Altered B-cell Research Articles

2,3,7,8-tetrachlorodibenzo-p-dioxin does not directly alter the phenotype of maturing B cells in a murine coculture system.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), acting through the aromatic hydrocarbon receptor (AhR), elicits numerous toxicological effects, including immunosuppression. Previous work from our laboratory has suggested that TCDD exposure in mice is associated with altered lymphopoietic development, in particular altered B-cell phenotype in the bone marrow. It remains to be determined which specific hematopoietic populations or subpopulations within the marrow cavity are directly targeted by TCDD. To examine the effects of TCDD on developing B cells in vitro, a stromal coculture model was used. Primary bone marrow cells from male, 6- to 7-week-old C57Bl/6 mice were cocultured separately on two AhR-containing stromal cell lines (M2-10B4 and S17) that support B-cell development in the presence of IL-7. The cocultures were treated with 0 to 10 nM TCDD. Shifts in phenotype were quantified by cell surface marker staining and flow cytometry. Four populations in the maturing B cell (very early pre-pro-B, pre-pro-B, pro-B, and pre-B) were defined for quantification. The results show that the only statistically significant effect of TCDD was within the pre-pro-B population in cultures with the S17 stromal cell line. The increase in number of cells with this phenotype was seen in cultures with both wild-type and AhR-/- primary bone marrow cells. These results suggest that the maturing B220+ B cell is not the direct target for TCDD-induced bone marrow B-cell alterations.

Epstein-Barr virus LMP2A-induced B-cell survival in two unique classes of EmuLMP2A transgenic mice.

Latent membrane protein 2A (LMP2A) is one of only two viral proteins expressed during latent Epstein-Barr virus (EBV) infections in human peripheral B cells. LMP2A blocks B-cell receptor (BCR) signal transduction in vitro by modulation of the Syk and Lyn protein tyrosine kinases. Five genetically unique LMP2A transgenic mouse lines (EmuLMP2A) with B-cell lineage expression of LMP2A were generated in this study to analyze the importance of LMP2A expression in vivo. These animals can be grouped into EmuLMP2A(BCR+) (TgB, Tg6, and TgC) and EmuLMP2A(BCR-) (Tg7 and TgE) lines based on B-cell phenotype. LMP2A expression in bone marrow cells of EmuLMP2A(BCR-) lines was associated with a bypass of normal B-lymphocyte developmental checkpoints inasmuch as immunoglobulin light-chain gene rearrangement occurred in the absence of complete immunoglobulin heavy-chain gene rearrangement. The resulting BCR-negative B cells were able to exit the bone marrow and colonize peripheral lymphoid organs. LMP2A expression in EmuLMP2A(BCR+) lines was not associated with altered B-cell development in a genetically wild-type background. When crossed into a recombinase activating null (RAG(-/-)) genetic background, LMP2A expression in either RAG(-/-) EmuLMP2A(BCR+) or RAG(-/-) EmuLMP2A(BCR-) animals was able to provide a survival signal to BCR-negative splenic B cells. Additionally, bone marrow cells from all EmuLMP2A animals were able to proliferate in response to interleukin-7-dependent developmental signals in vitro. These studies illustrate that LMP2A can provide a survival signal to BCR-negative B cells in two different groups of EmuLMP2A transgenic mice.

Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-B-ALL.

Immunophenotypic investigation of minimal residual disease (MRD) has traditionally been based on the investigation of phenotypic aberrants at diagnosis to be used later as a target for MRD detection. This approach has several shortcomings (it is only applicable to patients with aberrant phenotypes, requires a diagnostic sample, and is patient-specific) and therefore a search for simpler alternatives is warranted. The present study is based on the hypothesis that in precursor-B-ALL patients the persistence of residual leukaemic cells may induce abnormalities in the precursor-B-cell compartment in bone marrow (BM) and these could be used as a criteria to predict relapse. These abnormalities may include: (1) the presence of an increase in the frequencies of immature B cells (CD34+/CD19+ or CD20-/CD19+) or (2) the existence of an altered B-cell differentiation pathway due to a blockade or to the presence of B cells outside the normal pathway. A total of 180 BM samples from 45 consecutive precursor-B-ALL patients who achieved morphological complete remission (CR) were analysed by multiparametric flow cytometry. Our results show that a significant increase in immature B-cell subsets or an altered B-cell differentiation predicts a high relapse rate (P<0.01) and a shorter disease-free survival (P<0.01). Moreover, abnormalities in either of these two criteria detected at specific time points during follow-up (end of induction, maintenance, or after treatment) were associated with a significantly shorter disease-free survival (P<0.01). In summary, the investigation of abnormalities in B-cell differentiation is a relatively simple and cheap approach for predicting relapse in precursor-B-ALL patients.

