Laboratory of Virology, UPRES EA Viral Pathogenesis of Diabetes Type 1, University Hospital, and University Lille 2, 59037 Lille, France1
SEDAC Therapeutics, IBL-CNRS, 59021 Lille, France2
Pediatric Endocrine Unit, University Hospital, Lille, France3
Department of Medicine, Endocrinology and Metabolism, Charlon Hospital, Henin-Beaumont, France4
Department of Endocrinology and Metabolism, University Hospital, Lille, France5
Department of Pediatrics, Saint-Antoine Hospital, Catholic University, Lille, France6
Author for correspondence: Didier Hober. Correspondence address: Laboratoire de Virologie, Bât. Paul Boulanger, Hôpital Albert Calmette, Centre Hospitalier Régional et Universitaire (CHRU), 59037 Lille Cedex, France. Fax +33 3 20 44 52 81. e-mail dhober{at}chru-lille.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enteroviruses like polioviruses and CVB are referred to as weak IFN- inducers, as compared to strong IFN-
inducers like Sendai virus (SV) and herpes simplex virus type 1 (HSV-1) (Pitkaranta & Hovi, 1993
; Feldman et al., 1994
). However, it has been shown that human polyvalent IgGs enhance the IFN-
-inducing capacity of poliovirus in vitro (Palmer et al., 2000
). Furthermore, we have reported recently that CVB4-induced synthesis of IFN-
by PBMCs in vitro can be enhanced through interactions between CVB4, specific antibodies isolated from the plasma of healthy subjects, Fc
R and a receptor for CVB called CAR (coxsackievirus and adenovirus receptor) (Chehadeh et al., 2001; Bergelson et al., 1997
). This suggests that antibodies can play a role in the IFN-
response to CVB4.
Infection by a CVB serotype induces production of serotype- and CVB group-specific antibodies (Frisk et al., 1989 ). Epidemiological studies have indicated that CVB infection is frequent in patients with type 1 diabetes (Yoon, 1990
; Hyoty et al., 1995
); therefore, these individuals may have a higher prevalence of CVB antibodies compared with appropriate controls (Szopa et al., 1993
; Hyoty et al., 1995
; Hiltunen et al., 1997
). Thus, CVB-specific antibodies can be found in the circulating blood of diabetes patients and these antibodies may play a role in the interaction between CVB4 and PBMCs.
The mechanism of the increased expression of IFN- in patients with type 1 diabetes is unknown. In the current study, we investigated in vitro whether a preferential IFN-
response by PBMCs to CVB4 exists in patients with type 1 diabetes compared with healthy subjects and whether antibodies can be involved.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood samples were obtained from 20 healthy subjects: 10 adults (6 males and 4 females; median age 28 years, range 2345) and 10 children (5 males and 5 females; median age 12 years, range 716), who had not any suspected immunological, infectious or metabolic disease. These subjects were hospitalized or were outpatients at the hospital.
Viruses.
The JBV strain of CVB4, kindly provided by J. W. Almond (Aventis Pasteur, Marcy-LEtoile, France), was grown in Hep-2 cells (BioWhittaker) in Eagles minimum essential medium (MEM) (Gibco BRL) supplemented with 10% heat-inactivated foetal calf serum (FCS) (Eurobio) and 1% L-glutamine (Eurobio). Supernatants were collected 3 days post-infection (p.i.) and then clarified at 1000 r.p.m. for 10 min. Virus titres were determined by plaque-formation assay on Hep-2 cells and expressed as p.f.u./ml. Aliquots of virus preparations were stored frozen at -80 °C.
SV was kindly provided by D. Garcin (Department of Genetics and Microbiology, University of Geneva, Switzerland). HSV-1 (a laboratory strain) was cultivated in Vero cells (ATCC) in MEM supplemented with 5% FCS and 1% L-glutamine.
IFN-
induction
Whole blood cultures.
Venous blood was collected in sterile 7·5 ml tubes (Beckton Dickinson) containing 20 IU/ml heparin (Heparin Choay). Samples (25 µl) of heparinized whole blood were dispensed into each of triplicate wells of a 96-well tissue culture plate containing 225 µl RPMI 1640 (Gibco BRL) or 225 µl RPMI 1640 containing 107 p.f.u./ml CVB4. The blood cultures were incubated in a humidified incubator at 37 °C with 5% CO2.
PBMCs.
