Macrophages (phagocytic-histiocytic reticular cells) in reactive-inflammatory lesions of the bone marrow and in myelodysplastic syndromes (MDS). An immunohistochemical and morphometric study by use of a new monoclonal antibody (PG-M1).

An immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow in 52 patients (28 males/24 females; age 68 years) with various subtypes of myelodysplastic syndromes (MDS) to determine the number of macrophages (phagocytic-histiocytic reticular cells). Quantifications included the haemosiderin-storing subpopulation (Prussian-blue reaction) of this lineage as well as the iron-free compartment. The latter was identified by a new monoclonal antibody (PG-M1) which is specifically directed against histiocytic reticular cells. Bone marrow specimens of individuals without haematological disorders and those showing reactive lesions served as controls. In comparison with the normal bone marrow and inflammatory changes (i.e. rheumatoid arthritis) 23 of the 52 patients with MDS revealed a significant increase in macrophages. This increase encompassed not only the iron-laden subpopulation but also the total number of phagocytic reticular cells. Accumulation of macrophages in MDS was speculated to be due to a premature and enforced degradation of dysplastic cell elements leading to phagocytosis of haemosiderin and debris material. Moreover, cells of the monocyte-macrophage system could be involved in the complex pathomechanism of fibrillogenesis, since in a considerable percentage of patients with MDS, an increase in reticulin (argyrophilic) fibres was noticeable. Our finding of an expansion of the macrophage compartment in about half of the patients with MDS is in keeping with results of cell culture studies on colony formation of granulocyte-macrophage precursors (CFU-GM).     

Macrophages and their subpopulations following allogeneic bone marrow transplantation for chronic myeloid leukaemia

A morphometric and immunohistochemical study was performed on 354 bone marrow trephine biopsies derived from 126 patients with chronic myeloid leukaemia (CML) before and after allogeneic bone marrow transplantation (BMT). The purpose of this investigation was to evaluate the macrophage population, including several subsets and their dynamics in the posttransplant period. In addition to the total CD68⁺ resident (mature) macrophages the so-called activated fraction identified by its capacity to express α-d-galactosyl residues, the pseudo-Gaucher cells (PGCs) and the iron-laden histiocytic reticular cells were also considered. Following immuno- and lectin-histochemical staining morphometric analysis was carried out on sequential postgraft bone marrow specimens at standardized intervals. Compared to the normal bone marrow and calculated per haematopoiesis (cellularity) an overall decrease of about 40–50% in the quantity of CD68⁺ macrophages and the BSA-I⁺ subpopulation was detectable in the early posttransplant period (9–45 days after BMT). Noteworthy was the temporal recurrence of PGCs in the engrafted bone marrow, which was not associated with a clonally transformed cell population or leukaemic relapse. Reappearance of postgraft PGCs was most prominent in the first 2 months after BMT. This conspicuous feature was presumed to be functionally associated with a pronounced degradation of cell debris following pretransplant myelo-ablative therapy (scavenger macrophages). Evidence for an activation of the BSA-I⁺ macrophage subset was derived from the identical carbohydrate-binding capacity shown by the PGCs. In the regenerating haematopoiesis shortly after BMT a significant correlation between the number of BSA-I⁺ macrophages and erythroid precursor cells was determinable. This result implicates a close functional relationship between postgraft reconstitution of erythropoietic islets and centrally localized activated macrophages. In conclusion, findings emerging from this study included the reappearance of PCGs in the engrafted bone marrow independently of a leukaemic relapse and the significant association of the activated BSA-I⁺ macrophage subset with the recovery of erythropoiesis. Received: 31 January 2000 / Accepted: 6 March 2000     

A histological study and three-dimensional reconstruction of F4/80-positive reticular cells and macrophages at the onset of murine bone marrow hematopoiesis

