Evaluating the Humoral Immunity and Interleukin 18 Receptor 1 in some Patients with Molluscum Contagiosum Infection

The molluscum contagiosum virus (MCV) is a dermatotropic poxvirus. The causative agent of molluscum contagiosum (MC) is nonlethal, common and worldwide. Additionally, little inflammation is associated with MC papules. The present study aims to evaluate the immune status of MC patients by measuring the level of immunoglobulins IgG and IgM by using the radial immune diffusion assay (RIA) and the level of interleukin 18 receptor 1 (IL-18R1) by the Enzyme-linked immunosorbent assay (ELISA).The study is conducted during November 2013 to April, 2014 in outpatient clinic of Baquba Teaching Hospital. There are 75 patients, diagnosed with clinical lesions of MCV on different areas of the body, whose age is ranged between 2-50 years including 40(53.3%) males and 35(46.7%) females. The study includes 15 healthy persons age between 2-50 years. The level of IL 18R1 were significantly elevated in patients (677.15±874.22) compared with control (178.46±31.79 ng/ml). There is also a significant elevation in the mean level of serum IgM, where it is 1946.6±825.6 mg/dl while in control group is 140.1±68.7mg/dl. By contrast in patients with lower levels of IgG than the control, the mean serum IgG level in patient is 221.9±96.7 mg/dl while in the control is 1229.9±299.7 mg/dl. Finally, there is no significant difference between MC patients from rural area and urban area.


Introduction:
Molluscum Contagious Virus (MCV) is first described and later named by Bateman in the early nineteenth century [1].Additionally , there are various types of MCV and they are most commonly seen on humans, but have been found to be on animals such as chickens, horses, oxen and cows [2].Furthermore, Molluscum contagiosum is caused by up to four closely related types of poxvirus, MCV-1 to 4 and their variants.However, MCV-2 causes the majority of infections, (60%) that is [3].On other hand , humoral immunity and cellular immunity play an important role in the body's defense against molluscum infection.Most adults are more resistant to MCV infection than children because they have developed IgG antibodies against the viral antigen [4].Moreover Molluscum contagiosum virus contains an IL-18 binding protein gene that it apparently acquire from humans.This blocks the host's initial effective Th-one immune response against the virus by reducing local IFN-gamma production [3],also IL-18, a recently described member of the IL-1 cytokine Open Access super family ,is currently recognized as an important regulator of innate and adaptive immune responses.Furthermore, this little review will describe the basic biology of IL-18 and thereafter address its potential effector and regulatory role in several human disease states including autoimmunity and infection such as MCV infection.IL-18, formerly is known as interferongamma (IFN-γ)-inducing factor [5].The study of the immune status of patients infected with the Molluscum contagiosum virus is the first study whether in Diyala governorates or in Iraq, so the present study aims at assessing the immune status of patients infected with molluscum contagiosum through the measurement of the level of immunoglobulin (IgG,IgM) by the radial immune diffusion assay and measuring the level of interleukin 18 by Elisa assay which immune responses are key for the eventual resolution of MC .reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm± 10nm using Elisa reader.Finally, the concentration of IL18R1 in the samples is determined by comparing of the samples to the standard curve constructed from standard [6].Moreover, the detailed procedure is carried out as suggested by the leaflet supplied with the test kit Mybiosource(U.S.A).

IgG and IgM Level in the Serum :
The IgG and IgM levels in serum are detected by single radial diffusion assay (SRIA).Principle of the test includes: The concentration of IgG and IgM is measured by a single radial immunodiffusion (SRID) method in which equal volumes of reference sera and test samples are added to wells in agarose containing mono specific antisera.After that, the sample diffuses radially through this gel and the substance being assayed form a precipitin ring with the mono specific antisera.Ring diameters are measured and a reference curve is constructed on a graph paper.Unknown concentration is determined form the references standard curve [7,8].The detailed procedure is carried out as suggested by the leaflet supplied with the test kit Bussero (Milan) ITALY Statistical Analysis: Data Analysis are computer aided and the statistical analysis is done by using SPSS version 20 computer software.Also, frequency distribution and percentage for the selected variable were done.The independent t-test is used and the P-Value (less than 0.05) is considered as the level of significance [9].

Residence of the Studied Subjects in Both Groups
Forty seven (62.7%) patients are from rural area while 28 (37.7%) are from urban.There is no statistical difference between both groups as the p value is equal to 0.567.