PBMCs from heparinized blood were separated over a Ficollpaque solution (Diatrizoate Ficoll, Eurobio). Mononuclear cells were collected, washed three times with RPMI 1640, adjusted to a concentration of 2x106 cells/ml in RPMI supplemented with 10% FCS and 1% L-glutamine and then distributed as 0·1 ml aliquots into 96-well tissue culture plates. Thereafter, 0·1 ml of medium containing virus at an m.o.i. of 1 or medium containing virus at an m.o.i. of 1 and plasma at different dilutions were added to wells. Cultures were then incubated for 48 h at 37 °C in a 5% CO2 atmosphere.
After this incubation period, whole blood and PBMC cultures were irradiated with UV light for 1 h to inactivate residual viruses. Supernatants of culture were then harvested, clarified by centrifugation for 10 min at 1000 r.p.m. and stored at -20 °C until assayed for the presence of IFN-. Cultures not treated with viruses served as controls.
Immunoassay for IFN-
.
The concentration of IFN- was determined by a specific and sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), as described previously (Chehadeh et al., 2000b
). Antibodies were kindly provided by G. Alm, Biomedical Center, Uppsala, Sweden. The assay detection limit was 0·5 IU IFN-
/ml.
Bioassay for IFN-
.
Samples of whole blood and PBMC cultures were assayed for antiviral activity by protection of MDBK cells (BioWhittaker) against vesicular stomatitis virus-induced cytopathic effects, as described previously (Chehadeh et al., 2000b ). The assay detection limit was 2 IU IFN-
/ml. The antiviral activity of supernatant samples was neutralized with a rabbit antiserum to IFN-
(Biosource).
Protein A-affinity chromatography.
IgGs were obtained from the plasma of healthy subjects or patients using a protein ASepharose CL-4B column, as described previously (Chehadeh et al., 2001 ).
Antibody-dependent assays.
PBMCs were distributed into 96-well tissue culture plates as described above. Virusantibody complexes were made by incubating plasma or IgGs at various dilutions with CVB4 for 1 h at 37 °C. The optimal dilution of plasma differed between subjects and ranged from 1:100 to 1:1000, as described previously (Chehadeh et al., 2001). CVB4 or CVB4antibody complexes were added to cells at an m.o.i. of 1 before incubation for 2 h at 37 °C. In certain experiments, PBMCs were incubated with IgGs for 1 h and, after washing, were incubated with CVB4 at an m.o.i. of 1 for 90 min. The cells were then washed three times and cultured in RPMI 1640 containing 10% FCS for 48 h at 37 °C in a 5% CO2 atmosphere. After the incubation period, supernatants were harvested, clarified and stored until assayed for the presence of IFN-.
Isolation of cytophilic antibodies.
PBMCs (5x107/ml) were incubated for 30 min in a 56 °C water bath. Cells were removed by centrifugation at 300 g for 10 min. The cell-free supernatants generated (eluted fraction by PBMCs) were used in assays of CVB4-induced production of IFN- by PBMCs to detect the presence of cytophilic antibody dissociated from the cell surface during the 56 °C incubation.
Antibodies.
Monoclonal IgG1 neutralizing anti-human FcRI (CD64), clone 10.1 (Biotest), monoclonal IgG2a neutralizing anti-human Fc
RII (CD32), clone 2E1, and monoclonal IgG1 neutralizing anti-human Fc
RIII (CD16), clone 3G8, antibodies were purchased from Coulter Immunotech. Monoclonal IgG1 anti-human CAR antibody (RmcB) was kindly provided by J. Bergelson (Division of Infectious Disease, Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania, USA). Anti-D human monoclonal IgG1 and IgG3 immune sera were kindly provided by Etablissement de Transfusion Sanguine (Lille, France) and used as irrelevant human IgG antibodies. Murine IgG1 and IgG2a (Coulter Immunotech) were used as control antibodies.
Assays of CVB antibodies
Plaque neutralization assay.
The presence of anti-CVB4 neutralizing antibodies (NAs) in plasma were routinely assessed according to the method described elsewhere (Chehadeh et al., 2000a ). Results were expressed as the inverse of the final dilution (titre) of the sample that totally inhibited the cytopathic effect of CVB4 in Hep-2 cells.
ELISA.
The Serion ELISA Classic kit (Institut Virion-Serion) was used for quantitative detection of specific human anti-CVB IgG antibodies, according to the manufacturers instructions. Results were expressed in IU/ml.
