B-cell Regeneration Research Articles

A Technique for Inducing B-Cell Ablation in Chickens by in ovo Injection of Cyclophosphamide

The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of incubation. Blood samples were collected periodically from CY-treated and nontreated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 wk of age and booster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40%, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, B-cell responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by hemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. This study indicates the importance of monitoring the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.

Multiple myeloma related cells in patients undergoing autologous peripheral blood stem cell transplantation.

A high incidence of oligoclonal serum M-components is observed in multiple myeloma (MM) patients treated with autologous stem cell transplantation (ASCT). To determine whether these M-components are produced by myeloma clonally related cells or caused by an aberrant B-cell regeneration we analysed by semi-nested ASO-RT-PCR and DNA sequencing the immunoglobulin (Ig) variable genes (VH) obtained from bone marrow samples obtained before and after transplantation and peripheral blood stem cell (PBSC) samples from seven patients. Myeloma clonally related cells are identifiable by the expression of variant Ig heavy chain isotypes and were detected in two patients at presentation. No myeloma clonally related cells were found in post-transplantation samples (n = 7) in spite of the appearance of new serum M-components. However, in two cases we amplified sequences from post-transplantation bone marrow cells that were able to bind to the B-cell clone-specific CDR3 oligonucleotides but showed no further similarity regarding the VDJ rearrangement. These data indicate that serum oligoclonality post-transplantation is not caused by myeloma clonally related B cells but rather by the regenerating B-cell compartment.

Application of flow cytometry in the assessment of lymphoid reconstitution following BM transplantation

Lymphoid reconstitution following BM/stem cell transplantation or intensive chemotherapy is an area of intense interest. Investigating this has been facilitated by the availability of an extensive array of MAb reagents, together with the application of multi-color flow cytometry. These studies have also depended on a thorough understanding of normal lymphoid development, which is linked to the coordinated expression of specific surface proteins. This presentation reviews the current understanding of T-cell and B-cell regeneration as assessed by flow cytometry. T -cell reconstitution is recognized as a highly regulated process that appears to be distinctive for CD4þ T cells and CD8þT cells. It is clear that CD4þT cell-regeneration after intensive chemotherapy follows an age-dependent timetable. During childhood, the recovery of CD4þ T cells is dominated by thymic lymphopoiesis which is directly linked to thymic enlargement assessed radiographically. There is an inverse correlation between age and CD4þ T-cell number at 6 months post-therapy. The thymic-derived CD4þ T cell seen in children is characterized by the expression of CD45RA, the high molecular weight form of CD45. In contrast, older adults demonstrate CD4þ T cell recovery that is far more protracted, with evidence that many of the recovering cells represent progeny of peripherally-expanded T cells. These CD4þ T cells express CD45RO, the low molecular weight form of CD45, and appear to develop via a thymic-independent T cell regeneration pathway. This pathway requires antigen exposure by mature T cells for T-cell expansion and may result in a more limited T-cell repertoire. Taken together, these data suggest that the CD4þ T-cell reconstitution seen in adults does not recapitulate the CD4þ T-cell recovery observed in children. CD8þ T-cell reconstitution is distinctive from that seen for CD4þT-cell recovery. There is a very rapid expansion of CD8þ/CD57þ and CD8þ/CD28 T subpopulations following intensive chemotherapy (or radiation therapy). In contrast with CD4þ T cells, CD8þ T-cell reconstitution does not correlate with either the age of the host, or thymic enlargement. In addition, although the CD8þ T cells expressing CD45RA show a delayed recovery, this process has no relationship to age or thymic enlargement. These findings suggest that the CD8þ/CD57þ and CD8þ/CD28 T cells expand via a thymic-independent pathway which is not available to CD4þ T cells. This explains the marked difference in the kinetics of recovery between these two major T-cell subsets. B-cell reconstitution following BM transplantation appears to depend on the development of donor stem cells, or early B-cell progenitor. This pathway of recovery more closely recapitulates normal B-cell ontogeny and memory B cells from the donor appear to play a role only very early following the transplant. B-cell function remains defective for up to 1 year following the transplant in the absence of chronic GvHD, it fails to normalize when chronic GvHD is present. Furthermore, germinal center recovery in the lymph nodes appears months to years after the transplant. The recapitulation of normal ontogeny is also seen functionally, with normalization of serum IgM preceding IgG and IgA and recovery of antibody responses to protein antigens before responses to carbohydrate antigens. Cytotherapy (1999) Vol. 1, No. 4, 333

