T-cell receptor Vbeta repertoire after myeloablative and reduced intensity conditioning allogeneic haematopoietic stem cell transplantation.

T cells play an important role in the adaptive immune system. After haematopoietic stem cell transplantation (HSCT), T-cell function is impaired. This is reflected by the emergence of opportunistic infections, infections that are often difficult to treat because of the patient's insufficient immune function. T-cell receptor reconstitution was studied using CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) HSCT in 23 patients. Immune function in vitro was tested by lymphocyte stimulation at 3, 6 and 12 months after HSCT. Lower diversity in the CDR3 repertoire was demonstrated in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared with healthy donors. After myeloablative HSCT, lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Acute and chronic graft-versus-host-disease (GVHD) did not affect diversity. Responses to phytohaemagglutinin (PHA), Concanavalin A (Con A) and Staphylococcus aureus protein A were significantly lower compared with healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT, lymphocyte response to Con A was significantly lower at 3 months compared with healthy donors. Decreased responses to cytomegalovirus and varicella zoster virus antigens were seen in patients suffering from acute GVHD grade II or chronic GVHD. The T-cell repertoire is skewed under the first year after HSCT, and immune reconstitution after HSCT with myeloablative and RIC conditioning seems to be comparable. GVHD, infections and age are more important for immune reconstitution than type of conditioning.

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens.

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.

Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation.

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients.

Genetic markers for the efficacy of tumour necrosis factor blocking therapy in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a genetically complex disease where the response to different treatments varies greatly between different patients. This is the case with the tumour necrosis factor (TNF) blocking agents, where 20-40% of patients have been described as non-responders. No predictive markers exist as yet for the prognosis of response. OBJECTIVE: To analyse whether polymorphisms of several cytokine genes are associated with the responsiveness to TNF blockade with etanercept. METHODS: 123 patients with active RA were treated with etanercept and response rates were determined after three months using American College of Rheumatology (ACR)20 and disease activity score (DAS)28 response criteria. Genotyping was done for TNF (-308 TNFA), interleukin (IL)10 (-1087 IL10), transforming growth factor (TGF)beta1 (codon 25 TGFB1), and IL1 receptor antagonist (intron 2 IL1RN). RESULTS: 24 patients (20%) were defined as non-responders owing to their failure to fulfil any of the ACR20 or DAS28 response criteria. None of the recorded alleles was alone significantly associated with responsiveness to treatment. However, a certain combination of alleles (-308 TNF1/TNF1 and -1087 G/G) was associated with good responsiveness to etanercept (p<0.05). In addition, a combination of alleles influencing interleukin 1 receptor antagonist (IL1Ra) and TGFbeta1 production (A2 allele for IL1RN and rare C allele in codon 25 of TGFB1 gene) was associated with non-responsiveness (p<0.05). CONCLUSION: Genetic polymorphisms, which may influence the balance of pro- and anti-inflammatory cytokines of relevance for the course of RA, are associated with clinical responsiveness to etanercept treatment.

Altered expression of receptors for thyroid hormone and insulin-like growth factor-I during reconstitution after allogeneic hematopoietic stem cell transplantation.

Treatment with neuroendocrine hormones has been suggested to promote reconstitution of the immune system after hematopoietic stem cell transplantation (HSCT). We investigated the expression of genes encoding receptors for growth hormone (GH), insulin-like growth factor-I (IGF-I) and triiodothyronine (T3), at various time points after HSCT in 16 patients and 15 healthy controls. Peripheral blood mononuclear cells were isolated and RNA for GH receptor (GHR), IGF-I receptor (IGF-IR) and thyroid hormone receptor (TRalpha1) was amplified by RT-PCR. The expression of the genes was compared with the expression of beta-actin. We demonstrate increased expression of TRalpha1 RNA in patients at 1.5 months post HSCT, compared to a group of healthy controls, and decreased expression of IGF-IR RNA at 2 and 3 months post HSCT, compared to the controls. Serum from three of the patients was also analyzed for levels of T3, T4, TSH and IGF-I at several time points after HSCT. Serum levels for T3, thyroxine (T4), thyroid stimulating hormone (TSH) and IGF-I were within the normal range in all samples. Our results on the molecular level indicate a role for thyroid hormones and IGF-I in immune reconstitution after HSCT, even though the serum levels of T3, T4, TSH and IGF-I are normal.

Increased adipose angiotensinogen gene expression in human obesity.

