The subjects were 56 inpatients with positive HFRS-specific IgM and IgG antibodies, admitted to the Department of Infectious Diseases of this hospital and of the Xi’an Infectious Disease Hospital from 1992 to 2003. This study was conducted in accordance with the Declaration of Helsinki, with approval from the Ethics Committee of the First Affiliated Hospital of the Medical School of the Xi’an Jiaotong University. An informed consent was obtained from all participants. The diagnosis and classification were made according to the standard adopted during the National Epidemic Hemorrhagic Fever Symposium held in Nanjing in 1986. From the day the patients were admitted to the hospitals, blood and urine samples were collected twice a day, and a total of 195 blood samples and 186 urine samples were obtained, which were stored at negative 20°C. The control group consisted of 20 healthy blood donors. Their HFRS specific IgM and IgG antibodies and hepatitis virus infection indicators were all negative.

IL-6, TNF, IL-8 monoclonal antibodies, standard samples, and negative controls were provided by Professor Jin Boquan from the Department of Immunological Teaching and Research of the Fourth Military University. Fluorescent splits were made by the Shanxi Preventive Medicine Research Medical Institute. Fluorescent indicators (isosulfocyanic acid) containing IgG and IgM extracted from sheep blood and β2-MG kits were provided by the Shanghai Vaccine and Serum Institute.

Total serum globulins were determined by biuret. Specific IgM and IgG were detected by indirect immune fluorescence,4 and B2-MG by specific radiation fluorescence, according to the specifications of the kits. IL-6, IL-8, and TNF were determined by ELISA. The ELISA plate (96-well plate for ELISA reaction; Maxisorp, Denmark) was coated with 100μL/well of diluted capture antibody, incubated at 4°C for 48hours. On the third day, the plate was removed from 4°C and rinsed three times with wash buffer, for 5min each wash step. The standard curve and samples were added with 100μL/well and incubated at 37°C for 2h. After the plate was washed three times, diluted biotinylated detection antibodies were dripped with 100μL/well, and incubated at 37°C for 1h. The plate was washed three times. ABTS was added with 100μL/well, and incubated at 37°C for 30min. The absorption value (OD 410nm) was read by microplate spectrophotometer.

Results were expressed by the mean and standard deviation. Sample mean comparison was made using Student's t-test, square analysis and linear relative analysis. A p-value <0.05 was considered significant.

A total of 76 unrelated Chinese Han subjects (56 inpatients with HFRS, and 20 healthy control individuals) were included in the study. The patients were an average of 36.8 (18-61) years old, and consisted of 46 males and 10 females. The healthy controls had an average age of 34.9 (24∼55) years old, and 50% of them were males. All of the patients detected by RT-PCR were positive for viral RNA. In accordance with the 1986 Nanjing standard, ten cases were diagnosed as mild, 20 as moderate, 18 as severe, and eight as very severe. One of the very severe patients died of a lung infection, and the others survived after treatment.

In the fever stage, the concentration of serum IL-6 in patients with HFRS was higher than that in control group (p<0.001). The concentration of serum IL-6 continued to increase during hypotension stage, reached its peak during the oliguria stage, decreased significantly during the polyuria stage, and remained higher than the controls during the convalescent stage. Fifteen patients with HFRS were tested at the same time and showed the same pattern of change (Table 1).

As shown in Fig. 1, the concentration of urine TNF in patients with HFRS increased remarkably during the fever stage, reached its peak during the hypotension stage, decreased during the oliguria stage, and declined significantly during the polyuria stage. It also showed that the concentrations of IL-6 and IL-8 increased during the fever stage, and continued to increase during the hypotension stage, reaching their peak during the oliguria stage. Therefore, serum IL-6 and urine IL-6 were parallel (r=0.76, p<0.01).

Fig. 1.

Change of urine IL-6, IL-8, and TNF among different groups.

(0.1MB).

With increasing severity of the disease, the concentrations of urine TNF, IL-6, and IL-8 in patients with HFRS increased. The concentration of TNF differed significantly in each type. Except in severe and dangerous types, both the concentrations of IL-6 and IL-8 differed significantly in other types (p<0.05). IL-6 was positively related to both TNF and IL-8 (r=0.5609, p<0.005; r=0.3794, p<0.01, respectively) (Table 2).

During the course of HFRS, the concentration of IL-6 increased on the third day, peaked on the sixth day, and decreased significantly during the eighth and 12th day. The concentrations of IgM and IgG increased on the third day, and peaked on the ninth and 11th days, respectively. From the ninth day onwards, the concentration of IgM decreased significantly. The concentration of IgG remained high. The concentration of IL-6 was positively related to IgM decrease (r=0.42, p<0.05).

Serum globulins increased during the fever stage and peaked during the oliguria stage. Serum globulins were positively related to IL-6 (r=0.51, p<0.05).

116 samples collected during the fever, hypotension, and oliguria stages were analyzed. The analysis demonstrated that serum IL-6 was positively related to β2-MG, BUN, and Cr (r=0.56, 0.46, 0.61, respectively). During the fever, hypotension, and oliguria stages, IL-6 was positively related to β2-MG (r=0.53, 0.56, 0.67; p<0.01).

As shown in Table 3, with increasing severity of the disease, the concentration of IL-6 increased. The concentration in mild type differed greatly from that in moderate type, and also from those in severe and very severe types (p<0.005). The concentrations of β2-MG and specific IgG differed significantly in each type (p<0.01∼0.005). The three were highly related. The relative coefficients between IL-6 and β2-MG were 0.60, 0.66, 0.63, and 0.61 in mild, moderate, severe, and very severe, respectively. The relative coefficients between IL-6 and IgG were 0.58, 0.50, 0.55, and 0.52 in mild, moderate, severe, and very severe types respectively. The relative coefficients between β2-MG and IgG were 0.54, 0.5816, 0.60 and 0.56 in mild, moderate, severe, and very severe types respectively.