Adult bone marrow consists of two different compartments, a vascular compartment of sinusoid and a hematopoietic compartment consisting of stromal cells and hematopoietic cells. In the hematopoietic compartment, stromal cells play an important role in the formation of the microenvironment for hematopoiesis. To clarify the relationship between hematopoietic cells and stromal cells, particularly reticular cells and macrophages, we examined the femur bone marrow of ICR mouse fetuses and neonates using F4/80 immunostaining and three-dimensional reconstruction under light and electron microscopy. In the fetal femurs, the marrow cavity formed early from 15 days of gestation, and it showed a marked increase in volume thereafter. On the basis of the appearance of hematopoietic cells, marrow development could be classified into two stages, a pre-hematopoietic stage from 15 days of gestation to two days of age, and a beginning stage of hematopoiesis thereafter. The pre-hematopoietic bone marrow contains not only stromal reticular cells but also macrophages, and both types of stromal cells were strongly positive to F4/80 monoclonal antibody. These F4/80-positive reticular cells had a triangular cell profile with long and slender cytoplasmic processes. Reticular cells often contained large lysosomes of not only dying neutrophils but also erythroblast nuclei. A few erythroblasts accumulated around the processes, and the number of erythroblasts around reticular cells increased with bone marrow development. On the other hand, macrophages were located either close to sinusoids or in sinusoid lumen, and a close relationship to hematopoietic cells was hardly noticeable. At the beginning stage of hematopoiesis, F4/80-positive reticular cells extended their long and slender cytoplasmic processes, and the number and length of the processes appeared markedly increased. The three-dimensional cell surface of the F4/80-positive reticular cells became very complex. Numerous erythroblasts accumulated around the processes, and erythroblastic islands could gradually be recognized after four days of age. In the erythroblastic islands, central reticular cells were F4/80-positive and contained numerous large phagosomes originating from the expelled nuclei of erythroblasts. Although macrophages contained large phagosomes, the relationship between macrophages and hematopoietic cells could not clearly be elucidated even at the beginning stage of hematopoiesis. At the onset of bone marrow hematopoiesis, the hematopoietic compartment contained two kinds of F4/80-positive phagocytes, i.e., reticular cells and macrophages. In marrow erythroblastic islands, not macrophages but F4/80-positive reticular cells were located at the center of each island.     

Detection of the bcr/abl gene in bone marrow macrophages in CML and alterations during interferon therapy--a fluorescence in situ hybridization study on trephine biopsies

A fluorescence in situ hybridization (FISH) study was performed on trephine biopsies of the bone marrow in chronic myelogenous leukaemia (CML) to evaluate the bcr/abl translocation in macrophages before and during interferon (IFN) therapy. Mature macrophages and osteoclasts were identified by the monoclonal antibody PG-M1, which recognizes a fixative-resistant epitope of the CD68 molecule. In contrast to a control group, in 145 of 479 (30 per cent) macrophages of the CML bone marrow, a positive fusion signal was found, together with corresponding translocation sites in the surrounding myeloid cells. In patients following IFN treatment for approximately 12 months and with haematological/cytogenetic remission by clinical standards, this number was reduced to 10 of 136 (9 per cent) macrophages. In addition, the few multinucleated osteoclasts of the untreated CML bone marrow displayed positive translocation spots. This finding supports the hypothesis that stromal macrophages and osteoclasts are of haematogenic stem cell origin. Moreover, clinical remission and reduction of bcr/abl-positive macrophages under IFN therapy lend support to the hypothesis that the presence of malignant stromal macrophages may contribute to the selective expansion of leukaemic precursors and the suppression of normal haematopoiesis in CML.     