Flow cytometry of blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells and lymphocyte subsets after treatment with 2-chlorodeoxyadenosine

By flow cytometry and an extensive set of markers, we characterized leukemic cells from the blood and bone marrow of 68 symptomatic patients with hairy cell leukemia (HCL). Hairy cells identified in the large cell gate always expressed CD19, CD20, HLA-DR, CD45RA, and B-ly 7. Other markers were occasionally expressed, such as CD38, CD45RO, CD23, CD15, CD4, CD5, and CD10 (expressed on more than 20% of the hairy cells in 44%, 25%, 21%, 18%, 12%, 10%, and 5% of evaluated cases, respectively). During treatment with 2-chlorodeoxyadenosine (CdA), the median lymphocyte counts decreased from 2,000/microL to 300/microL. Flow cytometry was repeated at the nadir (n = 24) of lymphocyte counts, at 3 months (n = 46), at 6 months (n = 50), at 1 year (n = 39), and at 2 years (n = 12) after treatment. The initial decrease of CD8+ and CD20+ cells was greater than that of CD4+ and natural killer (NK) cells, leading to an increasing CD4/CD8 ratio. Median nadir values of CD4+, CD8+, CD20+, and NK cells were 128/microL, 78/microL, 10/microL, and 13/microL, respectively. The subsequent recovery was quicker for CD8+ and NK cells, leading to a normalization within 3 months, whereas CD20+ and CD4+ cells required 1 or 2 years to enter the normal range. The CD4/CD8 ratio thus decreased after the nadir and remained less than 1. CD45RA+ CD4 cells and CD45RA+/CD45RO+ double-positive cells were less affected by CdA. Activated T cells, ie, HLA-DR+ cells, rarely decreased below the normal range and often recovered with an overshoot. CD10+ cells increased in the bone marrow posttreatment as an indication of normal B-cell regeneration in 16 of 36 (44%) patients. The quick regeneration of certain lymphoid subsets might explain the lack of late infections in CdA-treated HCL patients.

Flow Cytometry of Blood and Bone Marrow Cells From Patients With Hairy Cell Leukemia: Phenotype of Hairy Cells and Lymphocyte Subsets After Treatment With 2-Chlorodeoxyadenosine

Abstract By flow cytometry and an extensive set of markers, we characterized leukemic cells from the blood and bone marrow of 68 symptomatic patients with hairy cell leukemia (HCL). Hairy cells identified in the large cell gate always expressed CD19, CD20, HLA-DR, CD45RA, and B-ly 7. Other markers were occasionally expressed, such as CD38, CD45RO, CD23, CD15, CD4, CD5, and CD10 (expressed on more than 20% of the hairy cells in 44%, 25%, 21%, 18%, 12%, 10%, and 5% of evaluated cases, respectively). During treatment with 2-chlorodeoxyadenosine (CdA), the median lymphocyte counts decreased from 2,000/microL to 300/microL. Flow cytometry was repeated at the nadir (n = 24) of lymphocyte counts, at 3 months (n = 46), at 6 months (n = 50), at 1 year (n = 39), and at 2 years (n = 12) after treatment. The initial decrease of CD8+ and CD20+ cells was greater than that of CD4+ and natural killer (NK) cells, leading to an increasing CD4/CD8 ratio. Median nadir values of CD4+, CD8+, CD20+, and NK cells were 128/microL, 78/microL, 10/microL, and 13/microL, respectively. The subsequent recovery was quicker for CD8+ and NK cells, leading to a normalization within 3 months, whereas CD20+ and CD4+ cells required 1 or 2 years to enter the normal range. The CD4/CD8 ratio thus decreased after the nadir and remained less than 1. CD45RA+ CD4 cells and CD45RA+/CD45RO+ double-positive cells were less affected by CdA. Activated T cells, ie, HLA-DR+ cells, rarely decreased below the normal range and often recovered with an overshoot. CD10+ cells increased in the bone marrow posttreatment as an indication of normal B-cell regeneration in 16 of 36 (44%) patients. The quick regeneration of certain lymphoid subsets might explain the lack of late infections in CdA-treated HCL patients.

