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A 61-year-old man presented with dyspnoea, chest pain, high fever and rigour. Chest X-ray revealed a combination of alveolar consolidations and abnormal nodular and interstitial markings. His clinical condition deteriorated despite treatment with antibiotics prescribed on a working diagnosis of pneumonia with an atypical pathogen. Finally, an open-lung biopsy specimen showed the characteristic picture of bronchocentric granulomatosis. Serological testing supported a primary infection with Mycoplasma pneumoniae. The patient responded well to treatment with prednisolone and erythromycin and five months after discharge, no radiological abnormalities were found. The combination of bronchocentric granulomatosis and mycoplasmal pneumonia has never been described in the literature and a causal relation can only be suggested. This case-report illustrates the importance of invasive diagnostic procedures if a patient with a clinical pneumonia fails to respond to adequate treatment.

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OBJECTIVE: To evaluate long-term immune reconstitution of children treated with highly active antiretroviral therapy (HAART). METHODS: The long-term immunological response to HAART was studied in 71 HIV-1-infected children (aged 1 month to 18 years) in two prospective, open, uncontrolled national multicentre studies. Blood samples were taken before and after HAART was initiated, with a follow-up of 96 weeks, and peripheral CD4 and CD8 T cells plus naive and memory subsets were identified in whole blood samples. Relative cell counts were calculated in relation to the median of the age-specific reference. RESULTS: The absolute CD4 cell count and percentage and the CD4 cell count as a percentage of normal increased significantly (P < 0.001) to medians of 939 x 106 cells/l (range, 10-3520), 32% (range, 1-50) and 84% (range, 1-161), respectively, after 48 weeks. This increase was predominantly owing to naive CD4 T cells. There was a correlation between the increase of absolute naive CD4 T cell counts and age. However, when CD4 T cell restoration was studied as percentage of normal values, the inverse correlation between the increase of naive CD4 T cell count and age was not observed. In addition, no difference in immunological reconstitution was observed at any time point between virological responders and non-responders. CONCLUSIONS: Normalization of the CD4 cell counts in children treated with HAART is independent of age, indicating that children of all age groups can meet their CD4 T cell production demands. In general, it appears that children restore their CD4 T cell counts better and more rapidly than adults, even in a late stage of HIV-1 infection.

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OBJECTIVE: To evaluate the clinical, immunologic, and virologic response to indinavir, zidovudine, and lamivudine in children with human immunodeficiency virus-1 (HIV-1) infection. STUDY DESIGN: Twenty-eight HIV-1-infected children (3 months to 16 years of age) with or without prior treatment with reverse-transcriptase inhibitors and a HIV-1 RNA >5000 copies/mL and/or a CD4 cell count less than the lower limit of the age-specific reference value were treated with indinavir, zidovudine, and lamivudine. Pharmacokinetics of indinavir were determined in each child. RESULTS: The combination treatment was well tolerated in the majority of patients. Clinical improvement was seen in all patients. After 6 months of therapy, 70% of the patients had an HIV-1 RNA load below 500 copies/mL, whereas 48% of the children had a viral load below 40 copies/mL. Relative CD4 cell counts in relation to the lower limit of the age-specific reference value increased significantly from a median value of 79% at baseline to 106% after 6 months of therapy. The doses of indinavir necessary to achieve area under the curve values comparable to adult values varied from 1250 mg/m(2)/d to 2450 mg/m(2)/d. CONCLUSIONS: Highly active antiretroviral therapy consisting of indinavir, zidovudine, and lamivudine in children reduced HIV-1 RNA to less than 500 copies/mL in 70% of the children within 6 months. Improved CD4 cell counts were observed in most patients, as was a better clinical condition (no invasive or opportunistic infections, increased weight gain). Side effects of the triple therapy were mild. Highly active antiretroviral therapy can be used as successfully in children as in adults.