Transplanted bone marrow cells repair heart tissue and reduce myocarditis in chronic chagasic mice

A progressive destruction of the myocardium occurs in approximately 30% of Trypanosoma cruzi-infected individuals, causing chronic chagasic cardiomyopathy, a disease so far without effective treatment. Syngeneic bone marrow cell transplantation has been shown to cause repair and improvement of heart function in a number of studies in patients and animal models of ischemic cardiopathy. The effects of bone marrow transplant in a mouse model of chronic chagasic cardiomyopathy, in the presence of the disease causal agent, ie, the T. cruzi, are described herein. Bone marrow cells injected intravenously into chronic chagasic mice migrated to the heart and caused a significant reduction in the inflammatory infiltrates and in the interstitial fibrosis characteristics of chronic chagasic cardiomyopathy. The beneficial effects were observed up to 6 months after bone marrow cell transplantation. A massive apoptosis of myocardial inflammatory cells was observed after the therapy with bone marrow cells. Transplanted bone marrow cells obtained from chagasic mice and from normal mice had similar effects in terms of mediating chagasic heart repair. These results show that bone marrow cell transplantation is effective for treatment of chronic chagasic myocarditis and indicate that autologous bone marrow transplant may be used as an efficient therapy for patients with chronic chagasic cardiomyopathy.     

Apoptosis and proliferation (PCNA labelling) in CML—a comparative immunohistological study on bone marrow biopsies following interferon and busulfan therapy

A comparative morphometric analysis was performed on smears and trephine biopsies of normal bone marrow and in chronic myelogenous leukaemia (CML) to assess the effects of therapy on apoptosis and cell proliferation. The in situ end-labelling (ISEL) technique was used for the demonstration of programmed cell death, in combination with the monoclonal antibody PG-M1 to identify macrophages. Cell proliferation was evaluated by employing the monoclonal antibody PC10 directed against proliferating cell nuclear antigen (PCNA). In CML (48 patients), significantly higher rates of apoptosis were observed than in normal bone marrow (smears, frozen sections, and paraffin-embedded samples) of 15 patients. In contrast, the PCNA labelling index of CML was not different from controls. In bone marrow tissue derived from CML patients, about 36 per cent of apoptotic bodies were ingested with CD68-positive macrophages. Study of the histotopographical distribution of labelled cells revealed that in CML, in contrast to the normal bone marrow, programmed cell death and PCNA activity were concentrated along the paratrabecular generation zone. In 28 patients with CML treated with interferon (IFN), sequential trephine biopsies displayed a significant enhancement of apoptosis which was associated with a decrease in PCNA reactivity. In contrast to this finding, no such alterations could be observed in 24 patients who received busulfan (BU) monotherapy. This study furthers the understanding of cell kinetics in CML. IFN therapy induces apoptosis and suppresses cell proliferation. The rate of programmed cell death prior to therapy and the extent of IFN-triggered apoptosis exert a significant predictive impact on survival. In this study, ISEL-positive (apoptotic) cells and bodies do not correspond to unscheduled cell repair as detected by PCNA immunoreactivity. © 1997 John Wiley & Sons, Ltd.     

Inosine 5′-phosphate dehydrogenase activity in normal and leukemic blood cells

Summary The enzyme inosinic acid dehydrogenase (EC 1.2.1. [14]) was measured and partially purified (10- to 15-fold) from normal and leukemic leukocytes. From the normal blood cells, the highest activities could be detected in lymphocytes and bone marrow cells. Dependent on the blast cell count, the leukemic IMP dehydrogenase had a higher mean specific activity than the enzymes of fractionated, immature bone marrow cells, or normal granulocytes. The partially purified enzymes from the various blood cells were apparently identical; they exhibited hyperbolic substrate saturation kinetics and were inhibited by a number of purine nucleotides. For the leukemic blast cell enzyme, theK _m values for the substrates, IMP and NAD⁺, were 28±11; 227±98 µM, and 34±10; 240±67 µM for the partially purified enzyme from normal, immature bone marrow cells.The hypoxanthine-guanine and adenine phosphoribosyltransferase activities increased in the leukemic cells when compared with mature granulocytes, but nearly always showed similar activities when compared with the fractionated bone marrow cells. Only one of the 30 investigated leukemic patients exhibited a marked decrease in hypoxanthine phosphoribosyltransferase activity of 0.5 nmol/mg/h. The phosphoribosyltransferase-specific activities of the leukemic cells are more variable than for the normal ones and no correlation of enzyme activities and blast cell count was apparent.