OBJECTIVE: Adipose angiotensinogen has been suggested as a stimulator of adipose tissue growth and development. Therefore, the association of subcutaneous adipose angiotensinogen gene expression with human obesity was studied. RESEARCH METHODS AND PROCEDURES: The study group consisted of 17 men, undergoing either gastric banding for obesity or elective laparoscopic cholecystectomy (7 obese, 10 non-obese men; body mass index 22 to 51 kg/m2; age 26 to 68 years). Subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured using competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Adipose angiotensinogen mRNA expression was about two times increased in obesity. The levels of 18S rRNA did not differ between the two groups. Body weight correlated independently and positively with adipose angiotensinogen mRNA expression after adjusting for differences in age and height. DISCUSSION: Adipose angiotensinogen gene expression is elevated in obesity in men.

The association of human adipose angiotensinogen gene expression with abdominal fat distribution in obesity.

OBJECTIVE: To investigate in obese subjects the relationship between angiotensinogen gene expression in the abdominal omental and subcutaneous adipose tissue on the one hand and body fat distribution as measured by waist-to-hip ratio (WHR) on the other hand and to compare angiotensinogen gene expression between the two adipose tissue regions. SUBJECTS: Twenty obese subjects undergoing weight reduction surgery with adjustable gastric banding (12 men, eight women; WHR 0.89-1.09; body mass index (BMI) 29-51 kg/m2, age 26-54 y). MEASUREMENTS: Omental and subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured by competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Angiotensinogen mRNA levels were one-third higher in the omental than in the subcutaneous adipose tissue region (P=0.02). The 18S rRNA levels did not differ significantly between the two adipose tissue regions. WHR correlated positively and significantly with angiotensinogen mRNA in both the subcutaneous and the omental adipose tissue (r=0.5). This relationship was independent of age and BMI. However, WHR did not correlate with 18S rRNA in any of the adipose tissue regions. CONCLUSION: The angiotensinogen gene in adipose tissue might be involved in the development of upper-body obesity.

Preoperative feeding does not reverse postoperative insulin resistance in skeletal muscle in the rat.

Metabolic studies on injured and postoperative patients have shown impaired glucose disposal in peripheral tissues after trauma. Using small-bowel resection as a model of surgical trauma, we investigated whether substrate availability could ameliorate the changes in muscle glucose uptake induced by trauma. We also studied the effect of preoperative feeding on postoperative insulin-stimulated insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity in both Wistar rats and genetically non-insulin-dependent diabetic Goto-Kakazaki rats (GK rats). Serum glucose, insulin, plasma epinephrine, lactate, and plasma nonesterified free fatty acids (NEFAs) were measured as indicators of the metabolic state and surgical stress. Insulin-stimulated glucose transport was significantly reduced in fed traumatized Wistar rats compared with fed nontraumatized rats (P < .05). Significant increases in in vivo insulin-stimulated IRS-1-associated PI 3-kinase activity were found in fed traumatized Wistar rats compared with fed nontraumatized Wistar rats and fasted traumatized Wistar rats, as well as fed traumatized GK rats compared with fed nontraumatized GK animals (all P < .017). Serum insulin concentrations were significantly reduced in fed traumatized Wistar and GK rats compared with the respective fed nontraumatized groups (both P < .01). Serum glucose levels were significantly elevated in fed traumatized GK rats compared with fed nontraumatized animals (P < .01). In the present study, preoperative feeding did not prevent a postoperative reduction in insulin-stimulated glucose transport in skeletal muscle. The finding that insulin-stimulated PI 3-kinase activity increased after trauma in both Wistar and GK rats indicates that postoperative insulin resistance is not caused by an impairment in the early steps of the insulin signaling pathway. The postoperative decreases in serum insulin despite high blood glucose suggest that trauma impairs the insulin response to hyperglycemia.

Long-term persistence of oligoclonal serum IgM repertoires in patients treated with allogeneic bone marrow transplantation (BMT).

Immunoglobulin gene rearrangements in patients treated with BMT have restricted repertoire diversity. Clonal variability remains low for 3 months and reconstitution of the humoral immune system appears to follow a wave-like pattern. In the present study we analysed serum IgM and IgG repertoires in 44 patients from 1 week to 3 years after transplantation. We applied a quantitative immunoblot technique in combination with a newly developed method for estimation of repertoire diversity in complex mixtures of antibodies. Our results demonstrate that 60% of BMT patients have severely reduced diversity in the IgM repertoire during and after the first year post-BMT, compared with healthy controls. In contrast, the majority of patients have a polyclonal IgG repertoire, similar to that of healthy controls. Serum IgM repertoires remain oligoclonal even though the serum concentration of total IgM is within normal range around 6 months post-BMT. During the first years after transplantation IgM as well as IgG repertoires are less diverse in patients receiving a BM graft from a sibling donor compared with those receiving a graft from an HLA-matched unrelated donor. Patients in the latter group show a higher incidence of infections and minor antigen mismatches which may promote the development of a diverse immunoglobulin repertoire post-BMT.