Insulin secretory capacity and the regulation of glucagon secretion in diabetic and non-diabetic alcoholic cirrhotic patients

Background/Aims: Insulin secretion is increased in cirrhotic patients without diabetes but decreased in cirrhotic patients with diabetes. Increased glucagon secretion is found in both groups. Our aim was to determine: 1) whether alterations in insulin secretion are due to changes in maximal secretory capacity or altered islet B-cell sensitivity to glucose, and 2) whether regulation of glucagon secretion by glucose is disturbed. Methods: Insulin, C-peptide and glucagon levels were measured basally and during 12, 19 and 28 mmol/l glucose clamps, and in response to 5 g intravenous arginine basally and after 35 min at a glucose of 12, 19 and 28 mmol/l in 6 non-diabetic alcoholic cirrhotic patients, six diabetic alcoholic cirrhotic patients and six normal controls. Results: Fasting insulin, and C-peptide levels were higher in cirrhotic patients than controls but not different between diabetic and non-diabetic patients. C-peptide levels at t=35 min of the clamp increased more with glucose concentration in non-diabetic cirrhotic patients than controls; there was little increase in diabetic cirrhotic patients. At a blood glucose of ≈ mmol/l the 2–5 min C-peptide response to arginine (CP ARG) was similar in all groups, but enhancement of this response by glucose was greater in non-diabetic cirrhotic patients and impaired in diabetic cirrhotic patients. Maximal insulin secretion (CP ARG at 28 mmol/l glucose) was 49% higher in the non-diabetic cirrhotic patients than controls ( p<0.05); in diabetic cirrhotic patients it was 47% lower ( p<0.05). The glucose level required for half-maximal potentiation of (CP ARG) was not different in the three groups. Cirrhotic patients had higher fasting glucagon levels, and a greater 2–5-min glucagon response to arginine, which was enhanced by concomitant diabetes ( p<0.001 vs controls). Suppression of plasma glucagon by hyperglycaemia was markedly impaired in diabetic cirrhotic patients (glucagon levels at 35 min of 28 mmol/l glucose clamp: diabetics, 139 ×/ ÷ 1.25 ng/l, non-diabetic cirrhotic patients, 24 × ÷1.20, controls, 21×/÷1.15, p<0.001). Suppression of arginine-stimulated glucagon secretion by glucose was also impaired in diabetic cirrhotic patients, and to a lesser extent in non-diabetic cirrhotic patients. Conclusions: Insulin secretory abnormalities in diabetic and non-diabetic cirrhotic patients are due to changes in maximal secretory capacity rather than altered B-cell sensitivity to glucose. The exaggerated glucagon response to arginine in alcoholic cirrhotic patients is not aboli0shed by hyperglycaemia/hyperin-sulinaemia. In diabetic alcoholic cirrhotic patients, the inhibitory effect of glucose on basal glucagon secretion is also markedly impaired.

Proinsulin, C-peptide, and insulin in normal subjects during an 8-h hyperglycemic clamp

Increased concentrations of proinsulin immunoreactive material (PIM) absolutely or relative to insulin is a characteristic finding in patients with non-insulin-dependent diabetes mellitus (NIDDM). The aim of this study was to test if 8 h or mild hyperglycemia (7-9 mmol/l) in healthy subjects could induce a preferential secretion of PIM from B cells. Serum concentrations of insulin, C-peptide and PIM were measured every 10 min during the 8 h of continuous glucose infusion in nine normal-weight healthy subjects without diabetes among their first-degree relatives. After a gradual rise in B-cell peptides, a steady state was reached. From 4 to 8 h no further difference in insulin, C-peptide or PIM concentration was found. Fasting PIM/C-peptide and PIM/insulin ratios of 0.5% and 2.3% increased during the glucose clamp to levels of 1.4% and 7.6%, respectively. Neither testing the regression slope nor comparing individual time points showed any significant difference for the PIM/C-peptide ratio from 2 to 8 h and for the PIM/insulin ratio from 3 to 8 h. These results do not support the hypothesis that an increased glucose drive per se results in an altered B-cell function with increasing PIM/C-peptide ratio. At least 8 h of mild hyperglycemia in healthy subjects does not progressively alter B-cell function.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (&gt; 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Fusobacterium nucleatum bacteraemia with multiple liver abscesses in an HIV-1 antibody positive man with IgG 2 deficiency