Statistical analysis.
Data are summarized as mean±SD. The significance of the differences in IFN- levels was determined by the MannWhitney U-test. The
2-test was used when appropriate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The enhancing effect of IgGs from controls and patients on CVB4-induced IFN- production in PBMC cultures was suppressed significantly when PBMCs were preincubated with 2 µg/ml of either anti-human Fc
RII IgG2a antibodies or anti-human Fc
RIII IgG1 antibodies but not with anti-human Fc
RI IgG1 antibodies (Table 1
). Irrelevant control antibodies did not inhibit IgG-mediated enhancement of CVB4-induced IFN-
production. To examine the role of CAR on the efficiency of IgG-mediated IFN-
production, PBMCs were preincubated for 1 h with anti-human CAR antibodies before challenging with CVB4 preincubated with IgGs from healthy subjects or patients. IFN-
production was inhibited in the presence of anti-CAR antibodies [80±12 (n=3) and 93±5% (n=3) inhibition in experiments with IgGs from donors and patients, respectively], whereas control isotypes did not inhibit IgG-mediated enhancement of CVB4-induced IFN-
production (data not shown). When PBMCs were preincubated with anti-Fc
RII, anti-Fc
RIII or anti-CAR antibodies before adding SV or HSV-1, the levels of IFN-
were not reduced (data not shown), which demonstrated that these antibodies did not give a negative signal to IFN-
-producing cells that blocked IFN-
synthesis.
|
Preincubation of PBMCs from healthy subjects with IgGs, after washing and before adding CVB4, resulted in the production of IFN- (Fig. 3a
). These results suggested that CVB4-specific antibodies bound to the surface of cells. To dissociate and recover antibodies from PBMCs, the PBMCs were heated at 56 °C for 30 min. Supernatants were generated by subsequent removal of cells and cell debris by centrifugation. CVB4-induced IFN-
production by PBMCs was used as a test to assess IFN-
-enhancing activity in these supernatants. As seen in Fig. 3(b)
, a significant IFN-
-enhancing activity was detected in supernatants of IgG-treated PBMCs heated at 56 °C. The preincubation of PBMCs used for testing supernatants with anti-Fc
RI, anti-Fc
RII and anti-CAR but not anti-Fc
RIII decreased by approximately 100 % the level of IFN-
(data not shown).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are several noteworthy considerations for the systems used in the study. The measurement of IFN--producing capacity by the whole blood method has been reported previously by us and other teams (Hober et al., 1998
; Uno et al., 1996
). The production of IFN-
per mononuclear cell was higher in whole blood than in PBMC cultures from healthy controls and patients with type 1 diabetes, although the cell number in whole blood cultures was about three times as low as that in PBMC cultures (data not shown). The discordant IFN-
levels in whole blood and PBMC cultures infected with CVB4, in particular the high values in whole blood cultures from patients, prompted us to study the role of plasma in CVB4-induced production of IFN-
.
The production of IFN- by PBMCs from healthy controls and patients with type 1 diabetes, obtained with a mixture of CVB4 and plasma, depended on the antibodies contained in the plasma, as evidenced by: (1) the eluted fractions containing IgGs obtained from plasma passed over a protein ASepharose column; and (2) the suppression of the enhancing effect of virus/eluted fraction by anti-Fc
RII and anti-Fc
RIII antibodies. The activity of plasma and IgGs obtained in our experiments suggested that the level of IFN-
-enhancing antibodies was higher in patients with type 1 diabetes than in healthy subjects. It may be assumed that IgGs from diabetics formed immune complexes with CVB4, since the adsorption of CVB4 complexed to patients IgGs on C1q-coated microwell plates, according to the procedure described previously (Chehadeh et al., 2001), inhibited the IFN-
-enhancing activity of IgGs (data not shown). Furthermore, our experiments with neutralizing anti-CAR antibodies suggested that CAR, a specific receptor for CVB4, in addition to Fc
RII and Fc
RIII, played a role in the induction of IFN-
by CVB4antibody complexes in PBMCs from patients and healthy controls. This result showed that IFN-
production was not just a consequence of cell activation by binding of CVB4IgG complexes but also depended on binding of CVB4 to a specific cell surface receptor. Our results agree with those of Palmer et al. (2000)
, suggesting that Fc
RII plays a role in the poliovirusantibody complex-induced production of IFN-
by PBMCs. However, in the present study, Fc
RIII played a role, whereas in their experiments, it did not; the difference could be related to the virus used and/or to the nature of IFN-
-producing cells. Indeed, previous works from our laboratory showed that the major IFN-
-producing cells in response to CVB4IgG complexes were CD14+ monocytes (Chehadeh et al., 2001
), whereas Palmer and colleagues suggested that poliovirusantibody complexes activated the synthesis of IFN-
by natural IFN-
-producing cells characterized as type 2 dendritic cell precursor (Siegal et al., 1999
; Palmer et al., 2000
). In an extension of the present work we will attempt to determine whether monocytes are involved in the production of IFN-
observed in vivo and in vitro in response to CVB4 infection in individuals with type 1 diabetes.
