Apoptosis during B lymphopoiesis in mouse bone marrow

To evaluate the magnitude of cell death and the critical stages at which it occurs during B lymphopoiesis in mouse bone marrow (BM), we have examined the kinetics of apoptosis at defined stages of B cell differentiation. FACS-sorted B220+ BM cells exhibited a low incidence of morphologically apoptotic cells by electron microscopy. In freshly prepared BM suspensions, the incidence of hypodiploid cells detected by multiparameter flow cytometry was greater among large dividing B220+ surface IgM- (sIgM-) precursor B cells and sIgM(low) immature B lymphocytes than among terminal deoxynucleotidyl transferase+ (TdT+) pro-B cells, small nondividing B220+ sIgM- precursors, and surface IgD+ mature B lymphocytes. During short-term culture, apoptotic cells, identified by both DNA content and in situ DNA strand break labeling, increased linearly with time without macrophage ingestion, providing an assay for the rate of entry into apoptosis. B220+ B lineage cells accumulated in apoptosis more rapidly than cells of other lineages. The apoptotic rate was greater among B220+ sIgM- precursor cells than sIgM+ B cells, and was highest among B220+ mu- pro-B cells. Coculture with stromal cells reduced the apoptotic rate of B220+ sIgM- precursors to a greater extent than that of sIgM+ B lymphocytes. The results lead to estimates of the actual number of B lineage cells undergoing apoptosis per unit time in successive differentiation compartments. The findings indicate that, although influenced by local microenvironmental factors, apoptotic cell death occurs most markedly at two developmental stages associated with Ig heavy chain gene rearrangement and Ag receptor expression, respectively.     

Identification of clonogenic common lymphoid progenitors in mouse bone marrow

The existence of a common lymphoid progenitor that can only give rise to T cells, B cells, and natural killer (NK) cells remains controversial and constitutes an important gap in the hematopoietic lineage maps. Here, we report that the Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) population from adult mouse bone marrow possessed a rapid lymphoid-restricted (T, B, and NK) reconstitution capacity in vivo but completely lacked myeloid differentiation potential either in vivo or in vitro. A single Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) cell could generate at least both T and B cells. These data provide direct evidence for the existence of common lymphoid progenitors in sites of early hematopoiesis.     

Ytterbium ion promotes apoptosis of primary mouse bone marrow stromal cells

One of the main target organs for the lanthanides(Ln) is bone. Previous studies revealed that ytterbium(Yb) produced damage to the skeletal system in vivo. But the effects of Yb3+ on bone marrow stromal cells(BMSCs) in vitro had not been reported. In this paper, cell viability, apoptosis, mitochondrial membrane potential(MMP), reactive oxygen species(ROS) and lactate dehydrogenase(LDH) were measured in order to study the effects of Yb3+ on BMSCs. The results indicated that Yb3+ displayed a slight positive effect on the BMSCs viability at concentrations of 1×10–6, 1×10–5, and 1×10–4 mol/L, but turned to decrease the viability of BMSCs at the highest concentration of 1×10–3 mol/L for 24, 48 and 72 h. Yb3+ at 1×10–3 mol/L promoted apoptosis of BMSCs, increased the levels of ROS and LDH, and decreased MMP in BMSCs. It suggested that the precipitate of Yb PO4 might decrease the viability of BMSCs. Yb3+ induced the apoptosis of BMSCs via mitochondrial pathway. The results might be useful for more rational application of Yb-based compounds in the future.     

Separation of megakaryocytes from mouse bone marrow by density gradient centrifugation

Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the "stomata" concept observed by early German and contemporary anatomists. "Occludens" junctions of the leaky type are seen though their macular or zonular nature is yet to be established.     