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INTRODUCTION: Regeneration of CD4+ T lymphocytes has been shown to be thymus-dependent in bone marrow transplant recipients and after intensive chemotherapy. The rate of CD4+ T cell regeneration is correlated positively with enlargement of the thymus, as shown on radiographs, and higher rates of CD4+ T lymphocyte regeneration were observed in children as compared with adults, consistent with thymic function diminishing with age. We hypothesized that in HIV infected patients CD4+ T cell recovery during highly active antiretroviral therapy (HAART) may also be thymus dependent. Therefore, repopulation of naive (CD45RA+), memory (CD45RO+) and total CD4+ T lymphocytes and total CD8+ T lymphocytes in peripheral blood was assessed in 13 HIV infected children during the initial 3 months of HAART. RESULTS: Significantly higher recovery rates of naive, memory and total CD4+ T cells were observed in children below the age of 3 years as compared with older children. Kinetics of total CD8+ T cells showed no relation to age. Moreover, recovery rates of naive CD4+ T cells in patients below 3 years of age were 10-40 fold higher as compared with previously reported naive CD4+ T cell recovery rates in adults on HAART. CONCLUSIONS: High recovery rates of naive, memory and total CD4+ T cells can be achieved in children below 3 years of age. Changes in CD8 counts did not correlate with age. These results indicate that regeneration of CD4+ T cells during HAART may be a thymus-dependent process.

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Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid-committed progenitor cells. Using the monoclonal antibodies (mAb) ER-MP12 and ER-MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony-stimulating factor (M-CSF)-responsive cells in the bone marrow have the ER-MP12hi 20- phenotype. In addition, we found that the ER-MP12hi 20- subset (comprising about 2 % of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER-MP12hi 20-subset, we used ER-MP58, a marker expressed at high level by all M-CSF-responsive bone marrow progenitors. With this marker the ER-MP12hi 20- cell population could be divided into three subfractions: one with absent or low level ER-MP58 expression, one with intermediate, and one with high ER-MP58 expression. These subfractions were isolated by fluorescence-activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER-MP58 on stem cell subsets was examined in the cobblestone area-forming cell (CAFC) assay. Our data indicate that in the ER-MP12hi 20- subpopulation myeloid-committed progenitors are characterized by high-level expression of the ER-MP58 antigen, whereas cells with other or broader differentiation capacities have an ER-MP58 negative/low or intermediate phenotype. These myeloid-committed progenitors have no significant repopulating ability in vivo, in contrast to the ER-MP58 intermediate cells. Primitive CAFC-28/35, corresponding to cells providing long-term hematopoietic engraftment in vivo, also did not express the ER-MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER-MP58 in combination with ER-MP12 and ER-MP20.

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At present, inhaled glucocorticoids are widely accepted as the therapy of choice in chronic asthma. Treatment with inhaled glucocorticoids significantly suppresses local airway inflammation in asthmatics, but may also have systemic effects, e.g. a reduction of the number of circulating hypodense eosinophils or a down-modulation of HLA-DR antigen (Ag) expression by T lymphocytes in peripheral blood. However, the effect of long-term therapy with inhaled glucocorticoids on peripheral blood monocytes (PBM), which are the precursors of the most numerous cell type in the lung, the alveolar macrophage, have not yet been evaluated. We therefore investigated the expression of various cell surface Ag on PBM from non-smoking patients with allergic asthma who were treated for 2.5 years with a beta(2)-receptor agonist plus either an inhaled glucocorticoid (beclomethasone dipropionate, BDP) (n = 4) or an anticholinergic or placebo (n = 8). We compared the results with healthy volunteers (n = 7). Long-term treatment of allergic asthmatics with inhaled BDP, but not anticholinergic or placebo therapy, was associated with a significantly lower CDllb Ag expression (p < 0.04) and higher expression of CD13, CD14 and CD18 Ag (p < 0.05, p < 0.02 and p < 0.04, respectively) when compared with the healthy control subjects (n = 7). Most interestingly, PBM of asthmatics treated with inhaled BDP expressed an almost two-fold higher level of CD14 Ag on their cell surface than PBM of patients treated with anticholinergic or placebo (p < 0.03). No significant differences in the expression of CD16, CD23, CD25, CD32 and CD64 Ag or HLA-DR were observed between PBM from the different patient groups or healthy controls. Taken together, this study shows that long-term local therapy with inhaled BDP coincides with an altered expression of at least one cell surface Ag on PBM from allergic asthmatics.