We demonstrated that PBMCs armed in vitro with cytophilic antibodies produced IFN- in the presence of CVB4. Moreover, we provide evidence of the presence of antibodies on the surface of freshly isolated PBMCs from patients producing IFN-
in response to CVB4. The role of antibodies was evidenced by the following experiments: (1) a 30 min incubation at 56 °C was used to recover cell surface-associated antibodies, as described previously (Tyler et al., 1989
); (2) the supernatants generated in this manner contained significant levels of IFN-
-enhancing activity in combination with CVB4, as measured by IFN-
assays. A role for IgGs in CVB4-induced production of IFN-
in these experiments was indicated by the ability of monoclonal antibodies against Fc
RI and Fc
RII to significantly inhibit the production of IFN-
. The anti-Fc
RII monoclonal antibody (clone 2E1) used in this study binds well to Fc
RIIa-expressing cells but is marginally reactive with cells expressing Fc
RIIb (Van de Winkel & Anderson, 1995
). Thus, the effect of the anti-Fc
RII monoclonal antibody in our experiments do not result from a possible activation of Fc
RIIb, which negatively regulates intracellular signalling passing through immunoreceptor tyrosine-based activation motifs and their cytoplasmic ligands like Fc
RI-mediated activation of IFN-
(Daeron, 1997
). IFN-
production was inhibited by anti-Fc
RII but not by anti-Fc
RIII antibodies, suggesting that Fc
RIII, which, like Fc
RII, binds immune complexes, is not involved in IFN-
production nor is it expressed on the IFN-
-producing cells in experiments with PBMCs armed with cytophilic antibodies. The presence of both Fc
R(I and II) and CAR molecules on cells was required for CVB4-induced IFN-
synthesis by PBMCs armed with cytophilic antibodies; otherwise, blocking of Fc
R or CAR independently could not inhibit IFN-
synthesis. The results of our experiments suggest that IgGs bound Fc
RI and Fc
RII, which is in agreement with the fact that these receptors bind monomeric IgGs. In contrast with Fc
RI, Fc
RII binds monomeric IgGs weakly but reacts mostly with IgG complexes (Sautès, 1997
; Carayannopoulos & Capra, 1993
). However, when isolated from blood, Fc
R-bearing cells are saturated by serum IgGs. In our system, one possible explanation for the effect of antibodies preincubated with freshly isolated PBMCs already saturated by IgGs is the following: IgGs added to cells bind to cell surface molecules (glycosyltransferases and/or lectin-like molecules) (Carayannopoulos & Capra, 1993
), then, when CVB4 is added, immune complexes formed compete with monomeric IgGs for receptors and displace bound monomeric IgGs. Indeed, conformational changes in the Fc region by virusantibody complex formation facilitates interactions with the Fc receptor (Frey & Einsfelder, 1984
); therefore, immune complexes are several times more effective than monomeric IgGs at competing for receptors.
The IFN- response mediated by the supernatants of heated PBMCs from patients producing IFN-
in the presence of CVB4 was reduced compared with the responses of these PBMCs to CVB4 (13±4 versus 128±64 IU/ml). This may be due to the relatively low concentrations of enhancing antibodies in the preparations. Moreover, the effects of supernatants of heated PBMCs from patients can be underestimated. Indeed, they were tested with cells from healthy controls; whether these cells can bind IgGs as well as cells from patients is not known.