Liver abscess formation due to Fusobacterium nucleatum is rare. We describe an HIV-I antibody positive man, with normal surrogate markers of cell-mediated immunity, who presented with F. nucleatum bacteraemia and liver abscess formation. He was found to have IgG2 subclass deficiency. This case illustrates the clinical importance of altered B-cell function in patients who are at an early stage of HIV disease.

Insulin secretion to glucose stimulus in pancreatic islets isolated from rats fed unbalanced diets

To verify the effect of different energetic sources on insulin secretion, just-weaned male Wistar rats were divided into four groups fed as follows: high carbohydrate (HC), high protein (HP), high lipid (HL) and balanced (C) diets during five weeks. Body weight gain and daily food intake were similar among the four groups. Insulin content of the isolated islets was not different; however, insulin release to a high glucose concentration (16.7 mM) stimulus was clearly higher in islets isolated from rats fed a balanced diet as compared to the other groups (HC, HP and HL). The rates of insulin secretion in islet perfusates from rats fed unbalanced diets were also decreased, although 45Ca 2+ outflow rate (FOR) was similar in all groups. Since the rate of U 14C-glucose oxidation was decreased in islets isolated from rats fed unbalanced diets, this could be one of the mechanisms for the reduced rates of insulin release observed. Therefore, the increased supply of specific fuels provoke metabolic alterations in B-cell leading to changes in insulin secretion.

Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice.

Interferons (IFNs) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation. In fact, IFNs inhibit growth of various normal and transformed cell types. Previously, a nuclear factor, IRF-1 (interferon regulatory factor 1), which binds to type I IFN and some IFN-inducible gene promoters, was identified and cloned. Since the IRF-1 gene is both virus and IFN inducible, an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation. In this study, we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer. In the transgenic mice, all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes (B cells). Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population. In fact, transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern.

Dynamics of Immunoglobulin Synthesis After Major Trauma

Major mechanical trauma causes a severe suppression of B-cell differentiation and IgM synthesis in mononuclear leukocyte cultures. In this study the effect of recombinant lymphokines and physiologic T-cell supernatants on B-cell differentiation was investigated. The influence of potentially suppressing monocytes and positive regulatory T-lymphocytes was eliminated using purified B-cell cultures. Antigen-induced IgM synthesis was reduced on all days following trauma. Addition of recombinant interleukin 2 or T-cell supernatant enhanced but did not restore IgM synthesis. Although recombinant interleukin 4 and recombinant interferon gamma had a suppressive effect on IgM synthesis in controls, both lymphokines were ineffective in the patients' IgM synthesis. Spontaneous IgG production in patients was dramatically elevated, and the addition of lymphokines did not show any enhancing effect in patients. These results demonstrate that the IgM/IgG shift observed in peripheral blood mononuclear cell cultures also exists in purified B-cell cultures. Interleukin 2 partially restored suppressed IgM synthesis, while interleukin 4 and interferon gamma were ineffective in patients' B-cell differentiation. Interleukin 2 was the most effective lymphokine for the induction of Ig synthesis. These results lead us to conclude that the altered B-cell metabolism might also be responsible for the suppression of humoral immunity following trauma.