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Monoclonal antibody ER-MP12 defines an antigen (Ag) on murine hematopoietic stem cells that is differentially expressed by the various subsets in the hematopoietic stem cell compartment. To test whether ER-MP12 could be an asset for further subfractionation of these subsets, we physically sorted our previously defined low-density ER-MP20- (i.e., Ly-6C-) Rhodamine-123dull (Rh123dull), and wheat germ agglutinindim (WGAdim) stem cell populations on the basis of ER-MP12 Ag expression. In addition, we determined the distribution of the ER-MP12 Ag on bone marrow 6 days after 5-FU treatment. Long-term and transiently repopulating stem cell subsets were both identified in vitro using the cobblestone area-forming cell (CAFC) assay. The data show that sorting on the basis of ER-MP12 improves the separation of primitive and more mature stem cell subsets in the Rh123dull but not in the WGAdim subpopulation. However, the combination of sorting cells on the basis of an intermediate ER-MP12 expression and a low WGA affinity (ER-MP12mediumWGAdim) allows an 840-fold enrichment for in vitro long-term repopulating cells (day-28 CAFC) when compared with unseparated bone marrow. The distribution of the ER-MP12 Ag on 5-FU-treated bone marrow stem cells was similar to that in normal bone marrow stem cells, suggesting that the level of Ag expression is not dependent on cell-cycle status. Together, the combination of ER-MP12 and WGA offers the advantage of a positive selection strategy for hematopoietic stem cells, allowing different stem cell subsets to be distinguished on the basis of their primitiveness. Since no mature bone marrow cells are found within the WGAdimER-MP12medium subpopulation, the combination of ER-MP12 and WGA enables hematopoietic stem cells to be highly enriched and thus makes the use of a cocktail of lineage-specific antibodies redundant.

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Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly-6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

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The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER-MP12 and ER-MP20, which in two-color flow-cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence-activated cell sorting followed by macrophage colony-stimulating factor-stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER-MP12hi20-, ER-MP12+20+ and ER-MP12-20hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER-MP12hi20- cells develop via the ER-MP12+20+ stage into ER-MP12-20hi monocytes.

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We searched for new cell surface markers that allow a positive identification of thymus-repopulating cells in the bone marrow (BM) of the mouse. Recently we raised two rat monoclonal antibodies (ER-MP12 and ER-MP20) that recognize cell surface antigens expressed by mouse haematopoietic progenitor cells, among which are progenitor cells of the macrophage lineage. Here we show that the ER-MP12 antigen, but not the ER-MP20 antigen, is also expressed by BM cells with thymus-repopulating ability. Using ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis six subpopulations of BM cells can be identified. The thymus-repopulating ability of each BM subpopulation was assessed after fluorescence-activated cell sorting and subsequent intrathymic injection into sublethally irradiated Thy-1 congenic recipient mice. Thymus-repopulating activity appeared to be exclusively confined to two subsets of BM cells expressing either high or intermediate levels of the ER-MP12 antigen, but lacking ER-MP20 antigen expression. These BM subsets comprised 1-2% and 30% of total nucleated BM cells respectively. The frequency of thymus-repopulating cells was maximal in the minor BM subpopulation with the highest level of ER-MP12 antigen expression. We conclude that ER-MP12 detects a hitherto unknown cell surface marker expressed by BM cells with thymus-repopulating ability.

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In the accompanying paper we showed that six distinct subsets of bone marrow (BM) cells can be identified using the mAb ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis. Upon intrathymic transfer into sublethally irradiated mice thymus-repopulating ability was restricted to ER-MP20- BM cells expressing either high or intermediate levels of the ER-MP12 antigen (1-2% and approximately 30% of BM nucleated cells respectively). The highest frequency of thymus-repopulating cells was found in the minor subset of ER-MP12(+)+20- BM cells. In the present study we demonstrate that upon intravenous transfer, thymus-homing and -repopulating BM cells are exclusively confined to the ER-MP12(+)+20- and ER-MP12+20- subpopulations, the highest frequency being detected among ER-MP12(+)+20- BM cells. Analysis of the peripheral blood leucocytes of reconstituted mice showed that not only prothymocytes but also progenitor cells of the B cell lineage as well as the myeloid lineage were present within both subsets. Three-colour flow cytometric analysis revealed that ER-MP12(+)+20- BM cells in particular were phenotypically heterogeneous with respect to the expression of the cell surface markers Thy-1, Sca-1, CD44, B220 and c-kit. Taken together our data demonstrate that ER-MP12 positively identifies BM cells with the ability to home to and repopulate the thymus. The phenotypic heterogeneity displayed by the ER-MP12(+)+20- BM subset, containing the highest frequency of thymus-homing and -repopulating cells, provides a basis for further separation of prothymocyte activity from other haematopoietic activities in the BM of the mouse.