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Abbreviations A-PRT adenine phosphoribosyltransferase - HG-PRT hypoxanthine-guanine phosphoribosyltransferase - PRPP 5-phosphoribosyl-1-pyrophosphate - ALL acute lymphocytic leukemia - AML acute myelogenous leukemia - CLL chronic lymphatic leukemia - CML chronic myelogenous leukemia - AMMOL acute myelomonocytic leukemia This study was supported by the Deutsche forschungsgemeinschaft Be 458/4     

Neuropilin-1基因在CML骨髓基质细胞中的表达及其意义

目的　了解Neruophlin -1基因在慢性粒细胞白血病 (CML)骨髓基质细胞中的表达情况 .方法　收集 14例CML和 2 0例正常对照骨髓标本 ,分离单个核细胞 .进行体外长期培养 ,收集贴壁细胞 (骨髓基质细胞 ) .利用RT -PCR方法分析两组骨髓基质细胞中的cDNA ,了解Neuropilin -1基因表达情况 .结果　建立了CML和正常人骨髓基质细胞培养方法 ,CML骨髓基质细胞中的Neuropilin -1基因表达率 (50 % )明显低于正常对照 (85% ) (p <0 .0 5) .结论　参与调控骨髓基质细胞的Neuropilin -1基因可表达于大部分正常人和部分CML患者骨髓基质细胞中 ,Neuropilin -1基因在部分CML骨髓基质细胞中的表达缺失可能与其调节造血功能异常有关     

体外培养骨髓基质细胞的类型及来源

用液体静置法体外培养小鼠骨髓基质细胞，对贴壁生长细胞进行倒置显微镜下的原位连续观察和数种酶的细胞化学观察。根据的细胞形态和酶蛋白质活性明显的移行特点，主要区分出三种类型的细胞，即网状细胞，巨噬细胞和成纤维细胞，观察到星形细胞和至少一部分巨噬细胞来源于网状细胞。     

Silver-stained nucleolar organizer regions in bone marrow cells and peripheral blood lymphocytes of Philadelphia chromosome-positive chronic myelocytic leukemia patients

Silver-stained nucleolar organizer regions (Ag-NOR) in bone marrow cells and/or phytohemagglutinin-stimulated peripheral blood lymphocytes were compared between six normal healthy persons as controls and 22 Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) patients, to examine if any disease associated changes occur in the expression of Ag-NOR. Although the frequency of Ag-NOR-positive cells and the number of Ag-NOR per cell were generally greater in lymphocytes than in bone marrow cells in both controls and CML patients, the Ag-stainability of these cell types in CML patients was considerably heterogeneous, compared with that found in controls. The peripheral lymphocytes of CML patients in the chronic phase, but not in the blastic phase, exhibited a significantly lowered Ag-stainability when compared with those of controls. while no such difference was observed between bone marrow cells of controls and leukemia patients in both phases of CML. In the blastic phase, however, the occurrence of Ag-NOR on the Ph of CML bone marrow cells was significantly less than expected. The present findings are discussed in relation to the existing data on the Ag-NOR expression in both normal and neoplastic cells.     

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.     

Stromal cell populations in necropsy bone marrow sections from allogeneic marrow recipients and non-transplant patients.

