B-cell Areas Research Articles

Chronic stress associated with spousal caregiving of patients with Alzheimer's dementia is associated with downregulation of B-lymphocyte GH mRNA.

It has been demonstrated that growth hormone (GH) is synthesized and secreted by human peripheral mononuclear cells (PBMC), and the expression of GH mRNA can be found throughout the human immune system. We studied a population of female caregivers of patients with Alzheimer's dementia (AD) who suffered from the stress of caring for these patients. We utilized quantitative RT-PCR to determine GH mRNA levels in T- and B-cell populations from PBMC. Subjects were nine caregivers of AD patients and nine age- and sex-matched controls. In the control group we found a threefold greater GH mRNA expression in B cells than in T cells. This finding was consistent with our previous in situ hybridization observation, suggesting GH mRNA in predominately B-cell areas of immune organs in humans. We also found that the expression of GH mRNA from total peripheral blood mononuclear cells and B cells in caregivers was 50% and 60% respectively less than that in the control group. Because the B-cell population is the source of antibody production, our findings suggest that the decrease in B-cell GH mRNA may contribute to the poor immune response to influenza virus vaccination that has been reported previously in chronically stressed caregivers.

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

Dendritic cells and Langerhans cells in the uptake of mucosal antigens.

Dendritic cells (DC) represent heterogenous populations of bone marrow-derived cells that have central roles in the initiation and regulation of immune responses (reviewed in Steinman 1991). The cells under consideration are quite distinct from the follicular DCs found in B-cell areas of lymphoid tissues. It is, however, now clear that there is not a single DC lineage, but that cells with DC properties may arise from at least three lineages and can exist in different states of maturation and activation. The relationships between these lineages and their functional significance in immune responses are far from clear.

Comparative pathogenesis of haemorrhagic enteritis virus (HEV) infection in Turkeys and chickens

The pathogenesis of haemorrhagic enteritis virus (HEV) infection in chickens 3-4 days post-infection was compared with that in turkeys. As expected, infected turkeys showed HEV-specific lesions that included enlargement and mottling of the spleen, as well as haemorrhagic enteritis. In infected chickens, only splenomegaly was observed. The number of HEV-infected cells in the spleen was significantly (P < 0.05) higher in the turkey than in the chicken. In both species, the immunohistochemical labelling of B-cell surface determinants was diminished and the splenic B-cell areas were undetectable after HEV infection. Infection with HEV resulted in an increase in nitric oxide production by macrophages in chickens but not in turkeys.

Sequential changes in CGRP-like immunoreactivity in NIDDM model Otsuka Long-Evans Tokushima Fatty (OLETF) rat pancreatic islets.

We describe the changes in B cells and calcitonin gene-related peptide (CGRP)-like immunoreactivity in the pancreatic islets of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of human non-insulin-dependent diabetes mellitus (NIDDM). In the OLETF rat pancreatic islets, CGRP immunoreactivity was seen in the nerve fibers with multiple varicosities and in endocrine cells that were identical to somatostatin-containing cells, but some somatostatin-immunoreactive cells lacked CGRP immunoreactivity. In the OLETF rats, plasma insulin levels were significantly higher than in the control rats (Long-Evans Tokushima Otsuka; LETO) only at 7 weeks of age. From 7 through 32 weeks of age, OLETF rats had a greater B-cell area than LETO rats. The length of CGRP-immunoreactive nerve fibers per area and the numbers of CGRP-immunoreactive cells per area did not differ between the groups at 7 weeks of age. After 16 weeks of age, both of these CGRP parameters in the OLETF rats became increasingly higher than in the LETO rats. These results suggest that CGRP is a B-cell growth factor and probably an inhibitory factor for insulin secretion.