Different effects of insulin and oral antidiabetic agents on glucose and energy metabolism in Type 2 (non-insulin-dependent) diabetes mellitus

Which therapy should be used in Type 2 (non-insulin-dependent) diabetic patients with "secondary sulfonylurea failure", insulin or a combination of sulfonylurea and metformin? To address this question, we have compared the effect of 6 months of insulin therapy twice daily with that of a combination of glibenclamide and metformin in 24 Type 2 diabetic subjects, who no longer responded to treatment with sulfonylureas. Both treatments resulted in an equivalent 30% improvement in mean daily blood glucose (p less than 0.001), without significant effect on serum lipids. Insulin improved glycaemic control primarily by reducing basal hepatic glucose production (p less than 0.05), but had no significant effect on peripheral glucose metabolism. The combination of glibenclamide and metformin enhanced significantly total body glucose metabolism (p less than 0.05), predominantly by stimulating the non-oxidative pathway. Neither insulin nor the combination therapy altered B-cell response to a test meal. Insulin therapy resulted in a 6% increase in body weight, 63% of which was accounted for by increased fat mass. Although body weight was unchanged during sulfonylurea/metformin therapy, lean body mass and energy expenditure decreased significantly (p less than 0.05). We conclude that insulin and glibenclamide/metformin have different long-term effects on glucose and energy metabolism in Type 2 diabetes.

Behavioral and autonomic responses to peer separation in pigtail macaque monkey infants

Brief maternal separations of young nonhuman primates have been used extensively to study the behavior and physiology of attachment, loss, and bereavement. The physiological responses to the loss of alternative attachment figures, such as peers, is less well documented in nonhuman primates. This study examined both autonomic and behavioral responses of peer-reared pigtail macaque infants to separation. Eight infants were removed from their mothers at birth and reared in four peer pairs. At 6 months of age, each monkey was implanted with a multichannel biotelemetry device which transmitted heartrate, body temperature, EEG, EMG, and EOG. Blood was collected twice weekly for immunological assessment. Behavioral and physiological data, including sleep, were collected for 1 week of baseline, 2 weeks of separation, and 1 week of reunion. Behavioral and physiological results indicated agitation but not depression following separation from their peer attachment figures. We found reduced mitogenic responses to pokeweed consequent to peer separation, suggestive of altered B-cell function. REM variables were the only sleep measures affected by the separation, and were suggestive of agitation but not depression.

Evaluation of B-cell secretion and peripheral insulin resistance during pregnancy and after delivery in gestational diabetes mellitus with obesity.

Nine pregnant women with gestational diabetes mellitus (GDM) were studied. Six normal pregnant women and six normal nonpregnant women were evaluated as control groups. All the women underwent oral glucose tolerance test and glucose clamp during the third trimester of pregnancy and two months after delivery. During OGTT, glucose, C-peptide and insulin plasma levels were determined. C-peptide and insulin values in the late phase of OGTT were higher during pregnancy than after delivery in both groups. In gestational diabetic women, the M-value in the second steady-state during glucose clamp was lower than in controls, both during pregnancy and after delivery. Nevertheless, in both groups the M-value during pregnancy was lower than after delivery. Moreover, in gestational diabetic women there was an inverse correlation between M-value in the second steady-state and ponderal excess index after delivery. In conclusion, the impaired peripheral glucose utilization and the pancreatic pattern of gestational diabetic women compared to normal suggested altered B-cell secretion response, increased peripheral resistance and overweight to be the main changes in GDM.

HTLV‐III seropositivity in symptom‐free Italian haemophiliacs. Correlation with consumption of commercial concentrate and abnormalities of T and B lymphocytes

Various immunological parameters exploring both T- and B-cell functions were determined in a group of 74 symptom-free Italian haemophiliacs treated with commercial concentrate imported from the USA and were correlated with the presence in their serum of antibody to HTLV-III. There was a strong correlation between HTLV-III seropositivity and the amount of concentrate consumed. A significant correlation between HTLV-III seropositivity and T-cell alterations, such as T4/T8 ratio less than 1 and reduction in the absolute number of T4+ lymphocytes, or B-cell alterations such as hypergammaglobulinaemia and enhanced spontaneous IgG synthesis in vitro, was also observed.

Effect of the removal of different endocrine glands upon insulin secretion and B-cell ultrastructure.