AIMS--To compare the numbers of alkaline phosphatase positive reticulum cells (AL-RC) and macrophages in bone marrow transplant (BMT) recipients with numbers in normal subjects and to look for correlations with clinical features. METHODS--Sections of femoral marrow were obtained at necropsy from 18 BMT recipients and nine normal subjects who had died suddenly. AL-RC were visualised through their endogenous alkaline phosphatase activity. Macrophages were stained by an immunocytochemical technique using the antibody EBM/11 (CD68) and through their endogenous acid phosphatase activity. The numbers of stained cells were counted and expressed as a percentage of total nucleated cells. RESULTS--In both sets of marrow tissue, more macrophages stained for CD68 than for acid phosphatase, indicating macrophage heterogeneity. The percentage value for CD68 positive macrophages was higher among the transplant recipients (p < 0.01). At least in part this was caused by a reduction in haemopoietic cell numbers. Percentage values for acid phosphatase and alkaline phosphatase positive cells did not differ between the two groups. To exclude the effect of changes in marrow cellularity, stromal cell ratios were compared. The AL-RC: CD68 and acid phosphatase:CD68 ratios were both lower in BMT recipients, indicating that after BMT either the absolute number of AL-RC and acid phosphatase cells decreases, or CD68 cells increase, or there is a combination of the two. There was no correlation between the number of each cell type and cell dose given at transplantation, time after transplantation, presence of graft versus host disease or infection, marrow erythroid:myeloid ratio, or peripheral white cell count. The ratio of AL-RC to macrophages in our intact marrow was 0.43, considerably higher than that reported in cultured marrow. CONCLUSIONS--AL-RC and acid phosphatase positive cells may be most important for supporting haemopoiesis and their reduction after BMT may contribute to depression of haemopoiesis. CD68 positive cells include macrophages with a wide variety of functions and these may be increased in response to marrow damage.     

Focal cerebral ischemia/reperfusion injury in mice induces hematopoietic prostaglandin D synthase in microglia and macrophages

Hematopoietic prostaglandin D synthase is a key enzyme in synthesis of prostaglandin D. Hematopoietic prostaglandin D synthase is expressed in microglia of the developing mouse brain. This study determined the serial changes and cellular localization of hematopoietic prostaglandin D synthase, and its role in cerebral ischemia/reperfusion injury using C57BL/6 mice (n=84) and bone marrow chimera mice (n=16). The latter mice were selected based on their expression of enhanced green fluorescent protein in bone marrow/blood-derived monocytes/macrophages. The middle cerebral artery was occluded for 60 min, followed by reperfusion. Hematopoietic prostaglandin D synthase expression was examined by immunohistochemistry and Western blotting. Hematopoietic prostaglandin D synthase-positive cells were mainly expressed in the peri-ischemic area at 12 h (P<0.05) and 24 h (P<0.001) after reperfusion, while they were mostly found in the transition area at 48-72 h postreperfusion (P<0.001). There was a significant increase in staining intensity as well as number of hematopoietic prostaglandin D synthase-positive cells in the ischemic core at 5-7 (P<0.001) days postreperfusion. Hematopoietic prostaglandin D synthase-positive cells also co-expressed ionized calcium-binding adapter molecule 1, a marker of microglia/macrophages, and cyclooxygenase-2, but not markers of neurons, oligodendrocytes and astrocytes. Until 72 h postreperfusion, many enhanced green fluorescent protein-positive cells were negative for hematopoietic prostaglandin D synthase, but the number of hematopoietic prostaglandin D synthase-enhanced green fluorescent protein coexpressing cells increased significantly at 5-7 days after reperfusion. Our results indicate that hematopoietic prostaglandin D synthase is mainly produced by endogenous microglia until 72 h after reperfusion, but at 7 days after reperfusion, it is also produced by migrating bone marrow/blood-derived macrophages in the ischemic brain tissue. We speculate that hematopoietic prostaglandin D synthase in the brain has different functions during early and late phases of ischemia.     

Quantitative observations on iliac bone marrow mast cells in chronic renal failure.