Long-term administration of a sucrose-rich diet to normal rats: Relationship between metabolic and hormonal profiles and morphological changes in the endocrine pancreas

The aim of the present investigation was to study normal rats fed a sucrose-rich diet (SRD) for a prolonged period (up to 30 weeks) (1) to obtain additional data on the hormonal and metabolic patterns induced by this treatment and (2) to provide information on changes taking place in the pancreatic islet cell populations. We found that long-term feeding with a SRD resulted in a steady state of hypertriglyceridemia and hyperglycemia in which insulin levels remained unchanged and unable to compensate for the increased demands of the developing metabolic changes. The endocrine pancreas showed a significant increase of both islet number and B-cell area, as well as changes in the profile of islet cell distribution. However, these changes were not accompanied by an increase in the pancreatic content of immunoreactive insulin (IRI). It may therefore be postulated that the newly emerged B-cell mass has some sort of derangement with the increased insulin demand resulting from insulin resistance induced by the long-term SRD feeding. Thus, feeding a SRD to normal rats may prove to be an attractive animal model for studying the role of environmental nutritional factors in the unsettled issue of the relationship between insulin resistance and relative insulin deficiency. The model might provide key information for understanding the pathophysiology of human diseases such as type II diabetes, dyslipidemia, and a number of entities included in so-called syndrome X.

Lack of effect of CS-045, a new antidiabetic agent, on insulin secretion in the remnant pancreas after 90% pancreatectomy in rats

We assessed the effect of CS-045, a new hypoglycemic agent, on B-cell function in partially pancreatectomized rats. At the age of 4 weeks, male Wistar rats were subjected to 90% pancreatectomy (Px). For 2 weeks starting at 6 weeks after surgery the Px rats were treated with CS-045 (CS rats) mixed with chow pellets in a proportion of 0.2% (w/w). To compare the efficacy of CS-045 with that of insulin therapy, an osmotic pump was implanted to release insulin (1.2 units/day) into the intraperitoneal cavity of the Px rats (Is rats). Plasma glucose levels in the CS and Is rats were significantly lower than in the control Px rats; however, no marked improvement in plasma glucose or insulin levels was observed in glucose tolerance test (2 g/kg, i.p.) in the CS rats. Insulin secretion by the isolated perfused pancreas in response to 16.7 mM glucose showed a biphasic pattern, but was slightly reduced in the Px and CS rats compared with the Is rats. Insulin secretion induced by 19 mM arginine was unaffected by the treatment. The insulin content of the CS rats was significantly greater than in the Px and Is rats. Histological observations suggested regranulation of the pancreatic islets of the CS rats. B-cell areas within the islet were restored to normal levels in the Cs and Is rats. These findings indicate that the hypoglycemic effect of CS-045, which is not mediated by insulin secretion from the residual pancreas, prevents destruction of the islet.

Effect of chronic vanadate administration in partially depancreatized rats

The effects of vanadate on B-cell function and replication in rats after 90% partial pancreatectomy (Px) were compared with insulin therapy. At the age of 4 weeks, male Wistar rats were subjected to sham operation or Px. Vanadate (0.2 mg/ml) was given in drinking water for 3 weeks starting at 2 weeks after surgery. Regular insulin (2.4 units/day) was administered as a continuous subcutaneous infusion through an osmotic pump. Plasma glucose levels were significantly higher in the Px rats than in the sham rats from 1 week after surgery. Vanadate lowered plasma glucose levels to near normal values in the Px rats as early as 2 days. The effect was sustained throughout the experiment. The hypoglycemic effect of insulin was less than that of vanadate. During an i.p. glucose tolerance test, plasma glucose levels were decreased in the Px rats treated with vanadate or insulin, while plasma insulin levels were not affected. The insulin content in the Px rats treated with vanadate was significantly ( P < 0.01) greater than in the insulin-treated Px rats. Histological examination showed fibrotic degeneration in the enlarged islets of Px rats, whereas the normal structure was retained in most islets of the Px rats treated with vanadate and insulin. In addition, B-cell areas within the islet were restored to normal levels not only in the insulin-treated Px rats but in the vanadate-treated Px rats. However, both vanadate and insulin failed to stimulate proliferative activity of the B-cells. These data suggest that vanadate is a new therapeutic option to ameliorate the diabetic state after Px.

Differential expression of two ICAM-1 epitopes and LFA-1 chains in B-cell non-Hodgkin's lymphomas.