Altered VH6-D-JH repertoire in human insulin-dependent diabetes mellitus and autoimmune idiopathic thrombocytopenic purpura.

We have characterized the peripheral B cell repertoire in T cell-mediated insulin-dependent diabetes mellitus (IDMM) and in B cell-mediated autoimmune idiopathic thrombocytopenic purpura (AITP). The VH6-containing repertoire in adult patients with IDDM or AITP and healthy control subjects was investigated by PCR amplification using VH6- and JH-specific primers. Nucleotide sequence analysis of VH6-D-JH rearrangements showed an abnormally high frequency of somatic mutations in non-functional rearrangements from diabetic (3. 58 %) as well as AITP patients (3.18 %), compared to controls (0.4 % and 1.43 %, respectively; p < 0.05). In contrast, the mutation frequency among functional rearrangements was 2.4 - 3 times lower in patients compared to controls ( p < 0.05). Detailed analysis of the VH6 genes carrying mutations showed that the underlying mechanism for this observation is probably different for the two diseases. Analysis of D- and JH gene usage revealed additional deviations from the normal pattern. Taken together, these results suggest defects in the mechanisms controlling selection of the B cell repertoire in patients with IDDM or AITP.

Prophylactic intravenous immunoglobulin treatment influences serum immunoglobulin M repertoire development after allogeneic bone marrow transplantation.

Patients treated with allogeneic bone marrow transplantation (BMT) suffer from a deficient humoral immunity during the post-transplant period. To prevent infections patients may receive prophylactic intravenous immunoglobulin (IVIG) therapy from 1 week before to 3 months after BMT. We have studied the effect of IVIG treatment on reconstitution of immunoglobulin repertoires in transplanted patients. Sera obtained from 13 IVIG-treated and 31 non-IVIG-treated patients before and at different time points after BMT, ranging from 3 days to 3 years, and from 18 healthy controls, were analyzed using a quantitative immunoblot system. The average immunoglobulin (Ig)M and IgG reactivity profiles against antigens derived from human liver, muscle and skin as well as Staphylococcus epidermidis protein extracts were similar in both patient groups and in controls. Both IgG and IgM reactivity profiles are, however, less heterogeneous among the individuals in the IVIG-treated patient group. Around 1 year after BMT the heterogeneity of the IgM reactivity profiles against allogeneic protein extracts is much lower in the IVIG-treated group compared to the non-IVIG-treated group and the healthy controls. This effect remains months to years after the IVIG treatment has been completed. Our results suggest that IVIG influences selection of the natural antibody repertoire mediated by the variable (V)-region during reconstitution after BMT.

Increased utilization of the VH6 gene family in patients with autoimmune idiopathic thrombocytopenic purpura.

The VH gene family utilization pattern among pokeweed mitogen-stimulated immunocompetent B cells (available repertoire) and naturally activated B cells (actual repertoire) from the spleen was analysed in a group of patients with autoimmune idiopathic thrombocytopenic purpura (AITP). For this purpose a non-radioactive RNA in situ hybridization technique was employed, allowing detection of VH gene family expression in single cells. The results show that the VH gene family expression pattern in patients with AITP does not correlate with genomic complexity of the VH genes. Furthermore, the pattern of VH gene family utilization in AITP patients is statistically different from that of healthy controls in the available, but not in the actual repertoire. The VH5 gene family is used at a frequency of 11.3% in patients' resting B lymphocytes, compared to 23.9% in controls. The VH6 gene family is used more frequently in patients (23.0% compared to 3.8% in controls). The increase in VH6 gene expression is not reflected in the actual repertoire, where the frequency of expression is 6.4%, and can therefore not be directly related to the presence of disease specific autoantibodies.

Skeletal muscle insulin resistance after trauma: insulin signaling and glucose transport.