Mast cells have been counted in sections of iliac bone from 61 control subjects at necropsy. Mast cells were found in all but three, and the range was 0-33-7, median 1-95 per mm2 marrow. The majority (82%) had less than 4-99 mast cells per mm2 marrow; in 37-7% there was less than 1 mast cell per mm2 marrow. In a group of 45 patients with chronic renal failure there was a significant increase in the numbers of mast cells (P less than 0-001) with a range of 0-96-55-63, median 9-55 per mm2 marrow. Mast cells were common in the areas of marrow fibrosis associated with osteitis fibrosa but this was not the sole cause of the increase since there was also an excess of mast cells in the non-fibrous parts of the marrow. There was a tendency towards greater numbers of mast cells in those cases with most marked osteitis fibrosa in association with the prominent marrow fibrosis, but there was no significant relationship between mast cell numbers and other features of oesteitis fibrosa such as the number of osteoclasts and the amount of woven bone formation. There was no relationship between the numbers of mast cells and the amounts of total bone, ostoid, percentage mineralization of cancellous bone, or the presence of osteomalacia.     

Observations on the haemopoietic response to critical illness.

Peripheral blood cytopenias are common in patients receiving intensive care, particularly in those with multiple organ failure. To assess the contribution of bone marrow hypoplasia in such patients 44 bone marrow samples from 24 patients under intensive care were studied by standard morphological techniques and by the granulocyte-macrophage colony forming cell (GM-CFC) assay. Frequently observed morphological abnormalities in the bone marrow included the following: (i) a reduction in overall cellularity in seven patients, with a progressive decrease in most patients studied sequentially; (ii) an increase in the number of actively phagocytic macrophages; and (iii) a disruption of normal bone marrow architecture with the accumulation of intercellular hyaluronic acid glycosaminoglycan. Mean GM-CFC growth was significantly reduced when compared with that in a group of normal controls. In four of five patients studied sequentially GM-CFC growth became subnormal in association with a reduction in bone marrow cellularity. Inhibitory serum factors were not identified. These morphological abnormalities are similar to the changes observed in gelatinous degeneration of the bone marrow. In both situations disruption of the haemopoietic microenvironment, with the accumulation of hyaluronic acid proteoglycan, may be an important factor in the inhibition of haemopoietic progenitor cell growth. The proliferation of macrophages, by the release of a variety of cytokines or reactive oxygen intermediates, may also be implicated in impaired haemopoiesis and the development of disordered erythropoiesis.     

Iliac bone marrow mast cells in relation to the renal osteodystrophy of patients treated by haemodialysis.

Mast cells have been counted in iliac bone biopsies from patients with chronic renal failure treated by haemodialysis, and attempts made to relate mast cells to the various features of azotaemic renal osteodystrophy. There was a significant increase in the number of marrow mast cells in comparison with controls, and haemodialysis has no favourable effect on the marrow mast cell hyperplasia which occurs in chronic renal failure. There was a general tendency towards greater numbers of mast cells in cases with most marked ostetis fibrosa but it was not possible consistently to relate hyperparathyroidism to mast cell numbers. Patients treated by parathyroidectomy, aluminium hydroxide, or the newer vitamin D metabolites failed to show any consistent fall in marrow mast cell numbers in spite of improvement in the ostetis fibrosa. The number of marrow mast cells was related to the duration of haemodialysis and to the amount of osteoid present and iversely to the percentage mineralization of bone. It was not possible to identify any causal relationship between the presence or severity of osteomalacia and the number of mast cells in the haemodialysed patients although mast cell hyperplasia was also observed in patients with osteomalacia unassociated with renal disease.     

