[Google Scholar] 23. lymphocytes with HPI/thymidine incorporation in unstimulated tonsillar lymphocytes) than controls ( 0.002). Lymphocytes from patients with IgAN also showed a significantly higher level of IgA antibody and IgA1 antibody against HPI antigens in culture supernatants than lymphocytes from controls (= 0.0002 and = 0.004, respectively). Our results suggest that HPI antigens stimulate tonsillar T and B lymphocytes in patients with IgAN and that an immune response to HPI antigens may play a role in the pathogenesis of this disease in some cases. ( 0.05). All patients required tonsillectomy because of adhesive tonsils characterized by a rough surface and pus in the deep crypts, as well as recurrent tonsillitis. After having informed consent, tonsillar tissues were obtained. HPI was isolated from the pharynx of all patients with IgAN. In this study, 16 patients with chronic tonsillitis, from whose pharynx HPI was isolated, were selected as controls. Cell preparation The tonsils were cut into small pieces with scissors , then pressed through 200 G stainless steel mesh. Tonsillar lymphocytes were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Flow Labs, McLean, VA), washed twice with minimal essential NGFR medium (MEM) supplemented with 7.5 m HEPES and 2% heat-inactivated new-born calf serum (NCS), Amyloid b-peptide (42-1) (human) then resuspended in PBS (0.1 m; pH 7.2) supplemented with 2% heat-inactivated NCS. Samples were stored in RPMI 1640 (Gibco, Chagrin Falls, NY) supplemented with 20% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO) at ?80C until use. Monocytes (detected as cells bearing CD116) comprised 4% of tonsillar mononuclear cells. HPI antigen HPI antigen was prepared from a strain isolated from the pharynx of a normal individual and subsequently cultured to stationary phase in brain heart infusion broth. Sonicate of HPI (SHP) was prepared by 5 min of high-power ultrasound pulses and stored at ?80C in PBS pH 7.4 at a protein concentration of 3.2 Amyloid b-peptide (42-1) (human) mg/ml . Tonsillar lymphocyte proliferation Isolated tonsillar lymphocytes were resuspended in culture medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco), l-glutamine 2 mm, penicillin 100 U/ml, and streptomycin 100 g/ml at 2 106 cells/ml. Cells were incubated for 72 h at Amyloid b-peptide (42-1) (human) 37C in a humidified atmosphere with 5% CO2, with or without SHP (0.1 g/ml) and phytohaemagglutinin (PHA; Sigma, St Louis, MO; 20 g/ml). Preliminary experiments established that these concentrations of SHP and PHA induced maximal lymphocyte proliferation. Lymphocyte proliferation was measured by thymidine uptake. After 72 h of incubation, 0.25 Ci of 3H-thymidine was added to each well of the microculture plates (Costar, Cambridge, MA), Amyloid b-peptide (42-1) (human) and the cultures were terminated after 16 h. The incorporated radioactivity, expressed as ct/min, was decided. Production of IgA against HPI antigen Isolated tonsillar lymphocytes were resuspended in culture medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine 2 mm, penicillin 100 U/ml, and streptomycin 100 g/ml at 1 106 cells/ml. Cells were incubated for 72 h at 37C in an humidified atmosphere with 5% CO2, with or without SHP (0.1 g/ml). Preliminary experiments established that this concentration of SHP induced maximal production of IgA against HPI antigen. Supernatant samples were stored at ?80C until use. ELISA for IgA, IgA1, and IgA2 against HPI antigens ELISA was done according to the modified method of Borradori carbonate buffer pH 9.5. After overnight incubation at 4C, wells were washed three times with PBSCTween (PBSCT) and shaken dry. Unoccupied absorption sites in the wells were blocked by overnight incubation at 4C with PBSCT made up of 0.5% (w/v) bovine serum albumin. Supernatant (100 l) diluted 1:100 with PBSCT was added to wells and incubated for 60 min at 37C. Amyloid b-peptide (42-1) (human) Plates were then washed three times with PBSCT, 100 l of peroxidase-conjugated rabbit IgG against human IgA diluted 1:1000 with PBSCT or peroxidase-conjugated MoAb against human IgA1 and IgA2 diluted 1:10 with PBSCT (Nordic Labs, Tilburg, The Netherlands) were added to each well and incubated for 60 min at 37C. Wells were washed, and 100 l of phosphate-citrate buffer pH 4.9 was added to each well. After incubation at room.