Surgical trauma induces peripheral insulin resistance; however, the cellular mechanism has not been fully elucidated. We examined the effects of surgical trauma on insulin receptor signaling and glucose transport in skeletal muscle, a tissue that plays a predominant role in maintaining glucose homeostasis. Surgical trauma was induced by intestinal resection in the rat. Receptor phosphorylation was not altered with surgical trauma. Phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase association was increased by 60 and 82% compared with fasted and fed controls, respectively (P < 0. 05). Similar results were observed for insulin receptor substrate-1-associated PI 3-kinase activity. Insulin-stimulated protein kinase B (Akt kinase) phosphorylation was increased by 2.2-fold after surgical trauma (P < 0.05). The hyperphosphorylation of Akt is likely to reflect amplification of PI 3-kinase after insulin stimulation. Submaximal rates of insulin-stimulated 3-O-methylglucose transport were reduced in trauma vs. fasted rats by 51 and 38% for 100 and 200 microU/ml of insulin, respectively (P < 0.05). In conclusion, insulin resistance in skeletal muscle after surgical trauma is associated with reduced glucose transport but not with impaired insulin signaling to PI 3-kinase or its downstream target, Akt. The surgical trauma model presented in this report provides a useful tool to further elucidate the molecular mechanism(s) underlying the development of insulin resistance after surgical trauma.

Oligoclonal dominance of immunoglobulin VH3 rearrangements following allogeneic bone marrow transplantation.

Normal numbers of circulating B lymphocytes are reached during the first 6 months following allogeneic BMT, but humoral immunity remains poor. The molecular basis for this lack of function in the first appearing B lymphocytes has not been clarified. Accordingly, we have studied the reconstitution of the VH3 containing Ig repertoire in two CML patients transplanted with allogeneic BM and one healthy control. PBMCs were isolated at several time-points after BMT and mRNA was prepared. VH3 containing Ig rearrangements were amplified with RT-PCR and then cloned and analyzed with colony hybridization using complementary determining region 3 (CDR3)-specific oligonucleotide probes. Four weeks after BMT, two individual clones together represented 52% of the analyzed CDR3 regions. At 6, 8 and 12 weeks after BMT the corresponding probes hybridized with 2-6% of the colonies. A similar pattern was obtained for the other patient. In samples from the healthy control no clones were detected using CDR3-specific oligonucleotide probes from the control. We conclude that the VH3 containing Ig repertoire after BMT is oligoclonal and that specific rearrangements dominate at different time-points. This restriction of the B cell repertoire may contribute to the impaired humoral immunity observed in BMT recipients.

Age-related impaired affinity maturation and differential D-JH gene usage in human VH6-expressing B lymphocytes from healthy individuals.

To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we analyzed a panel of 179 polymerase chain reaction-derived VH6-D-JH rearrangements from cord blood, peripheral blood, and spleen. Nucleotide sequence analysis of the CDR3 region shows that there is a difference in D and JH gene usage in functional rearrangements between lymphocytes from peripheral blood and spleen. Analysis of the VH6 gene shows that the mutational frequencies rise from 0.81% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decrease to 0.80% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20-49 years, and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. These data show an age-related impaired affinity maturation which might relate to the decrease in immunological responsiveness among the elderly.

Aberrant V(H) gene utilization in patients with established insulin dependent diabetes mellitus.

We have compared the B-lymphocyte repertoire in seven IDDM patients with 12 healthy controls by examining the variable heavy (V(H)) gene expression. The V(H) gene representation in the pool of pokeweed mitogen (PWM) stimulated, immunocompetent B cells and in the pool of naturally activated plasma cells (actual repertoire) was analysed by RNA-RNA in situ hybridization. Differences between IDDM patients and normal controls in the relative expression of several V(H) gene families were observed. In IDDM patients, the V(H)3 was significantly underrepresented in the PWM stimulated repertoire. In the actual B cell repertoire the V(H)5 clones were underrepresented among diabetic patients. Moreover, the altered distribution of V(H) gene usage between the PWM stimulated repertoire and the actual repertoire observed in normal controls was found to be less pronounced in the IDDM patients. This observation suggests a defect in the V-gene directed cellular selection occurring between resting, immunocompetent B cells and naturally activated plasma cells. The possible implication of the observed aberrations in the B cell selection process for the pathogenesis of autoimmunity is discussed.

Uremic serum enhances scavenger receptor expression and activity in the human monocytic cell line U937.