Islet cell defence and repair mechanisms in insulin-dependent diabetes: a role for the pancreatic regenerating (Reg) gene?

Introduction The characteristic inability to maintain glucose homeostasis in diabetes mellitus reflects an absolute or partial insulin deficiency. This lack of insulin is due to an overall reduction in the mass of properly functioning B-cells. Such a reduction occurs as a result of a combination of an increased rate of cell destruction and a decreased rate of cell repair and renewal. While considerable attention has been focused on the elucidation of the factors resulting in €3-cell loss, less information is available regarding possible common genetic control mechanisms, thus implicating alterations in islet cell repair and/or adaptive B-cell regeneration as causative factors in diabetes. There are obvious difficulties in performing studies on islet cell repair and renewal in diabetes in man. However, earlier investigations performed in models of diabetes have provided some insight into the defence and repair mechanisms activated in the islet cells following injury and onset of disease [ 11. The hypothesis that pancreatic B-cells possess defence mechanisms against cell injury gained support from observations that islets are able to survive, and even recover functional activity, after exposure to cytotoxic attack or disturbed metabolism [2, 31. The nature of the islet cell defence process is unclear but is probably due to a combination of a repair/recovery response of surviving cells and an adaptive growth response to a reduced B-cell mass. However, the magnitude and subsequent effectiveness of the islet cell defence and repair mechanisms is dependent on the extent and nature of the cytotoxic insult [4]. A short exposure of rat

Species specificity of serum factors from rabbits with acute pancreatitis in stimulation of B-cell regeneration in pancreatic islets in experimental diabetes

Species specificity of serum factors from rabbits with acute pancreatitis in stimulation of B-cell regeneration in pancreatic islets in experimental diabetes

Nicotinamide Increases C‐peptide Secretion in Patients with Recent Onset Type 1 Diabetes

Nicotinamide, a poly-(ADP-ribose) synthetase inhibitor, has been shown in animal models to induce islet B-cell regeneration. An open controlled trial was therefore carried out for 1 year in 36 patients with recent onset (less than 4 weeks symptoms) Type 1 diabetes. Twenty-three patients were treated with nicotinamide (200 mg daily) in addition to insulin, and 13 control patients were treated with insulin alone. Metabolic and immunological variables at entry were similar in the two groups. A significant increase of stimulated plasma C-peptide levels compared to diagnosis was observed only in the nicotinamide treated group (1.4 +/- 0.3 (+/- SE) micrograms l-1 at diagnosis vs 2.4 +/- 0.4 at 6 months, p less than 0.04; and 3.0 +/- 0.5 at 1 year, p less than 0.01). Patients receiving nicotinamide had lower glycosylated haemoglobin levels at 6 months and 1 year compared to the control group (p less than 0.04 and p less than 0.03, respectively) although insulin dose was lower. Small doses of nicotinamide may be successful in improving metabolic control in recent onset Type 1 diabetes, probably by increasing residual islet B-cell function.

B-Cell Reconstitution After Autologous Bone Marrow Transplantation: Increase in Serum CD23 (“IgE-Binding Factor”) Precedes IgE and B-Cell Regeneration

AbstractThe serum levels of IgE and the soluble cleavage product of CD23 (sCD23) were prospectively monitored for up to 1 year after transplantation in 34 patients who underwent autologous (n = 33) or syngeneic (n = 1) bone marrow transplantation (BMT). In 25 patients (74%), a transient IgE peak (two- to 2,750-fold increase) appeared in the serum 3 to 4 weeks after BMT. In 18 patients (51%), a two- to 125-fold increase in sCD23 coincided with the IgE peak. In only three patients was a sCD23 peak observed without a concomitant increase in IgE. The sCD23 increment pre- ceded the IgE peak in each individual case. During the period of increased sCD23 serum levels, the absolute numbers of circulating B cells and other cell types expressing surface CD23 were extremely low. The biologic significance of these findings is discussed in light of present knowledge of regulation of B-cell growth and differentiation with special reference to the role of sCD23 as a multifunctional cytokine.© 1989 by Grune & Stratton, Inc. 0006-4971/89/7308-0016$3.00/0