B-cell non-Hodgkin's lymphomas (B-NHL) and B-cell areas of reactive lymphadenopathies were investigated immunohistochemically for expression of two distinct ICAM-1 epitopes, Me14/D12 and P3-58, and the LFA-1 alpha and beta chains. Partial or total loss of expression of one or both epitope(s) and/or chain(s) was evident in all B-NHL in function of increasing Working Formulation (WF) malignancy grade, with most defects in the high-grade tumors, namely the lowest detectability of the ICAM-1 Me14/D12 and LFA-1 alpha chain, the lowest co-expression of ICAM-1 epitopes and LFA-1 chains, and the most frequent simultaneous loss. The ICAM-1 and LFA-1 profiles overlapped within the low- and intermediate-grades, whereas striking differences between the high-grade subtypes were detected. Specifically, Burkitt's and lymphoblastic tumors always lost both epitopes and both chains. Large cell, immunoblastic tumors occasionally did so, and also showed either uncoordinated expression or co-expression of these constituents. It is suggested that expression defects of this type may help differentiate malignant from benign lymphoproliferations, and also be involved in the progression of B-NHL, since most are observed in high-grade tumors, whose ICAM-1 and LFA-1 profiles indicate that their subtypes are the expression of distinct normal B-cell differentiation stages.

Natural history of B-cell dysfunction in spontaneously diabetic Chinese hamsters

To elucidate the pathogenesis of diabetes in spontaneously diabetic Chinese hamsters (CHAD strain), a longitudinal study from just after weaning to overt diabetic state was performed. Fasting and non-fasting plasma glucose, non-fasting plasma insulin and pancreatic hormone contents (insulin, glucagon and amylin) were measured, and light microscopic examination of pancreatic islets by immunohistochemical technique and pancreas perfusion study were performed. No insulitis was found in the islets of the CHAD strain. In animals aged 1 month, there was no significant difference in the percentage of B-cell area to islet area between the CHAD strain and the control. At this stage, hyperinsulinemia was observed despite normal plasma glucose levels both in fasting and non-fasting states. In the animals of the CHAD strain aged 2–4 months, insulin secretion from the pancreas, pancreatic insulin content and non-fasting plasma insulin level decreased in proportion to the decrease of B-cell mass. In animals aged about ten months, severe hyperglycemia and hypoinsulinemia were observed. We demonstrated the existence of amylin-like immunoreactivity in the B-cells of Chinese hamsters. However, no amyloid deposit was observed in the islets of the CHAD strain. After the onset of diabetes, amylin secretion from the pancreas and pancreatic amylin content in the CHAD strain were significantly lower than those in the control. We demonstrated the natural history of B-cell dysfunction in the CHAD strain. It could mean the process of B-cell exhaustion. The profile of the CHAD strain is similar to some types of human NIDDM. Therefore, the CHAD strain is a useful diabetic model in the study of NIDDM.

Ultrastructure of pancreatic B-cells of Atlantic salmon ( Salmo salar) during smoltification and seawater adaptation

Pancreatic B-cells of Atlantic salmon ( Salmo salar) were identified by immunostaining and the ultrastructure and morphology of these cells were investigated during smoltification and acclimation to seawater. Fish were classified as smolts based on length, condition factor, silvering, branchial Na + K + - ATPase activity and salinity tolerance. Individuals measuring less than 15 cm long were used as age-matched, non-smolting controls. Tissue samples were collected from juvenile salmon in February, April and May and, also, in June from post-smolts retained in fresh water or transferred to seawater for 1 month. Pancreas islet size was measured and B-cells were identified by immunohistochemistry and immunocytochemistry. The surface areas of B-cells, nuclei and granules within the cells were quantified, allowing comparison of nucleus: cell (N:C) and total granule: cytoplasm (G:Cyt) ratios. Additionally, the ratio of the number of homogeneous granules: total granules (Hom:G) was determined. No significant changes in size of islets were detected in parr and smolts. In fish undergoing smoltification a slight increase in G:Cyt and a significant decrease in Hom:G ratios ( P<0.05) were observed, whereas in non-smolting parr the G:Cyt ratio increased significantly ( P<0.05) and the Hom:G ratio remained significantly higher than in smolts. A decrease in B-cell area ( P<0.05) was observed in post-smolts retained in fresh water or transferred to seawater. Results are consistent with decreased insulin synthesis or secretion during parr-smolt transformation. In parr during this same period, enhanced B-cell activity was indicated by increases in G:Cyt and elevated Hom:G ratios compared to smolts.

