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Lai et al. Imbalance of NK and B cell subsets in GMG exacerbation

for MG activity and for monitoring effectiveness of MG symptoms were upper respiratory tract infection
immunotherapy. (14 cases), inappropriate drug use (4 cases), surgery
(1), exhaustion (1), and stress (1) [Table 2].
METHODS
Flow cytometric analysis
Patients The peripheral blood samples from all the
In this study, 54 patients with MG who met the participants were obtained by venipuncture and
standard clinical criteria [37] and 10 healthy controls stored in tubes containing ethylene diamine tetraacetic
(HC), 4 men and 6 women, aged 38.50 ± 16.37. All the acid (5.4 mg/tube). Each sample was divided into 2
participants signed the informed consent prior to the tubes, each one containing 100 µL fresh whole blood
enrollment. The study was performed in accordance which was further incubated with 5 µL of mAbs (Becton
with the 1964 Declaration of Helsinki (including Dickinson) for 20 min in dark at room temperature.
amendments) and has been approved by the Ethics Breg cells were stained with FITC conjugated anti-
Committee of Huazhong University of Science and human CD19, PE conjugated anti-human CD1d,
Technology. Exclusion criteria were severe heart APC conjugated anti-human CD5, and NK cells were
diseases, severe kidney diseases, severe mental analyzed after staining with PE conjugated anti-human
illness, acute or chronic hepatitis, tuberculosis or HIV CD16, PerCP conjugated anti-human CD3, APC
infection. The mean ± standard deviation of the ages conjugated anti-human CD56, respectively. Isotype-
of the patients was 40.30 ± 14.81 years. The sex ratio matched immunoglobulin served as control for analysis
(male:female) was 28:15. The clinical features of + + + - + +
the participants were summarized in Table 1. Eleven of CD1d CD5 CD19 B cells and CD3 CD16 CD56
patients presented with ocular MG (OMG) (age: 35.36 NK cells. After incubation, erythrocytes were lysed
± 4.57; sex ratio: 5:6), and 43 with generalized MG with FACS lysing solution (Becton Dickinson). The
(GMG). The GMG patients were further divided into remaining cells were resuspended in PBS then at
two groups according to the clinical status. Group least 30,000 lymphocyte events were acquired from
I included 25 patients who were in exacerbation each tube to determine their proportions using the
stage (PE), presenting symptomatic exacerbation FACS Caliber flow cytometer (Becton Dickinson).
or myasthenic crisis (age: 44.52 ± 2.58; sex ratio:
8:17). Group II consisted of 18 patients who were Statistical analysis
in remission (PR), manifesting slight limb weakness All statistical analyses were performed using the
or ocular symptoms for at least three months (age: SPSS software application. The data were expressed
35.44 ± 3.66; sex ratio: 7:11). Overall, 43 patients as the mean ± standard error. For a comparison of
were on prednisone and trans-sternal extended the different subgroups of the MG patients and the
thymectomy was performed on 30 patients, including healthy controls, the data were analyzed using two-
8 with thymic hyperplasia and 22 with thymoma. The tailed Student’s t-test. P-values lower than 0.05 were
identified factors which provoked the deterioration of considered to be statistically significant.

Table 1: Clinical characteristics of the MG patients
Patients in exacerbation Patients in remission Ocular MG
Characteristic
(number) (number) (number)
Gender
Female 17 11 6
Male 8 7 5
Age (year, mean ± SD) 44.52 ± 2.58 35.44 ± 3.66 35.36 ± 4.57
Osserman’s classification
Class I 11
Class IIa 1 6
Class IIb 21 12
Class III 1
Class IV 2
Thymoma 9 11 2
With thymectomy 6 8 1
Thymic hyperplasia 1 5 2
With thymectomy 3 1
Use of prednisone 24 17 2
Immunosuppressant
Tacrolimus 1
MG: myasthenia gravis; SD: standard deviation
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ September 18, 2017 181