The macrophage scavenger receptor (SR) plays a leading role in atherogenesis, but little is known about the relevance of SR to atherosclerosis in uremia. In this study, the impact of uremic serum on SR expression and activity was examined in the human monocytic cell line U937. The cells were cultured with serum from ten healthy subjects, ten hemodialysis (HD) and ten continuous ambulatory peritoneal dialysis (CAPD) patients. SR mRNA expression was examined using reverse transcriptase-polymerase chain reaction followed by Southern blot. SR protein amount was evaluated by ligand blot. SR activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using flow cytometry. Uremic serum dose-dependently enhanced SR activity primarily by increasing the amount of receptor protein. Heat-inactivated uremic serum had a stimulatory effect, but ultrafiltrate of uremic serum, which included molecules with a molecular weight less than ten kDa, had no effect. The serum levels of macrophage-colony stimulating factor (M-CSF), an activator of SR, were fourfold higher in uremia and significantly correlated with SR activity in cells treated with uremic serum. Pre-treatment of uremic serum with a neutralizing antibody to M-CSF abolished the effect of uremic serum on SR activity. In conclusion, uremic serum contains a factor(s) that enhances SR expression and activity in U937 cells. Elevated M-CSF in uremic serum could be responsible for this enhancement.

Selective usage of VH genes in adult human B lymphocyte repertoires.

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1-6 and for the constant region genes C mu and C gamma. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.

Elevated expression levels of an Ig transgene in mice links the IgH 3' enhancer to the regulation of IgH expression.

To delineate the role of the IgH 3' enhancer in the regulation of Ig heavy (IgH) chain gene expression, mice harbouring rearranged IgH transgenes, with (PSVmu3) and without (PSVmu1) this element, were produced. RNA and protein analysis from the different transgenic lines revealed a 5- to 7-fold increase in the expression level of the transgene containing the IgH 3' enhancer. This difference is also reflected at the protein level in hybridomas generated from the two transgenic lines. The elevation of transgene Ig expression in the PSVmu3 lines is restricted to activated B lymphocytes, an observation which is further supported by the ability of this transgene to be reactivated upon immunization. Interestingly, although the up-regulation of transgene expression in PSVmu3 animals is considerably higher in comparison to the PSVmu1 animals, a significant response is still observed in the PSVmu1 mice. We speculate therefore that the IgH locus is subject to transcriptional modification in late B cell development. Our data suggest that both the Emu enhancer and the IgH 3' enhancer can up-regulate transgene Ig expression, but the presence of the 3' enhancer results in elevated levels of transgene Ig production. It therefore appears that the expression level of IgH genes is subject to transcriptional modification during B cell development. Additional control elements are most likely required for optimal Ig expression, since our expression data from the transgene in PSVmu3 animals are incompatible with endogenous Ig levels. The recent identification of additional enhancer elements in the far 3' end of the IgH locus supports this possibility. The data presented here provides a sound basis for the production of high levels of mAb, possibly tailored to suit the needs of the researcher.

Inhibition of immunoglobulin production in vitro by IgG and F(ab')2 fragments, but not by the Fc portion.

Antibody-secreting B cells were measured as plaque-forming cells (PFC) in a modified haemolysis-in-gel assay, using protein A coupled sheep erythrocytes as targets. Human lymphocytes from blood (PBL), bone marrow or spleen were stimulated in vitro by various polyclonal B-cell activators and incubated with intravenous immunoglobulin (IVIG) or peptide fragments of IVIG. IgG and IgM production from PBL and bone marrow cells, measured as PFC, was inhibited more than 50% by IVIG 2.5 mg/ml, compared to controls without IVIG. Inhibition of the IgG and IgM response of spleen cells by IVIG varied depending on the stimuli. Using Staphylococcus aureus protein A (SPA), inhibition was almost 90% (P < 0.001). The inhibition of the IgG and IgM responses to lipopolysaccharide from Escherichia coli (LPS) were 70% (P < 0.01) and 28% (P < 0.05), respectively. IgG stimulation by pokeweed mitogen (PWM) was inhibited by 57% (P < 0.01), but the IgM response was inhibited only by the higher IVIG concentration of 5.0 mg/ml. In mixed lymphocyte cultures of spleen cells, IgG and IgM production were inhibited by more than 60% (P < 0.05). The effect of IgG, IgG-F(ab')2 and IgG-Fc on LPS or PWM-stimulated spleen cells were compared, using equimolar concentrations of the various preparations. IgG- and IgM-producing PFC were significantly (P < 0.05) inhibited in a dose-dependent fashion by IgG and F(ab')2, but not by Fc. LPS-induced IgG and IgM production was inhibited also when IgG and F(ab')2 were added up to 48 h after the stimulator. A comparison of IgG, F(ab')2 and Fc products from different companies showed that all IgG and F(ab')2 preparations significantly inhibited IgG and IgM production of LPS-stimulated spleen cells. No significant inhibition was obtained with any of the purified Fc products.