Discussion
The present study reveals that most of patients are found in ages ranging between (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) years.This result might agree with the study done by [10], who reports that most of the patients of the ages ranging between (11-30) years, whereas it disagree with the study done by [11] where the range between (31-40) years.This difference in results may be due to the difference in social living levels.The present study shows that male to female ratio is 1:1.14.as it resembled to most studies in the world, where in study done by [12] the male to female ratio was 1:6.1.The same result of 1:1.6 is obtained from the study done by [13] .The other study reported by [14] the male to female ratio is 1:4.2.IL-18R1 increases in the serum of 41.3% of infected patients which could be assigned to the critical role of IL-18R1 in defense against virus infections and provides a mechanism for evasion of the immune system by MCV [15].These results are in concurrence with the study done by [16] who has noted an increased level of IL-18R1 in the serum of patients with molluscum contagiosum which is between (20-40%) higher level.Another study also demonstrates an increased levels of IL-18R1 in patients with molluscum contagiosum [17].When IL-18 is first described, it is as an IFN-gammainducing factor during endotoxemia in mice preconditioned with a prior injection of heat-killed P. acnes, a known stimulator of the reticuloendothelial system, particularly of Kupffer cells in the liver.Because of its property to induce IFN-gamma, interleukin -18 is by default a member of the Th1-inducing family of cytokines (IFN-γ, IL-2, IL-10 , IL-12, and IL-15).However, there is also are indication that IL-18 plays a role in hypersensitivity reactions, a characteristic of T-helper 2 responses.Indeed, the biologic activities of IL-18 are clearly related to host response to virus infection (MCV) [18] .Like all interleukin responses to infections, there are 2 sides to the coin, IL-18 protects the host by its capacity to induce IFN-gamma and other immunestimulatory cytokines in a non-specific fashion (commonly called -innate‖ immunity), which assists the immune system in a specific T and B cellmediated response (commonly called -adaptive‖ immunity).Moreover , one can conclude that some of the pathologic significances of infection are, in part, mediated by IL-18 in somewhat the same fashion as mediated by IL-1 and TNF-alpha.These are clearly harmful to the host and are the targets of therapeutic intervention [19].Through the development of the innate immune response, cytokines and cytokine receptors evolved early.Several mechanisms are intrinsic to the capacity of a cytokine to cause inflammation.These are identical to those that assist the host in fighting the infection.In the case of interleukin -18, the IFN-gamma-inducing property and the induction of endothelial adhesion molecules facilitate the containment and killing of the invading microbe.For example, IL-18 increases the expression of some adhesion molecules such as ICAM-1 [19,20] and VCAM-1 [20,21], which facilitate the migration of neutrophils and lymphocytes in containing a nidus of infection.Migration of neutrophils from the vascular compartment into the tissue spaces is also a primary process in inflammatory diseases.The capacity of interferon-gamma to increase the synthesis of inducible nitric oxzide (NO) is essential for killing organisms (intracellular).In this regard, IL-18 as an IFN-γ-inducing factor serves a key role in controlling infections due to molluscum contagiosum ,Salmonella, Cryptococcus, Toxoplasma, Candida, and Mycobacterium organisms, since the production of nitric oxzide is important for intracellular killing.Earlier studies show that MCV encodes a family of proteins with homology to mammalian interleukin -18 binding proteins.Interleukin -18 is a pro-inflammatory cytokine that induces synthesis of interferon-gamma, activates natural killer cells, and is essential for a T-lymphocyte helper type 1 response.The reliance of MCV on IL-18 inhibitors supports another evidence that IL-18 is important for IFN-gamma induction by microbial organisms and viruses.Moreover, inhibition of IFN-gamma alone would not block the induction of the other cytokines by IL-18.Therefore, targeting IL-18, MCV can prevent the cascade of downstream effects that follow the activation of the IL-18 receptor.[15,22].IL-18 performance a major role in the production of interferon-gamma from T-cells and natural killer cells [23] .The level of IgG and IgM, among MC patients , It has been shown that all patients with MC have low levels of IgG and high levels of IgM which indicates acute infections [24] and there results are consistent with many reports [25,26,27] ,in addition to the finding of this study in contrast to the study done by [28] where IgG is high and IgM is low.The immunoglobulins (humoral immunity) plays an important role in the body's defense against molluscum infection.Most adults are resistant to MCV infection because they have developed IgG antibodies against the viral antigen.However, patients with weakened cellular immunity, such as in HIV infection or post-transplant immunosuppression, are more likely to develop widespread infections that are solid to treat.It has been reported that up to 24 % of patients with molluscum have a concomitant diagnosis of atopic dermatitis, and these children also experience more difficulty in clearance.While it is accurate that immunosuppressed patients are more likely to develop more severe Molluscum Contagious virus infections, recent data have shown that contrary to common belief, the prevalence of immunosupression among children with molluscum contagiosum is low [29].In conclusion :The IL-18R1 is increased in the serum of 41.3% of infected patients ; all patients with MC have low levels of IgG and high levels of IgM, finally most of the patients are found in ages ranging between 17-30 years.

Table - 5
).The mean±SD of IgM in patients is 1946.6±825.6 mg/dl while in the control it is 140.1±68.7mg/dl.All patients have high IgM level and all the control have normal values.This result is highly significant which indicates that the patient with MC has a higher level of IgM than the control (p value= 0.0001) (Table -4, Table -5).By contrast, patients have lower levels of IgG than the control.The mean±SD of IgG in patients is 221.9±96.7 while in the control it is 1229.9±299.7.All patients have low IgG level and all the control have normal values.This result is highly significant (p value= 0.0001).