Electrophoresis of the PCR products was performed on 2% agarose gels and the PCR fragments were visualized by ethidium bromide staining under UV illumination. == Serum levels of IL-12, IFN- and TNF- in patients with KD == To determine the effect of PHIG therapy, serum levels of IL-12, IFN- and TNF- in patients with KD (n= 28, 3.2 2.0 years old) in the acute phase and in the convalescent phase after PHIG therapy were compared with those of patients with bacterial infections (n= 13, 4.7 3.9 years old), age-matched healthy controls (n= 10, 2.5 1.8 years old) and healthy adult volunteers (n= 8). amounts of IFN- if exogenous IL-12 was introduced. KD patients in the acute phase had higher levels of serum IFN- than did controls and patients with bacterial infection. Although IL-12 levels of children with or without KD were not significantly different, IL-12 levels of children were much higher than those of adults. However, serum levels of IL-12 of KD patients were transiently but significantly decreased by PHIG therapy and IFN- amounts subsequently reverted to basal levels thereafter. These findings indicate that PHIG inhibits IL-12 production of SPE-A-activated monocytes and thereby decreases IFN- synthesis by T cells and suggest that inhibition of IL-12 and IFN- production is an important part of the mechanisms underlying PHIG therapy on KD. Keywords:Streptococcus pyrogenic exotoxin A, pooled human immunoglobulin, Kawasaki disease, IL-12, interferon-gamma == INTRODUCTION == Intravenous administration of pooled human immunoglobulin (PHIG) is usually reportedly effective against not only severe bacterial and viral infections [1] but also certain diseases such as idiopathic thrombocytopenic purpura [2], some autoimmune diseases [3,4] and KD [5,6]. However, the mechanism underlying its effect has not been fully elucidated. It was reported that PHIG inhibits bacterial superantigen-induced production of proinflammatory cytokines, such as interferon-gamma (IFN-) and tumour necrosis factor-beta (TNF-) [7,8] from PBMCin vitro. On the other hand, IL-12 is usually a recently reported cytokine [912] which activates natural killer (NK) cells and T cells and is considered to be critical against bacterial infections. IL-12 is produced by phagocytic cells and antigen-presenting cells (APC) [11,12], such as monocytes, macrophages and dendritic cells when these cells are activated by bacterial components, including lipopolysaccharide (LPS) [1114]. IL-12 is also known as an inducer of IFN-, IL-2 and others [12,15,16]. Particularly, IL-12 is an essential and potent inducer of IFN- from NK cells, T cells and NK-type Goat Polyclonal to Rabbit IgG T cells [12,17,18] and is also critically involved in anti-tumour immunity Carisoprodol [15,16,18,19]. KD is an acute multisystem vasculitis which primarily affects young children under 6 years of age [20]. Approximately 20% of KD patients develop coronary artery abnormalities and some of them suffer from myocardial ischaemia and even myocardial infarction [21,22]. Although the aetiology is still unknown, the possibility that bacteria, their components and bacterial superantigens of staphylococcus or streptococcus, are involved in this disease has been considered [2325]. Bacterial superantigen-induced toxic shock syndrome mimics KD in some aspects and some patients with infective enteritis due toYersinia pseudotuberculosismeet the diagnostic criteria of KD [26]. No specific virus has been identified in KD patients; instead, marked granulocytosis, which is not usually seen in viral infections, is observed in KD. Among therapies tested, i.v. injection of PHIG is empirically effective for KD and improving coronary artery complications [5,6]. However, the mechanism of this treatment remains largely unknown. In the present study, we demonstrate that PHIG inhibits IL-12 production from monocytes and suppresses IFN- production by T cells stimulated with Streptococcus pyrogenic exotoxin A (SPE-A). In addition, we show that PHIG therapy decreases serum IL-12 levels in patients in the acute phase of KD and seems to initiate reduction of serum IFN-. == MATERIALS AND METHODS == == Reagents == SPE-A was purified from culture filtrate Carisoprodol ofStreptococcus pyogenesstrain NY-5 [27]. PHIG (Venoglobulin IH, lot no. B025VH) was provided by the Green Cross Corp. (Osaka, Japan). Human recombinant IL-12 (p70) (1.7 107U/mg) was kindly provided by Hoffman-LaRoche (Nutely, NJ), and recombinant IL-1 and TNF- were purchased from R&D Systems (Minneapolis, MN). == Cell culture == PBMC were obtained from six healthy human adult volunteers by FicollPaque gradient centrifugation and suspended in RPMI 1640 containing 10% fetal bovine serum (FBS). SPE-A (100 ng/ml)-stimulated PBMC (1 106/ml) were cultured for 9 days at 37C and 5% CO2, with or without PHIG (5 mg/ml), and cell culture supernatants were harvested at each 24 h and were subjected to ELISA. In some experiments, SPE-A-stimulated PBMC were incubated with PHIG and/or IL-12 (20 U, 1000 pg/ml) for 144 h. Cell culture supernatants were harvested each 48 h. == High performance liquid chromatography analysis of SPE-A after incubation with PHIG == Carisoprodol SPE-A (100 ng/ml) was incubated at 37C and 5% CO2with or without 5 mg/ml PHIG for 2 h and then the decrease of.