Prevention of cyclophosphamide-induced micronucleus formation in mouse bone marrow by indole-3-carbinol

Indole-3-carbinol (I3C) is a glucobrassicin derivative isolated from cruciferous vegetables. In this study, the protective effect of 13C is reported against cyclophosphamide (CP)-induced micronuclei formation in mouse bone marrow cells. The three test doses, namely 500, 250 and 125 mg/kg body weight of 13C provided protection when given 48 hr prior to the single ip administration of cyclophosphamide (50 mg/kg). The efficacy of the test doses of 13C was also evaluated using a lower dose of CP (25 mg/kg body weight). A significant inhibition in micronuclei formation was noticed with 13C at 250 and 125 mg/kg body weight dose. 13C could not induce micronuclei formation at the test doses 500 and 250 mg/kg body weight. 13C, therefore seems to have a preventive potential against CP-induced micronuclei formation in Swiss mouse bone marrow cells.     

A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures

Long-term bone marrow cultures provide a model for the study of hematopoiesis. Both an intact, adherent stromal layer and hematopoietic stem cells are necessary components in these cultures. Mycophenolic acid treatment of mouse long-term bone marrow cultures depletes them of all assayable hematopoietic precursors. The residual stromal cells are functional and support hematopoiesis if new progenitor cells are supplied. We now show that these mycophenolic acid-treated stromal cell cultures contain cells capable of hematopoietic differentiation without the addition of new progenitors. When treated with tumor necrosis factor alpha (20-200 units/ml), the apparently pure stromal cultures undergo an intense burst of hematopoietic activity. After 4 days such cultures contain approximately 2 x 10(6) hematopoietic cells and, by 1 week, they are indistinguishable from control long-term cultures that were not treated with mycophenolic acid. These results suggest that the stromal cultures either contain hematopoietic stem cells that are maintained quiescent and mycophenolic acid-resistant, perhaps by intimate contact with the stroma, or contain adherent cells that can be induced to differentiate into hematopoietic stem cells. These stem cells are primitive, in that they are capable of multilineage development in the long-term cultures, but are unable to form spleen colonies or myeloid colonies in semisolid medium. These data demonstrate that the adherent fraction of cultured bone marrow contains very primitive hematopoietic cells and that tumor necrosis factor alpha activates their proliferation and differentiation. They also suggest a strategy for obtaining the earliest progenitors free of other, more mature cell types.     

A simple model of the optimal time for allogenic bone marrow transplantation in chronic granulocytic leukemia

Allogenic bone marrow transplantation is the treatment of choice in chronic granulocyte leukemia patients, while the best results are achieved when it is performed in the chronic phase of the illness. That is why time optimization for bone marrow transplantation in chronic granulocyte leukemia means making priority lists for transplantation according to medical indications. This study comprises a very simple model of optimal time for bone marrow transplantation in chronic granulocyte leukemia. It is based on data of the International Bone Marrow Transplant Registry (IBMTR) on bone marrow transplantation results in different phases of chronic granulocyte leukemia and prognostic model for survival of younger leukemic patients according to which there are three groups of patients. The mathematical method estimated cumulative risks of the final therapeutic results. This model has shown that the time limit for transplantation is the fourth year of the disease in the low risk group; the third year of the disease in the medium risk group and the second year in the high risk group of patients.     

Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow

The frequency of micronuclei (micronucleated polychromatic erythrocytes, MPCE and micronucleated normochromatic erythrocytes, MNCE) was studied at 12, 24 and 36 h post-treatment in the bone marrow of mice treated with 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt of hydroquinone (HQ). Treatment of mice with various doses of HQ resulted in a dose dependent increase in the frequency of both MPCE and MNCE at all the post-treatment time periods. The frequency of MPCE was significantly higher after administration of 3.125 mg/kg HQ at 24 h post-treatment, except 12 and 36 h, where a significant increase in the frequency of MPCE was observed only after administration of 6.25 mg/kg drug dose. Similarly, a significant increase in the frequency of MNCE was observed after 12.5 mg/kg HQ treatment at all the post-treatment time periods. The dose effect relationship between various HQ doses and MPCE and MNCE induction was linear and linear quadratic, respectively at all the post-treatment time periods. The PCE/NCE ratio declined in a dose dependent manner at all the post-treatment time periods and this decline was significant when compared to non-drug treated controls. The dose effect relationship was linear quadratic at all the post-treatment time periods studied.     