Identification and characterization of a mouse monoclonal antibody (M10) directed against canine (dog) CD8+ lymphocytes

Monoclonal antibodies (mAb) were produced by immunizing BALB/c mice with non-adherent dog lymphocytes. M10 was specific for a subset of dog lymphocytes. M10 belonged to the IgG1 subclass and reacted with 26% of dog peripheral blood lymphocytes, 24% of spleen lymphocytes, 81% of thymus cells, 1.2% of bone marrow cells (5.8% of bone marrow lymphocytes) and 23% of PHA-stimulated lymphocytes. Immunohistology of snap-frozen thymus and spleen showed that the spleen B-cell area stained negative, whereas the spleen T-cell area and the thymus medulla exhibited positive reaction in 20–30%. The thymus cortex was strongly positive. M10 diminished cell lysis by 58% in cell mediated lysis assays (CML). Immunoblot assays revealed that M10 recognized an antigen with a molecular weight of 76 kD under non-reducing and 33 kD under reducing conditions. Finally, M10 bound to a canine CD8α transfected rat T-cell line (NB2). These findings characterize M10 as an antibody directed against the dog CD8 antigen.

Increase in somatostatin to glucagon ratio in islets of alloxan-diabetic dogs: effect of insulin-induced euglycemia.

We have previously shown that acute insulin-induced normalization of glycemia in alloxan-diabetic (A-D) dogs results in marked inhibition of total pancreatic glucagon content, but normalization of somatostatin content. We suggested that this glucagon deficiency might account for A-cell unresponsiveness in diabetes. To examine these changes in detail at the islet level, morphometric and immunologic analyses were carried out on pancreata from four normal (N), four hyperglycemic A-D dogs (HD), and four A-D dogs after acute normalization of glycemia with insulin (ND). The total number of islets per pancreas (3.9 x 10(6) +/- 0.5 x 10(6); determined from the number of islets per square millimetre) was reduced by 60% (p < 0.001) in HD, and this was not affected by acute normalization of glycemia. Insulin content per islet was 1247 +/- 205 pg in N, and this was reduced in both HD and ND to 2 and 5%, respectively (p < 0.001). Similarly, insulin-containing B-cell area was 76 +/- 1% of the total islet area in N, and was unmeasurable in HD and ND. Glucagon content per islet was 89 +/- 6 pg in N, and this was increased by 215% (p < 0.001) in HD, but was normalized in ND. The A-cell area increased concomitantly by 170% from 17 +/- 1 to 46 +/- 2% (p < 0.01) of islet area in HD, and remained elevated in ND.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of alternate-day hydrocortisone therapy on the immunologically immature rat. II: Changes in T- and B-cell areas in spleen.

The histologic effect of 400 mg/M2/day of hydrocortisone, given alternatively from day 7 to day 19 after birth, was studied on the spleen of the immunologically immature rat. Two days after the cessation of treatment, the periarteriolar lymphatic sheaths were found to be largely depleted of small lymphocytes. Immunoperoxidase studies confirmed a depletion of T lymphocytes. The effects of hydrocortisone on the thymus seem to be more important than its direct lymphocytolytic effect in producing this splenic lesion. In contrast, no apparent change in the number of medium-sized B lymphocytes in the marginal zone was detected. Medium-sized B lymphocytes of the spleen, reported to be responsible for IgM synthesis, appeared to be subjected to a different mechanism of hydrocortisone action, other than lysis, resulting in a decrease in antibody production. Primary follicles were not seen in spleens of hydrocortisone-treated rats. Twenty-three days after treatment, spleens had a histologically normal appearance.