B-cell reconstitution after autologous bone marrow transplantation: increase in serum CD23 ("IgE-binding factor") precedes IgE and B-cell regeneration

The serum levels of IgE and the soluble cleavage product of CD23 (sCD23) were prospectively monitored for up to 1 year after transplantation in 34 patients who underwent autologous (n = 33) or syngeneic (n = 1) bone marrow transplantation (BMT). In 25 patients (74%), a transient IgE peak (two- to 2,750-fold increase) appeared in the serum 3 to 4 weeks after BMT. In 18 patients (51%), a two- to 125- fold increase in sCD23 coincided with the IgE peak. In only three patients was a sCD23 peak observed without a concomitant increase in IgE. The sCD23 increment preceded the IgE peak in each individual case. During the period of increased sCD23 serum levels, the absolute numbers of circulating B cells and other cell types expressing surface CD23 were extremely low. The biologic significance of these findings is discussed in light of present knowledge of regulation of B-cell growth and differentiation with special reference to the role of sCD23 as a multifunctional cytokine.

Induction of islet B-cell regeneration in partially pancreatectomized rats by poly(ADP-ribose) synthetase inhibitors.

In order to clarify the mechanism of the prevention of diabetes mellitus developing after subtotal pancreatectomy, we examined regenerative activities of islet B-cells in 90% pancreatectomized and poly (ADP-ribose) synthetase inhibitor-treated rats by using autoradiographic and stathmokinetic techniques. Thirty days after 90% pancreatectomy, islets of rats without 3-aminobenzamide treatment were decreased in number, small in size and had irregular contour. Degranulation of the B-cells and fibrotic degeneration were frequently encountered. On the contrary, islets of remaining pancreas in rats receiving 3-aminobenzamide were increased in number, and their diameters ranged from 0.3-0.6 mm, being about two fold larger than those of normal rats. The labeling index of 3H-thymidine autoradiography and the mitotic indices in islet B-cells were increased in a temporally correlated manner in the 3-aminobenzamide treated rats. The mitotic indices of the 3-aminobenzamide-treated group on the 5th, 7th, 10th and 15th days were significantly larger than those in the control group. These results indicate that poly (ADP-ribose) synthetase inhibitors can induce self-replication or regeneration of B-cells in partially pancreatectomized rats.

Dynamics of glucose-induced insulin release during the spontaneous remission of streptozocin diabetes induced in the newborn rat.

Neonatal rats injected with streptozocin (STZ, 100 mg/kg) at birth exhibited an acute diabetes followed by a spontaneous remission. We have previously shown that this recovery from neonatal diabetes is due to B-cell regeneration and reaccumulation of pancreatic insulin stores starting from 3 to 5 days after birth. The B-cell population during this period is heterogeneous with both surviving B-cells that have escaped the toxic effect of STZ and newly formed B-cells. To evaluate to what extent this B-cell population is functionally normal, we have measured in vitro the dynamics of glucose-induced insulin secretion from pancreatic fragments of rats treated with STZ at birth. The insulin responses were tested at intervals after STZ treatment, from day 1 to day 21 using perifusion of pancreatic fragments, and on day 21 and at 5 mo using perfusion of isolated pancreas. While the glucose-induced insulin release was completely obtunded (2% of the normal response) on day 1 after STZ, it could be demonstrated after day 3. Moreover, it increased as a function of age (6% and 36% of the normal responses on day 5 and day 14, respectively). This restoration of the insulin response to glucose closely paralleled the recovery of pancreatic insulin stores (6% and 51% of normal values, respectively, on day 5 and day 14). In sharp contrast with the lack of glucose response in vitro in the adult, glucose-induced insulin release was still detected on day 21 regardless of the in vitro system used (perifusion or perfusion). Furthermore, on day 21 the B-cells of the STZ rats exhibited a tendency toward enhanced insulin response to arginine, which is a prominent feature in adult rats previously treated with STZ at birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly(adp-ribose) synthetase inhibitors also induce islet b-cell regeneration

