pediatr
Allergy Immunol 2010; 21(8):1171-8.
31.
Pramono LA, Setiati S, Soewondo P, Subekti I,
Adisasmita A, Kodim N, et al. Prevalence and predictors of undiagnosed diabetes
mellitus in Indonesia. Acta Med Indones 2010; 42(4):216-23.
32.
Campus G, Cagetti MG, Senna A, Blasi G, Mascolo A,
Demarchi P, et al. Does smoking increase risk for caries? a cross-sectional
study in an Italian military academy. Caries Res 2011; 45(1):40-6.
33.
Coelho C, Julio C, Silva G, Neves A. Tobacco and male
infertility: a retrospective study in infertile couples. Acta Med Port 2009; 22(6):753-8.
34.
Nagaya T, Yoshida H, Takahashi H, Kawai M. Heavy
smoking raises risk for type 2 diabetes mellitus in obese men; but, light
smoking reduces the risk in lean men: a follow-up study in Japan. Ann Epidemiol
2008; 18(2):113-8.
35.
Meric A, Ozucer B, Gedikli O, Yildirim Y, Korkut AY,
Kahya V, et al. Impact of Smoking on p65 Nuclear Factor kB, p38
Mitogen-Activated Protein Kinase, and Inducible Nitric Oxide Synthase
Expression Levels in Oral Mucosa. J Craniofac Surg 2012; 23(4):970-3.
36.
van Greevenbroek MM, Jacobs M, van der Kallen CJ, Blaak
EE, Jansen EH, Schalkwijk CG et al. Human plasma complement C3 is independently
associated with coronary heart disease, but only in heavy smokers (the CODAM
study). Int J Cardiol 2012; 154(2):158-62.
37.
Vaccarella S, Herrero R, Snijders PJ, Dai M, Thomas JO,
Hieu NT et al. Smoking and human papillomavirus infection: pooled analysis of
the International Agency for Research on Cancer HPV Prevalence Surveys. Int J
Epidemiol 2008; 37(3):536-46.
38.
Centers for Disease Control and Prevention (CDC).
Neonatal tetanus--Montana, 1998. MMWR Morb Mortal Wkly Rep 1998; 47(43):928-30.
39.
Kuehn BM. CDC panel recommends vaccine for smokers;
reviews HPV safety data. JAMA 2008; 300(23):2713-4.