Effect of Mucosal and Systemic Immunization with Pneumococcal Polysaccharide Type 3, 4 and 14 in the Rat

Four immunization routes were investigated to induce an immune response against three structurally different types of pneumoccoccal polysaccharide (PPS) in the rat. In particular, the contribution of the IgA isotype in these immune responses was studied. Six days after administration of PPS type 3, 4 or 14, the localization of specific antibody-containing cells (ACC) in different lymphoid tissues and the antibody titres in serum were studied. All four routes induced anti-PPS ACC in the spleen. After intraduodenal, intravenous and especially intraperitoneal administration of PPS, many IgA-specific anti-PPS ACC were also found in parathymic and mesenteric lymph nodes and in the lamina propria of intestinal tissue. Several anti-PPS ACC were found in Peyer's patches, located peripheral of the B-cell areas. The intratracheal immunization elicited only a local immune response, in bronchus-associated lymphoid tissues and paratracheal lymph nodes. The localization of these anti-PPS ACC was influenced by the route of immunization. After all four investigated routes, specific antibodies were found in serum against PPS. However, some remarkable differences between PPS-3, 4 and 14 were found in the magnitude of the immune response and the distribution of the isotypes. Both route of immunization and structure of the PPS have a profound influence on the immune responses in rats.

Histochemical and immunohistochemical study of the mucosal lymphoid system in swine

Summary Enzyme histochemical and immunohistochemical techniques were used to examine palatine tonsils and aggregated lymphoid follicles (Peyer's patches) of the ileum in 6- to 9-day-old and in 6-month-old pigs. Histochemical techniques were used to detect α-naphthyl-acetate esterase (anae), α-naphthyl-butyrate esterase (anbe), β-glucuronidase, adenosine triphosphatase (ATPase), and acid phosphatase (AcP). Nonspecific esterases (anae, anbe) were detected in macrophages, T-cell area lymphocytes, eosinophils, fibroblastic reticular cells (frc), follicular dendritic cells (fdc), and interdigitating cells (idc). β-Glucuronidase reactivity was strong in macrophages, eosinophils, fdc, and idc, and weaker in frc. Adenosine triphosphatase reactivity was detected in B-cell area lymphocytes, fdc, frc, and idc. Cell types with acid phosphatase reactivity were macrophages, fdc, frc, and idc. Nonepithelial cells of tonsils and aggregated lymphoid follicles of the ileum had similar enzymatic reactions. In Peyer's patches, however, epithelial cells were positive for all enzymes studied; in tonsils, only nonspecific esterases were detected. Immunoperoxidase techniques were used to detect S-100 protein and cytoplasmic immunoglobulins (IgG, IgM, and IgA). The S-100 protein was detected in lymphocytes, fdc, and frc of tonsils and Peyer's patches; in tonsillar epithelial and endothelial cells; and in idc of Peyer's patches. Cytoplasmic immunoglobulins were detected in lymphoblastoid and plasmacytoid cells of follicles and diffuse lymphoid tissue in both organs and in ileal epithelial cells. Compared with those of 6-month-old pigs, cells of 6- to 9-day-old pigs stained less intensely by all enzyme histochemical techniques, and fewer cells were reactive for immunoglobulins.

Ultrastructural and immunohistochemical studies on the ontogenic development of bronchus-associated lymphoid tissue (BALT) in the rat: special reference to follicular dendritic cells

The ontogenic development of lymphoid and non-lymphoid cells in bronchus-associated lymphoid tissue (BALT) of the rat was studied ultrastructurally and immunohistochemically. In the late foetal period, only the alveolar macrophages showed a weak positivity for leucocyte common antigen, but no immune region associated (Ia) antigen was detected by monoclonal antibody, MAS 043. Mast cells were present. At 6 days of age, Ia-positive cells were observed in the alveolar wall and peribronchial interstitial tissue, and ultrastructurally there was an aggregation of the fibroblastoid mesenchymal cells. By 10 days of age, the aggregation of lymphoid cells together with S-100-positive reticulum cells had formed a BALT-like periarterial lymphoid sheath. In the adult animals, an obvious B-cell area was present in the central part and subepithelium of BALT, whilst in this area, S-100-positive, strongly Ia-positive cells with a dendritic form were observed. These dendritic cells appeared to be identical to the follicular dendritic cells (FDC) seen in the secondary follicles of lymphoid organs. Those cells may be derived from the fibroblastic reticulum cells, and may function to present antigen to lymphocytes.