Granulocyte-macrophage colony stimulating factor suppresses lipopolysaccharide-induced osteoclast-like cell formation in mouse bone marrow cultures

Lipopolysaccharide (LPS) is a potent bone resorbing factor. We investigated the effect of LPS on osteoclast formation in three types of cultures. LPS inhibited osteoclast formation induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in a dose-dependent manner, in cultures of whole bone marrow cells without dexamethasone. LPS increased the amount of granulocyte-macrophage colony stimulating factor (GM-CSF) in the culture supernatant, and anti-GM-CSF antiserum almost abolished the inhibition of osteoclast formation by LPS, thereby indicating that GM-CSF generated by treatment with LPS may be responsible for the inhibition of osteoclast formation. In cultures with dexamethasone, the amount of GM-CSF was decreased to one-third of that with 1,25(OH)2D3 alone and was not changed by treatment with LPS. In this culture system, LPS enhanced osteoclast formation. In the coculture system of nonadherent bone marrow cells and a stromal cell line in the presence of 1,25(OH)2D3 and dexamethasone, where no detectable GM-CSF was present in the supernatant, LPS markedly enhanced osteoclast formation, whereas exogenously added GM-CSF (100 pg/ml) almost completely inhibited osteoclast formation. LPS stimulated pit formation on dentin slices by the osteoclast-like cells formed by in vitro culture system.     

A novel growth-factor-dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential

We describe two modified methods for median-to-ulnar motor conduction comparison in the diagnosis of median neuropathy at the wrist: the median-thenar to ulnar-thenar latency difference (TTLD), and the median-thenar to ulnar-hypothenar latency difference (THLD). We also describe an F-wave ulnar-to-median comparative test, the F-wave latency difference (FWLD). The abnormal cutoffs based upon 34 normal controls are: TTLD, 0.8 ms; THLD, 1.2 ms; FWLD, 0.6 ms. In 50 patients (79 hands) with clinically defined carpal tunnel syndrome and electrophysiological evidence of median neuropathy at the wrist (based upon a prolonged median nerve palm-wrist latency), the diagnostic sensitivities were: 95-98%, 85-88%, and 75-78%, respectively. These tests are therefore highly sensitive. They are easily performed and require minimal additional effort to incorporate into commonly used clinical electrodiagnostic routines. They may be advantageous when a concomitant polyneuropathy is present, and they may also help avoid technical pitfalls and aid in identification of anatomic variants.     

Proton dosimetry in bone using electron spin resonance

We have studied the ESR response of proton-irradiated (in vitro) bone. The ESR response as a function of proton (E = 105 MeV) dose to bone was linear from 0 to 50 Gy and similar to the photon (E = 6 MV) dose response. The ESR depth response (Bragg) curve was depressed as compared to a depth-response curve determined with a parallel plate ionization chamber (PPIC). There was a short-term ESR signal fade in the Bragg peak region, likely attributable to the organic component in bone. We are continuing to investigate these latter two effects.     

Allogeneic bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) after high-dose, marrow-ablative chemoradiotherapy has been established as the treatment of choice for various hematologic, neoplastic, and congenital disorders. The most common type of marrow graft is an allogeneic one from a sibling donor who has compatible human leukocyte antigen (HLA). Only 30% of patients requiring allogeneic BMT have an HLA-compatible sibling donor. Over the past few years, marrows from unrelated HLA-compatible donors have been used with increasing frequency and promising outcome in certain hematologic malignancies. Despite the morbidity and mortality associated with this treatment modality, allogeneic BMT may provide a 20% to 90% chance of long-term, disease-free survival to patients with a wide variety of neoplastic and abnormal marrow disorders.     