Monoclonal Antibody Against Bone Marrow Stromal Cells Its Production and Characterization

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1,2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line HI derived from long-term bone marrow culture (LTBMC) of C57BL/6 mice (3,4). A newly established monoclonal antibody, designated R4-A9, was obtained from a hybridoma prepared by fusion of Y.B2/ 3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with HI cells. The immunofluorescence of live HI cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4-A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4-A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4-A9 selectively reacted with the sinusoidal endothelium, peri-sinusoidal adventitial cells (5) (adventitial reticular cells (6)) and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4 A9. SDS-PAGE analysis showed that R4-A9-immuno-precipitated proteins had a molecular mass of 100 kDa under reducing conditions. These data show that R4-A9 is highly specific for reticular cells and the endothelium of murine bone marrow and spleen, providing a new approach for detailed structural analysis of the constituents of bone marrow stroma. Acta Pathol Jpn 41: 499–506, 1991.     

Polycythemia rubra vera versus secondary polycythemias. A clinicopathological evaluation of distinctive features in 199 patients

To determine parameters of distinctive value in polycythemia rubra vera (PV) versus secondary polycythemias (SP), a clinicopathological study was performed on 199 patients. These presented with a borderline to marked elevation of the hemoglobin level (> 18 g/dl in men and > 16 g/dl in women). Evaluations of clinical features and bone marrow histopathology were carried out independently. According to the results derived from laboratory data and representative pretreatment trephine biopsies, three groups of patients emerged: group I presenting with the concordant clinical and morphological findings of early to manifest PV (136 patients), group II consisting of 55 patients with the congruent signs and symptoms of SP mostly caused by various chronic bronchopulmonal disorders, and finally eight patients (group III) with divergent findings. Between group I and II patients (PV versus SP), a number of clinical parameters proved to be significantly different. With the exception, of the red cell mass, platelet count, leukocyte alkaline phosphatase, LDH, spleen size, and the erythropoietin level had a significantly discriminating impact. Morphological features of distinctive value consisted of a set of specific lesions. Contrasting SP with an only borderline to slight increase in cellularity associated with a moderate enlargement of the erythroblastic islets, PV was always characterized by a significant increase in hematopoiesis, revealing a trilinear proliferation (panmyelosis). Megakaryopoiesis was strikingly different in PV as compared to SP by displaying clustering and a pleomorphous appearance. i.e., very small and giant megakaryocytes with staghorn-like nuclei were neighboring each other. Moreover, conspicuous alterations of the interstitial compartment were recognizable in SP. These consisted of deposits of cell debris in histiocytic reticular cells, iron-laden macrophages, and a plasmacytosis, implying an inflammatory reaction. These changes were only very rarely observed in PV, as opposed to a minimal to slight increase in reticulin fibers in about 12% of patients. In conclusion, a more elaborate evaluation of bone marrow features resulted in a set of diagnostic criteria with discriminating capacity that should be considered in prospective clinical trials.     

Defective CSA-dependent granulopoiesis in patients with chronic drug-induced neutropenia

Colony stimulating activity (CSA) and granulocyte-macrophage progenitor cells (GM-CFC) were assayed in the bone marrow and peripheral blood of 17 patients with drug-induced chronic neutropenia. Leukocyte-derived and monocyte/macrophage-derived CSA from the neutropenic patients was found to be significantly decreased compared to normal control. However, bone marrow and peripheral blood GM-CFC were within normal limits. These data suggest that in neutropenic patients monocyte/macrophages exhibit most likely a qualitative defect in CSA production, which may account at least in part, for the impaired granulopoiesis observed in drug-induced neutropenia.     

The Hermansky-Pudlak Syndrome: Ultrastructure of Bone Marrow Macrophages

The present investigation has explored the fine structure of the lipid inclusions which fill the bone marrow macrophages of patients with the Hermansky-Pudlak syndrome. Red blood cells are the major substrate of the reticular macrophages, and incomplete digestion of erythrocytes leads to formation of the massive inclusions. Progressive transformation of the macrophages results in an end-stage cell whose damaged cytoplasm is nearly replaced by huge lipid-containing vacuoles surrounded by particulate debris. This type of cell has not been found in bone marrows from patients with hemolytic, thrombocytopenic or lipid storage diseases.