Alteration in plasma glucose levels in Japanese encephalitis patients

Abstract

A unique factor, human T cell hypoglycaemic factor (hTCHF), has been shown to produce hypoglycaemia during the convalescent stage in the plasma of patients with Japanese encephalitis virus (JEV) infection. The present study was undertaken to investigate the ability of T cells from fresh peripheral blood mononuclear cells (PBMC) of such patients to produce hTCHF. The PBMC, as well as the individual subpopulations, were cultured for 24 h and the culture supernatants (CS) were assayed for hypoglycaemic activity. The activity was observed in the CD8+ T cells. The hypoglycaemia in JE-confirmed patients coincided with the gradual rise in circulating glucagon level, with no significant alterations in insulin, growth hormone and cortisol levels. The hTCHF was purified by ion exchange chromatography and the purified protein was observed as a approximately 25 kDa band on SDS-PAGE. Secretory hTCHF in the sera of patients and T cell CS was present in 88% of convalescent serum samples. We conclude that during the convalescent stage of JEV infection, a unique factor, hTCHF, is secreted by activated CD8+ T cells from patients and that this is responsible for the development of hypoglycaemia.

Figure 1

Plasma glucose levels in JE-confirmed patients (♦) and normal healthy controls (⋄) on different days. Blood samples were collected in the morning approximately at the same time each day. Children over 5 years old were fasted overnight before blood collection, while children below 5 years were fasted for 4 h prior to sample collection. Data are presented as mean ± SD of triplicate experiments.

Figure 2

Insulin (•), growth hormone (♦), glucagon (▴) and cortisol (▪) levels in JE-confirmed patients and normal healthy controls on different days. Data are presented as mean ± SD of triplicate experiments.

Figure 3

Plasma glucose levels in mice on different days post-inoculation. Groups of mice were inoculated (i.v) with culture supernatants (CS) of macrophages (♦), T (▪) and B (•) cells from JE-confirmed patients at the 20th day of infection or normal healthy individuals as controls. Data are presented as mean ± SD of triplicate experiments.

Figure 5

SDS-PAGE of human T cell hypoglycaemic factor (hTCHF). Lane 1 shows molecular weight markers and position of hTCHF in the gel is indicated by the arrow in Lane 2. Anti-hTCHF antibodies reacted specifically with hTCHF in Western blot analysis (data not included).

Figure 4

Elution profile for culture supernatant (CS) of peripheral blood T-cells from JE-confirmed patients by ion exchange chromatography.

Figure 6

Dot blot test for detection of hTCHF in sera and the T cell culture supernatants (CS) of PBMC of the JE cases and normal healthy controls. The sera and 24 h T cell CS cases were blotted on nitrocellulose membrane and blocked. Blots were treated with anti-hTCHF antibody followed by antimouse IgG + horseradish peroxidase and then developed with substrate diamino-benzidine as described in Patients and methods. The controls included CS from normal healthy individuals. Convalescent serum samples (lane A-B); purified T cell CS (lane C), acute sera (lane D) and lane E shows negative control (NC) and positive control (PC).