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Monoclonal antibodies (mAbs) directed against the transferrin receptor are known to inhibit proliferation of cells due to iron deprivation. Some cell types, however, escape from growth inhibition by a mechanism which is unclear at present. This mechanism is the subject of the present study. We investigated the differential growth inhibition caused by anti-transferrin receptor mAb ER-MP21 in connection with the differentiation of murine macrophages (M phi). Therefore, we applied two models of M phi differentiation, namely, culture of bone marrow cells in the presence of M-CSF and a panel of M phi cell lines ordered in a linear differentiation sequence. In both models we observed that proliferation of M phi precursors was strongly inhibited by ER-MP21. In contrast, proliferation of more mature stages of M phi differentiation was hardly affected. Remarkably, iron uptake by M phi precursor and mature M phi cell lines was inhibited by ER-MP21 to the same extent. However, mature M phi cell lines showed an iron uptake two- to threefold higher than that of M phi precursor cell lines. These observations strongly suggest that mature M phi escape from ER-MP21-mediated growth inhibition, because these cells take up more iron than is actually needed for proliferation. Furthermore, we found that enhanced iron uptake by mature M phi is not necessarily accompanied by a higher cell surface expression of transferrin receptors, thus suggesting an increased recycling of transferrin receptors in mature M phi.

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This study reports on the earliest stages of mononuclear phagocyte differentiation. A crucial question in this developmental process is whether mature macrophage (M phi) heterogeneity is already appointed at the precursor cell level. For this purpose, we produced clonal populations of mononuclear phagocytes from bone marrow culture by somatic cell hybridization with two hypoxanthine, aminopterin, thymidine-sensitive myeloid cell lines. A panel of 22 stable hybrids was obtained from these fusions. Differentiation stage analysis of the hybrids indicated that all cell lines had immature mononuclear phagocyte characteristics. The hybrids exhibited typical myeloid morphology and mainly nonadherent growth. Mature M phi features, such as expression of the cell surface antigens Mac-1, Mac-2 and F4/80, phagocytosis of latex beads, and expression of nonspecific esterase and acid phosphatase activity, were virtually absent. The immature M phi markers Thy-1, MIV25 and MIV52, on the other hand, were readily expressed, although heterogeneity was observed among different hybrid cell lines. We then analyzed the differentiation potential of seven hybrids by culture of the cells in the presence of post-lipopolysaccharide serum supplemented with interferon-gamma and found that the expression of mature M phi characteristics was induced. However, the various hybrids showed divergent patterns of mature M phi marker induction. R0C2 cells, for instance, showed extensive morphological and phenotypical differentiation without concomitant induction of phagocytosis. In contrast, W1C4 cells showed significant induction of phagocytosis without simultaneous increase of phosphatase and esterase activity. R1C1 cells were unique in the strong induction of Ia antigen expression. Together, our data indicate that (a) early M phi differentiation stages can be rescued by somatic cell hybridization, and that (b) the obtained cell lines are able to mature according to divergent differentiation programs.

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The aim of the present study was the phenotypical analysis of early stages in macrophage (M phi) differentiation. For this purpose, we produced a panel of syngeneic rat hybridomas, which secreted antibodies (mAb) against M phi precursor antigens. As immunogens we used immortalized M phi precursors (P.J. M. Leenen et al., Eur. J. Immunol. 1990, 20: 15). We screened the obtained mAb in the following in vitro models of M phi differentiation: (a) a panel of M phi cell lines ordered in a linear differentiated sequence; (b) immature and mature mononuclear phagocytes obtained from bone marrow (BM) culture; (c) a panel of M phi precursor hybrids, and (d) differentiated and control M phi precursor hybrid cells. Four mAb, ER-MP12, ER-MP20, ER-MP54 and ER-MP58, were selected. These mAb recognize antigens which disappear in the course of M phi differentiation. Next, we investigated whether these mAb also recognized M phi precursors in normal BM. For this purpose, ER-MP-positive and -negative BM fractions were isolated using a fluorescence-activated cell sorter. Fractions were cultured in M phi-colony-stimulating factor-containing conditioned medium, and the resulting mature M phi progeny was quantified using the MTT assay. The present experiments indicate that ER-MP12 and ER-MP20 detect a subpopulation of BM M phi precursors, whereas ER-MP58 stains virtually all M phi precursors. Biochemical analysis of radioiodinated antigens revealed that these mAb recognize different molecules. ER-MP12 and ER-MP20 bound to single-chain (glyco)proteins of 140 kDa and 14 kDa, respectively. ER-MP54 precipitated multiple polypeptides, of which the major chains have an apparent molecular mass of 90, 80-85 and 70-75 kDa. Based on the molecular mass of the recognized antigens and the mAb specificities we conclude that ER-MP12, ER-MP54 and ER-MP58 recognize hitherto unknown antigens of murine M phi precursor cells. The antigen detected by ER-MP20 is most likely identical to Ly-6C.

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