Human Germinal Centre B Cells Inhibit Mitogen‐Induced proliferation of Mantle Zone B Cells

Lymphoid follicles are the main B-cell areas in peripheral lymphoid tissues. These structures commonly consist of germinal centre (GC) and mantle zone (MZ) regions. In the present work, human tonsillar B cells belonging to these two compartments were purified by a combination of density centrifugation and separation techniques involving the recognition by monoclonal antibodies of specific surface molecules followed by panning and magnetic bead selection. These purified populations were identified as GC and MZ cells by three different criteria: (1) GC cells showed the phenotype IgD- CD20+bright CD38+ CD44- and peanut agglutinin (PNA)+, and MZ cells were IgD+ CD20+dim CD38- CD44+ and PNA-; (2) morphologically, MZ cells appeared as small resting lymphocytes whereas GC cells consisted of large blastic cells of the germinal centre; (3) functionally, most GC, but not MZ, cells underwent apoptosis early in culture. The isolation of GC and MZ cells allowed the study of their proliferative response. As a result of these studies, GC cells were demonstrated to inhibit the proliferation of MZ cells in response to B-cell mitogens (Staphylococcus aureus Cowan I and anti-mu plus BCGF) in a concentration-dependent way; 50% inhibition was reached at a GC/MZ cell ratio of 1/2. This effect did not require GC-cell DNA synthesis since similar results were obtained with irradiated GC cells. Neither was it due to a non-specific toxic effect since GC cells did not alter the proliferative response of autologous T cells to mitogens (phytohaemagglutin and anti-CD3). The inhibition required cellular contact between GC and MZ cells, and was not restricted by histocompatibility barriers. These data suggest the possible existence of a new regulatory pathway within peripheral B-cell areas.

Characterization of the inflammatory infiltrate in ovarian dysgerminoma: an immunocytochemical study.

The inflammatory infiltrate has been characterized in 10 cases of ovarian dysgerminoma using a panel of antisera to T-cells, B-cells and macrophages. The expression of Class II major histocompatibility complex (MHC) antigens and the distribution of interferons alpha and gamma were also examined. T-lymphocytes were present in all tumours, often closely admixed with neoplastic elements. T-cells in most areas were immunoreactive with gamma interferon. B-cells were generally scanty although germinal centres were present in three tumours. Immunocytochemistry revealed greater numbers of macrophages than had been appreciated on routinely stained sections. Macrophages were closely related to both lymphoid and tumour cells, and many macrophages were immunoreactive for alpha interferon. Class II MHC expression was mainly restricted to macrophages and B-cell areas although occasional T-cells were also stained. Dysgerminoma cells did not express Class II MHC antigens.

Influence of the decrease of intracellular antigenic content on morphometric analysis: effect of the type and dilution of the first antibody.

The aim of the study is to determine the effect degranulation of B cells on their immunohistochemical detection and to evaluate whether this effect depends on the technique of immunological detection. The biological model is the pancreatic insulin-containing B cell. To decrease the insulin content of the pancreas, insulin release was stimulated by five intraperitoneal injections of glibenclamide (2 mg/kg). Specimens of the pancreas were taken for insulin extraction and quantitation by radioimmunoassay (RIA), ultrastructural analyses and immunocytochemistry. The sections were treated either by polyclonal or monoclonal anti-insulin serum at various concentrations and peroxidase-antiperoxidase (PAP) technique eventually followed by silver amplification. The B-cell insular fraction or relative B-cell area (RBA) was measured with an automatic image analyser. The reference value for the relative B-cell area was established in control rats and corresponds to the ratio of the insular area occupied by immunostained B cells to the islet area. Its value was confirmed by calculation of the area occupied by non-B cells. RIA indicates a decrease of 83% of the insulin content in treated rats while the number of B granules decreases by 72% at the ultrastructural level. In usual conditions (polyclonal serum, 1/1500) the degranulation leads to a 16% underestimation of the B-cell insular fraction. This underestimation increases when monoclonal antibodies are used and further increases when higher dilutions are tested. The silver amplification does not prevent this underestimation and, in this particular model, exclusively acts by increasing the contrast. The only means of restoring the correct level of detection is to use the serum at a higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)