When PBMCs from patients with type 1 diabetes were exposed to CVB4 in the absence or presence of their own IgGs, the levels of IFN- were higher than those obtained with cells and IgGs from healthy subjects. The high IFN-
response to CVB4 in diabetic patients was not related to age of onset of diabetes, duration of diabetes or metabolic decompensation (data not shown), which indicates intrinsic hyperresponsiveness to CVB4 in type 1 diabetes. It has been reported that there is an hyper-IFN-
responsiveness by PBMCs to IFN-
inducers like polyinosinic:polycytidilic acid [poly(I:C)] in type 1 diabetes (Toms et al., 1991
); however, in the current work we found no difference between patients and healthy subjects in response to IFN-
inducers like HSV-1 and SV. The difference observed in our study of CVB4 response in patients and healthy subjects can be explained by a history of CVB infections responsible for the presence of higher levels of IFN-
-enhancing anti-CVB antibodies in patients than in healthy subjects. Nevertheless, there was no correlation between the titres of anti-CVB antibodies detected in patients by neutralization assay or by ELISA and the production of IFN-
by their PBMCs. These results are in agreement with our previous studies showing that IFN-
-enhancing antibodies directed towards CVB4 detected in healthy controls were different from NAs against that virus (Chehadeh et al., 2001
) and a priori suggest that IFN-
-enhancing antibodies do not bind H antigen, which is incorporated into the immunoenzymatic assay used in our experiments for detecting anti-CVB antibodies. Together these data are consistent with the existence of anti-CVB antibodies in plasma from patients with type 1 diabetes not detected by methods usually available.
The preferential IFN- response by PBMCs from patients with type 1 diabetes to CVB4 supports the hypothesis that CVB4 can play a role in the development of type 1 diabetes, although the mechanism has not been elucidated. Further studies are needed to know whether the observed IFN-
hyperactivity towards CVB4 is an event prior to (predisposing) or post clinical onset of type 1 diabetes.
In conclusion, the current studies demonstrate that CVB4 can strongly induce the in vitro production of IFN- by PBMCs from patients with type 1 diabetes in comparison with healthy subjects. The IFN-
-inducing activity of CVB4 is strongly enhanced by IgGs from patients with type 1 diabetes in comparison with IgGs from healthy subjects. Moreover, it has been shown that the CVB4-induced production of IFN-
by PBMCs from patients can result from the presence of IFN-
-enhancing-specific antibodies on the cell surface. Studies are needed to characterize the antibodies involved in the CVB4-induced production of IFN-
and to define their role in the pathogenesis of CVB4 infection. Future works will be directed along this line in our laboratory.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carayannopoulos, L. & Capra, J. D. (1993). Immunoglobulins: structure and function. In Fundamental Immunology , pp. 283-314. Edited by W. E. Paul. New York: Lippincott Williams & Wilkins.
Chehadeh, W., Weill, J., Vantyghem, M. C., Alm, G., Lefèbvre, J., Wattré, P. & Hober, D. (2000a). Increased level of interferon- in blood of patients with insulin-dependent diabetes mellitus: relationship with coxsackievirus B infection. Journal of Infectious Diseases 181, 1929-1939.[Medline]
Chehadeh, W., Kerr-Conte, J., Pattou, F., Alm, G., Lefèbvre, J., Wattré, P. & Hober, D. (2000b). Persistent infection of human pancreatic islets by coxsackievirus B is associated with alpha interferon synthesis in cells. Journal of Virology 74, 10153-10164.
Chehadeh, W., Bouzidi, A., Alm, G., Wattré, P. & Hober, D. (2001). Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon- by human peripheral blood mononuclear cells in vitro. Journal of General Virology 82, 1899-1907.