Poly(adp-ribose) synthetase inhibitors also induce islet b-cell regeneration

Amelioration of Diabetes Mellitus in Partially Depancreatized Rats by Poly(ADP-ribose) Synthetase Inhibitors: Evidence of Islet B-Cell Regeneration

Ninety percent depancreatized rats received daily intraperitoneal injection of 0.5 g/kg nicotinamide and 0.05 g/kg 3-aminobenzamide, potent inhibitors of islet poly(ADP-ribose) synthetase. One to three months after the partial pancreatectomy, urinary and plasma glucose levels in nicotinamide- and 3-aminobenzamide-treated rats were markedly lower than those in saline-treated control rats. Morphologic examination of the remaining pancreata revealed that islets in the poly(ADP-ribose) synthetase inhibitor-treated rats were markedly enlarged and consisted largely of B-cells. These results suggest that poly(ADP-ribose) synthetase inhibitors induce islet B-cell regeneration, thereby preventing or improving diabetes mellitus in partially depancreatized rats.

Amelioration of diabetes mellitus in partially depancreatized rats by poly(ADP-ribose) synthetase inhibitors. Evidence of islet B-cell regeneration

Amelioration of diabetes mellitus in partially depancreatized rats by poly(ADP-ribose) synthetase inhibitors. Evidence of islet B-cell regeneration

Effects of aging on the regenerative capacity of the pancreatic B-cell of the rat.

Since an increased insulin resistance could be expected to lead to a compensatory B-cell hyperplasia, it may be speculated that human type II diabetes becomes manifest only in those individuals who are unable to respond to an increased insulin demand with a higher rate of B-cell proliferation. To test this hypothesis experimentally, the cell cycle and growth regulation of the pancreatic B cell has been studied in vitro. Islets of fetal, 1-wk, 3-wk, and 3-mo-old rats were isolated and maintained in tissue culture at different glucose concentrations. The proliferating islet cells were synchronized with hydroxyurea and their progression through the cell cycle studied by pulse labeling with 3H-thymidine. From the cell cycle data it was possible to calculate the rate of formation of new B-cells. The cell cycle of the B-cells was similar in all donor age groups. When the glucose concentration of the culture medium was raised from 2.7 to 16.7 mM, the rate of B-cell proliferation increased 2.5-fold in all age groups, but there was no further increase in proliferation at 33.3 mM. At each of the glucose concentrations tested the rate of B-cell formation decreased with increasing age of the donor. Results indicate that only a fraction of the islet cells are capable of entering the cell cycle and undergoing mitosis. This fraction composed about 10% of the fetal islet cells but was less than 3% in the adult islets. The small pool of proliferating cells in adult islets could explain why B-cell multiplication, although present in the aging rat, is insufficient to increase the insulin output to levels at which normal glucose tolerance is maintained. This forms an interesting parallel to the development of type II diabetes in man, in which an inherited low capacity for B-cell regeneration may predispose to the disease.

Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia

Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia

Studies on islet cell regeneration, hyperplasia and intrainsular cellular interrelations in long lasting streptozotocin diabetes in rats.

Diabetes was induced in rats with various single i.v. doses of streptozotocin. The rats were killed 4–10 months thereafter. Marked hyperglycaemia, ketonaemia and a rise of plasma free fatty acids resulted. The weight increase was markedly reduced. There was a clear reciprocal correlation between the injected dose of the diabetogenic agent and plasma IRI. The same was true for the B-cell counts. There was a marked B-cell hypertrophy, whereas we found only slight indication for B-cell regeneration. In one animal a functioning B-cell adenoma developed and almost all the parameters tested were normalized. In the diabetic animals the A2-cells were predominant in the islets of Langerhans and their mean nuclear diameters were increased. In the duodenal mucosa no increase in the number of glucagonocytes was found. We interpret this activity of the glucagonocytes as a consequence of permanent and long-lasting hyperglycaemia.