The Serum Level of CD16 + 56 in Zona

Objectives: To study the change of CD16 + 56 in zona. Materials and Methods: 62 zona patients admitted at Inpatient Department of Dermatology, Venereology Allergy, 108 Military Central Hospital, and 30 healthy volunteers who were matched for age and sex participated in this study. Prospective, cross-sectional study with controls was conducted. We measured the number of CD16 + CD56 by flow cytometry using FACS Callibur. Results: The mean level of CD16 + 56 in zona patients was 253 ± 200 (106 cells/l), which was lower than that of control group but then increased after treatment. The change of CD16 + 56 did not correlate with patients’ age. Patients who were in severe condition or had affected size from 3% or more had a lower number of CD16 + 56 than control group. Conclusion: The number of CD16 + 56 in zona patients were lower than that of healthy people.


Introduction
Zona is a common disease caused by an epidermotropic and neurotropic virus named Varicella Zoster virus (VZV). All ages and both sexes can develop zona but the age of peak incidence is from 50 or more. In immunocompromised patients, a higher rate of infection and a more severe course of disease are seen [1].
Prolonged sequela is a burden for patients' quality of life.
NK cell has a diameter reaching 12 -15 µm, which is larger than B cell and T cell. It is produced and differentiated in bone marrow and accounts for 5% -10% of all monocytes in peripheral blood. Main surface markers are CD2, CD16, CD45, CD56, CD57, CD11a/CD18, CD11b/CD18 and CD11c/CD18. NK cell is distinguished from other monocytes by the presence of CD16 and CD56 [2]. The development of NK cells depends on the interaction between their progenitors and bone-marrow stromal cells, under the effect of cytokines such as IL- 15,Flt3 ligand and stem-cell factor [3].
The critical function of NK cell is destroying cells with pathogenous antigens such as cancer or virus-infected cells. They secret cytokines such as IFN-γ, TNFα, and interleukin (IL)-10 … First response of host under invasion of VZV is the activation of NK cell against viruses when they penetrate the mucosal barrier and launch adaptive immune response [4]. There were some studies on the role of NK cells in confining the inflammatory process caused by Herpes viruses [5].
Counting the number of CD16 + 56 can evaluate the change of NK cell.
It is surprising that there has been little study on the interaction between VZV and NK cells to demonstrate the involvement of them in VZV pathogenesis. A research of Mireille T. M. Vossen et al. on 5 severe herpes zoster children found that NK cells are decreased in acute phase but restored during convalescence [3].
Some studies also indicated the decline in NK cells can make the Herpes infection worse, even cause fatality [6]. These reports confirm the association of NK cells during VZV infection. However, to the best of our knowledge, it has not been investigated whether NK cells play an important part in the infection of VZV. Subsequent reports were able to identify the role of VZV-infected lymphocytes in viremia. However, the third major lymphocyte population in peripheral blood-natural killer (NK) cells has never been identified adequately due to the delayed development of the NK cell field in comparison to our understanding of T cell and B cell immunology. Moreover, there is a limitation in number of scientific investigations on the role of NK cells under reactivation of VZV in zona. For that reason, we conducted this study with the objective to measure the change of CD16 + 56 in zona so as to progress our understanding of immune interactions during VZV reactivation. Moderate and severe zona patients, older than 18 years old, present ≤ 5 days after the onset of skin symptoms, not using any medication such as corticoid or zona treatment drugs, having normal immune system, HIV/AIDS (−), no contraindication for the drugs used in this study, can follow treatment procedures and agree to participate in our research.

Materials
Exclusion criteria: patients are not qualified according to inclusion criteria, do not agree to participate in the study or cannot follow treatment procedures.
-Control groups: 30 healthy people came for periodic health examination in J. Cosmetics, Dermatological Sciences and Applications matology. The age and sex were matched with patients group.
Severe: affected area ≥ 2% BSA; Likert ≥ 7; swollen regional lymph nodes might present; injured peripheral nerves might present; sleep disorder: severe; general symptoms: fever might present or not.

Methods
Study design: prospective, cross-sectional study with controls. Steps: -Study group: collecting first blood sample (before treatment) to count the number of CD16 + 56 by flow cytometry using FACS Callibur (Becton Dickinson-America).
Collecting second blood sample (after day 20 of treatment) to count the number of CD16 + 56.
-Control group: collecting blood sample only one time to count the number of CD16 + 56.
Data analysis: Data was analyzed by using SPSS software statistical computer package version 18. For quantitative data Student's t-test was used and the means and standard deviation were calculated. P-value less than 0.05 was considered significant.   Note: There was no significant difference in the number of CD16 + 56 between three age groups (p > 0.05).