A case of kala-azar diagnosed by bone marrow aspiration

A group of 30 consecutive patients with iatrogenic vascular injury were studied to determine the aetiology of the condition and to investigate its possible prevention. Of the patients, 18 were males and 12 females; mean age was 37.0 years (range 2 weeks to 70 years). Most injuries (n = 25) involved the arterial system; eight cases (27%) were recognized during the operative procedure. The most common presentations were bleeding and chronic ischaemia. Cannulation of vessels was the cause of injury in 14 cases (47%); two-thirds of these were iatrogenic in nature. A conservative policy was adopted in nine patients; surgery was planned but not performed in two. A total of 19 patients were operated on (17 reconstructions, one ligation, one fasciotomy); two died from causes related to their original condition. In order to reduce the incidence of iatrogenic injury, medical personnel should be informed of possible vessel damage, especially during cannulation. All surgical and radiological procedures should be carefully audited.     

A model of the prostate gland for use in internal dosimetry

Several radionuclides or radiolabeled pharmaceuticals may be taken up by the prostate gland. METHODS: A model of the prostate gland has been developed and implemented in the adult male mathematical phantom within software which calculates absorbed fractions of energy from activity in source regions within the phantom. RESULTS: Specific absorbed fractions are reported for all target regions within the phantom for 12 discrete source energies from 0.01 to 4.0 MeV. S-values for all target regions for six radionuclides are also reported. CONCLUSIONS: This work provides another organ useful for internal dose calculations within the 70-kg phantom.     

The FE-lspd model for electron beam dosimetry

The FE-lspd model is a two-component electron beam model that distinguishes between electrons that can be described by small-angle transport theory and electrons that are too widely scattered for small-angle transport theory to be applicable. The two components are called the primary beam and the laterally scattered primary distribution (lspd). The primary beam component incorporates a simple version of the Fermi-Eyges model and dominates dose calculations at therapeutic depths. The lspd component corrects erros in the lateral spreading of the primary beam component, thereby improving the accuracy by which the FE-lspd model calculates dose distribution in blocked fields. Comparisons were made between dose profiles and central-axis depth dose distributions in small fields calculated by the FE-lspd, Fermi-Eyges and EGS4 Monte Carlo models for a 10 MeV beam in a homogeneous water phantom. The maximum difference between the dose calculated using the FE-lspd model and EGS4 Monte Carlo is about 6% at a field diameter of about 1 cm, and less than 2% for field sizes greater than 3 cm diameter. The maximum difference between the Fermi-Eyges and Monte Carlo calculations is about 18% at a field diameter of about 2.5 cm. A comparison was made with the central-axis depth dose distribution measured in water for a 3 cm diameter field in a 10 MeV clinical electron beam. The errors in the dose distribution were found to be less than 2% using the FE-lspd model but almost 18% using the Fermi-Eyges model. A comparison was also made with pencil beam profiles calculated using the second-order Fermi-Eyges transport model.     

Endothelium and bone marrow transplantation

Thrombotic complications may occur early after marrow transplantation and many data suggest that endothelial injury plays a pivotal role in their pathogenesis. Since plasma thrombomodulin and P-selectin are thought to be of value as markers of vascular endothelial cell membrane injury, we investigated their plasma concentration in bone marrow transplant patients aiming better to clarify the degree of endothelial involvement. Plasma thrombomodulin and P-selectin were monitored in 25 patients without thrombotic complications before transplant, on day 0 and weekly for 1 month thereafter, while in three patients who developed VOD monitoring continued until day +52. These proteins were in the normal range in all the uncomplicated patients and in two with reversible VOD, while they were always very high in the only patient who developed very severe and lethal VOD. In conclusion, we suggest that endothelial activation/damage occurs rarely in the course of BMT for hematological malignancies; we were able to document endothelial injury in only one patient with very severe thrombotic complication.