Daeron, M. (1997). Fc receptor biology. Annual Review of Immunology 15, 203-234.[Medline]
Feldman, S. B., Ferraro, M., Zheng, H. M., Patel, N., Gould-Fogerite, S. & Fitzgerald-Bocarsly, P. (1994). Viral induction of low frequency interferon- producing cells. Virology 204, 1-7.[Medline]
Flowers, D. & Scott, G. M. (1985). How useful are serum and CSF interferon levels as a rapid diagnostic aid in virus infections? Journal of Medical Virology 15, 35-47.[Medline]
Frey, J. & Einsfelder, B. (1984). Induction of surface IgG receptors in cytomegalovirus-infected human fibroblasts. European Journal of Biochemistry 138, 213-216.[Abstract]
Frisk, G., Nilsson, E., Ehrnst, A. & Diderholm, H. (1989). Enterovirus IgM detection: specificity of mu-antibody-capture radioimmunoassays using virions and procapsids of coxsackie B virus. Journal of Virological Methods 24, 191-202.[Medline]
Green, J. A., Charette, R. P., Yeh, T. J. & Smith, C. B. (1982). Presence of interferon in acute- and convalescent-phase sera of humans with influenza or influenza-like illness of undetermined etiology. Journal of Infectious Diseases 145, 837-841.[Medline]
Hiltunen, M., Hyoty, H., Knip, M., Ilonen, J., Reijonen, H., Vahasalo, P., Roivainen, M., Lonnrot, M., Leinikki, P., Hovi, T. & Akerblom, H. K. (1997). Islet cell antibody seroconversion in children is temporally associated with enterovirus infections. Childhood Diabetes in Finland (DiMe) Study Group. Journal of Infectious Diseases 175, 554-560.[Medline]
Hober, D., Benyoucef, S., Chehadeh, W., Chieux, V., De La Tribonnière, X., Mouton, Y., Bocket, L. & Wattré, P. (1998). Production of interleukin-4, interferon(IFN)- and IFN-
in human immunodeficiency virus-1 infection: an imbalance of type 1 and type 2 cytokines may reduce the synthesis of IFN-
. Scandinavian Journal of Immunology 48, 436-442.[Medline]
Hyoty, H., Hiltunen, M., Knip, M., Laakkonen, M., Vahasalo, P., Karjalainen, J., Koskela, P., Roivainen, M., Leinikki, P., Hovi, T. and others (1995). A prospective study of the role of coxsackie B and other enterovirus infections in the pathogenesis of IDDM. Childhood Diabetes in Finland (DiMe) Study Group. Diabetes 44, 652657.[Abstract]
Palmer, P., Charley, B., Rombaut, B., Daeron, M. & Lebon, P. (2000). Antibody-dependent induction of type I interferons by poliovirus in human mononuclear blood cells requires the type II Fc receptor (CD32). Virology 278, 86-94.[Medline]
Pitkaranta, A. & Hovi, T. (1993). Induction of interferon in human leukocyte cultures by natural pathogenic respiratory viruses. Journal of Interferon Research 13, 423-426.[Medline]
Sautès, C. (1997). Structure and expression of Fc receptors (FcR). In Cell-mediated Effects of Immunoglobulins , pp. 29-66. Edited by W. H. Fridman & C. Sautès. Heidelberg: Springer-Verlag.
Siegal, F. P., Kadowaki, N., Shodell, M., Fitzgerald-Bocarsly, P. A., Shah, K., Ho, S., Antonenko, S. & Liu, Y. J. (1999). The nature of the principal type 1 interferon-producing cells in human blood. Science 284, 1835-1837.
Szopa, T. M., Titchener, P. A., Portwood, N. D. & Taylor, K. W. (1993). Diabetes mellitus due to viruses: some recent developments. Diabetologia 36, 687-695.[Medline]
Toms, G. C., Baker, P. & Boucher, B. J. (1991). The production of immunoreactive - and
-interferon by circulating mononuclear cells in type 1 diabetes. Diabetic Medicine 8, 547-550.[Medline]
Tyler, D. S., Nastala, C. L., Stanley, S. D., Matthews, T. J., Lyerly, H. K., Bolognesi, D. P. & Weinhold, K. J. (1989). GP120 specific cellular cytotoxicity in HIV-1 seropositive individuals. Evidence for circulating CD16+ effector cells armed in vivo with cytophilic antibody. Journal of Immunology 142, 1177-1782.
Uno, K., Nakano, K., Maruo, N., Onodera, H., Mata, H., Kurosu, I., Akatani, K., Ikegami, N., Kishi, A., Yasuda, Y., Tanaka, K., Setoguchi, J., Kondo, M., Muramatsu, S. & Kishida, T. (1996). Determination of interferon--producing capacity in whole blood cultures from patients with various diseases and from healthy persons. Journal of Interferon and Cytokine Research 16, 911-918.[Medline]
Van de Winkel, J. G. J. & Anderson, C. L. (1995). CD32 cluster workshop report. In Leucocyte Typing V , pp. 823-826. Edited by S. F. Schlossman. New York: Oxford University Press.
Yoon, J. W. (1990). The role of viruses and environmental factors in the induction of diabetes. Current Topics in Microbiology and Immunology 164, 95-123.[Medline]
Received 11 March 2002;
accepted 7 May 2002.