Results (Tables 2-9)
p 1-2 : The difference between group with age < 50 and group with age from 50 to 69 (See Table 2); p 1-3 : The difference between group with age < 50 and group with age ≥ 70 (See Table 3); p 2-3 : The difference between group with age from 50 to 69 and group with age ≥ 70 (See Table 4).   Table 5).  Table 6). (p > 0.05). However, the number of CD16 + 56 in patients with influenced acreage from 3% or more were lowest in three groups and significantly lower than that of control (See Table 7). Notes: after treatment, the number of CD16 + 56 were significantly increased (p < 0.05) (See Table 8). Note: After treatment, no significant difference was observed between study group and control (See Table   9).

Discussion
In 62 zona patients, the percentage of male was larger than that of female, because this study was operated in Inpatient Department of Dermatology, Venereology Allergy, 108 Military Central Hospital in which more male patients visit annually. Patients in 50 -69 years old took the largest proportion and there were more severe patients than moderate ones, which are demonstrated in Table 2. 2-rising in number in both peripheral blood and bone marrow; 3-local accumulation by the role of inositol phosphates and increased intracellular calcium(activated Phospholipase A2 and arachidonic acid have a key role in activating NK cells); 4-cellular lysis due to particles in cytoplasm containing protease, nucleases and perforin. These substances make holes on target cell membrane, leading to the change in ions entering the cells, which in turn makes them swelling and lysing. Moreover, some studies demonstrate that lifelong NK cells with immunological memory can contribute to immunity in fighting against Herpes reinfection [10]. In varicella children, the activity of NK cells was enhanced in places where VZV intruded and higher than that of healthy ones. Immunocompromised children with zona had a decreased activity of NK cell in the first 3 days and then escalated in recovery period [5]. When counting the number of immune cells in a 2-year-old girl died from varicella, Etzioni discovered that the number of T and B lymphocyte were normal but the NK cells' activity was dropped. cells [3]. The second explanation is that when injecting antigens of VZV in the skin of healthy people who used to be infected, NK cells are rapidly recruited to the infected site. Last but not least, in some in vitro experiments, VZV infected cells become more sensitive to granulysin-a cytotoxic molecule manufactured by NK cells [11]. The activity of NK cells relies on perforin and granzyme. The function of NK cells in exterminating VZV infected cells through CD16 Fc receptor and cytokines they secrete, mainly interferon [12].
However, VZV has many mechanisms to slow down and suppress NK cells.
First, activated VZV can penetrate the host immune system and limit the activity of NK cells by decreasing the expression of ligands binding NKG2D-a receptor activating NK cells [13]. Second, VZV can retain and increase NK cell-inhibitory signals. Moreover, there are some other strategies such as inhibiting the expression of TRAIL death receptor in infected cells which is necessary for the NK cell's TRAIL-induced apoptosis, interfering with NK cells migration or the genesis of an immunological synapse helping NK cells recognize and kill infected host cells [6]. Cauda realized in the first week of disease, activity of NK cells is slowed down due to virus reactivation but higher than that of controls in the second week [14]. Some researches indicated a reduction in IFN-γ production from VZV infected NK cells, which is an important cytokine restraining the replication of viruses. Furthermore, VZV can manipulate the downstream signals after IFN-γ binds its receptor [10]. The study of Choon Kwan Kim also demon-N. L. Anh et al.
strated the level of IFN-γ in zona patients is lower than that of healthy controls, reflecting the attenuation in function of NK cells [11].
In our study, the number of CD16 + 56 were indifferent between 3 groups of age, although patients > 70 years old had the lowest quantity (Table 3), which is suitable to the explanation in which immune response is weakened through age.
The amount of CD16 + 56 in zona patients were lower than that of controls (Table 5), and after 20 days of treatment, they increased significantly (Table 8), which was equivalent to control group (Table 9), meaning that after our therapy, the volume of CD16 + 56 returned to normal. No significant difference was observed between study group and control group in gender and age ( Our research had some limitations. We did not count the number of CD16 + 56 in patients before infection, so the measurement of change in quantity of these cells was not objective. We also evaluated the severity of zona basing on some criteria: affected area, pain intensity (Table 1), symptoms, … which is not mentioned in domestic or international documents for zona, making our results relative.

Conclusions
After studying on 62 zona patients and 30 healthy volunteers, we draw some conclusions: In zona patients, the number of CD16 + 56 were lesser than that of controls and recovered after treatment.
The change in the concentration of CD16 + 56 was independent of patients' age.
Patients who were in severe condition or had affected size from 3% or more had a lower level of CD16 + 56 than control group. No significant difference was observed in other groups.