Glucose-induced insulin secretion and B-cell ultrastructure were studied in islets obtained from normal, adrenalectomized, radiothyroidectomized, ovariectomized and orchidectomized rats. Both parameters were also studied in the same experimental groups submitted to specific substitutive therapy. Insulin secretion in response to high glucose was significantly diminished in adrenalectomized, hypothyroid and male castrated rats. Conversely, this secretion was enhanced in ovariectomized rats. These abnormal insulin responses were restored to normal range by specific substitutive therapy. B-cell ultrastructure was markedly altered in hypothyroid and in female and male castrated rats. No significant changes were observed in the adrenalectomized rats. No conspicuous alterations were depicted in the other islet cell populations. The features of the morphological alterations were mainly related to changes in the B-granules and the rough endoplasmic reticulum. Modifications of the other B-cell organelles were less frequent. In the castrated rats, a distinctive feature was the appearance of a finely granulated colloid material. These B-cell alterations, consecutive to changes in the circulating levels of a given hormone, seemed to depend on the chemical structure of the hormone itself rather than on the changes induced in the B-cell secretory function. The ultrastructural changes described were reversed, as in the case of insulin release, by specific substitutive therapy. It is concluded that changes in the circulating levels of the hormones studied are followed by specific alterations in both B-cell secretion and ultrastructure.

In vitro pancreatic B-cell response of hyperinsulinemic rats. Differences between pregnancy and VMH-lesions.

We investigated in vitro the pancreatic B-cell response of hyperinsulinemic animals compared to that of controls with the aim of clarifying if the hyperinsulinemia is related to an alteration of the islet insulin secretion. Hyperinsulinemia was induced by pregnancy (observation at 19.5 days) and electrolytic lesions of the hypothalamic nucleus ventromedialis (2 days after lesion). The kinetics of the glucose dose/response curves (measured during a 60 min incubation period) differed markedly in the two hyperinsulinemic conditions. Whereas the islets from pregnant rats were characterized by a lowered glucose threshold, an enhanced half-maximal and maximal secretion rate, those of lesioned rats showed only an increased half-maximal and maximal secretion response in spite of a comparable islet insulin content. When culturing islets of hyperinsulinemic animals up to 6 days, the insulin secretion of those prepared from VMH-lesioned rats behaved identically to controls, whereas the islets of pregnant rats maintained their enhanced sensitivity at a glucose concentration of 5 mmol/l. These results confirm that the pancreatic B-cell response contributed significantly to the hyperinsulinemia, but different mechanisms are likely to be responsible for the altered B-cell secretion.

Genetic control of B- and T-lymphocyte abnormalities of NZB mice in crosses with B10.D2 mice.

Parenteral NZB and B10.D2, F1 and F1 x B10.D2 mice were studied to determine the genetic control of (1) altered B-cell IgD expression, (2) plasma cell frequency, (3) IgM secretion per plasma cell, (4) primary in vitro cytotoxic T-cell responses to H-2-compatible cells, (5) production of thymocyte-binding antibodies, and (6) production of red-cell-specific antibodies. The results demonstrate that, in this cross, IgD abnormalities and production of red-cell-specific antibodies were recessive traits. There was a common genetic influence on plasma cell frequency, IgM secretion per plasma cell and production of thymocyte-binding antibodies which was distinct from the genes governing the ability to generate a cytotoxic T lymphocyte response to H-2-compatible cells.

Abnormalities of immunoregulation in progressive systemic sclerosis. Evidence for excess helper-cell function and altered B-cell function

Abnormalities of immunoregulation in progressive systemic sclerosis. Evidence for excess helper-cell function and altered B-cell function

EFFECTS OF 1.25‐DIHYDROXYCHOLECALCIFEROL, PARATHORMONE AND Ca2+ ON THE PANCREATIC B‐CELL SENSITIVITY TO ALLOXAN

The sensitivity to alloxan was investigated by blood and urine glucose determination and light and electron microscopic study of the endocrine pancreas in groups of mice differing from each other with respect to food ingestion and treatment before alloxan administration. Because of differences in occurrence of glucosuria, degree and duration of hyperglycemia, and severity of structural lesions, it was concluded that starvation increases the alloxan sensitivity and that pre-treatment with 1.25-dihydroxycholecalciferol (DHCC) or parathormone (PTH), but not with Ca2+, enhances the alloxan effect. The serum-calcium concentration determined 10 minutes after pre-treatment was significantly increased in the group given Ca2+, but not in the groups injected with DHCC or PTH. Starved mice injected with DHCC or PTH 10 minutes before alloxan administration exhibited a pronounced second hyperglycemia of long duration, and extensive, selective B-cell necrosis. Starvation and increased serum concentration of DHCC and PTH are believed, directly or indirectly, to induce B-cell alterations which increase the alloxan sensitivity.