Lysozyme turnover in the rat.

Lysozyme turnover in the rat was studied with (125)I-labeled rat lysozyme. It was found that plasma lysozyme has a rapid disappearance rate with a half-life of 75 min. The rate of synthesis was calculated at 3.4 mug/min per 100 g rat. This rate of synthesis was compared with figures from the literature for the turnover rate of neutrophilic granulocytes, and the data were consistent with the concept that disintegrating neutrophils are the main source of plasma lysozyme. The distribution of enzymatic lysozyme activity and of radioactive lysozyme was studied in several organs. Very high enzymatic activity was found in leukocytes as were considerable activities in lungs, kidneys, bone marrow, spleen, and intestine; little enzymatic activity was found in the urine. High radioactive levels as compared with plasma radioactivity were demonstrated only in the kidneys. This indicates that of the organs studied, the kidney is the predominant site of storage and destruction of plasma lysozyme. Lysozyme was found to disappear only slowly from the kidneys over a period of 4 days. The data obtained seem to indicate that lysozyme or a lysozyme degradation product precipitable by trichloroacetic acid was released in small amounts from the kidneys to plasma throughout this period.

Lysozyme turnover in man.

Lysozyme turnover studies with (125)I-labeled human lysozyme were carried out on 22 patients, viz. nine control patients, seven nephrological patients with varying degrees of renal insufficiency, including three bilaterally nephrectomized patients, and six hematological patients with disturbed turnover of the neutrophilic granulocytes. It was found that plasma lysozyme has a rapid turnover with a fractional catabolic rate of 76%/hr of the plasma content. Lysozyme catabolism varied with the endogenous creatinine clearance; in addition however, extrarenal sites of catabolism were demonstrated since lysozyme could be broken down in the anephric patients, although only at a rate amounting to about 15% of the rate found in persons with intact kidneys. In the uremic patients the increased plasma lysozyme concentration was due to decreased rates of catabolism; in the hematological patients the increased plasma lysozyme level was due to increased rates of synthesis which supports the hypothesis that plasma lysozyme mainly stems from disintegrating neutrophilic granulocytes. Furthermore, it was shown that in the nonhematological patients examined, the rate of synthesis varied with the endogenous creatinine clearance.

Runoff water quality from turfgrass established using volume-based composted municipal biosolids applications.

Municipal programs for turfgrass establishment recommend large volume-based application rates of composted municipal biosolids (CMB). This study compared runoff water quality among combinations of two common turfgrass establishment practices and two CMB sources. Bryan- or Austin-CMB were incorporated into 5 cm of soil at a rate of 12.5 or 25% by volume (v/v) on an 8.5% slope. Tifway bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy, var. Tifway] sprigs were planted and established; sod, produced at a separate site using either CMB amendment at the 25% v/v rate, was transplanted to the runoff plots on the same day. A mature stand of bermudagrass was used as a control. Runoff water was collected after each of eight natural rain events during the sampling period. Total runoff water loss (mm) was similar for the CMB-amended sprigged and transplanted sod stands. The concentration of total dissolved P (TDP) in runoff water was greatest from the transplanted sod in the first seven rain events (4.1 to 7.5 mg L(-1)). The concentration of TDP in runoff water was similar at both the 12.5 and 25% v/v incorporation rates. Regression analysis indicated Mehlich-3-extractable soil test P concentrations in soil amended with CMB were positively correlated to concentration and mass loss of dissolved P in runoff. At similar application rates, dissolved P loss in runoff water was reduced by incorporating CMB into the soil on site rather than transplanting sod produced with CMB.

Trends in the incidence of leukemia in Denmark, 1943-77: an epidemiologic study of 14,000 patients.

An epidemiologic study of the total population of patients with leukemia in Denmark during 1943-77 was performed with special emphasis on time trends. The material stemmed from the national Danish Cancer Registry and was believed to be virtually complete. Of the 13,813 patients, 40% had acute leukemia; 40%, chronic lymphocytic leukemia; patients, 40% had acute leukemia; 40%, chronic lymphocytic leukemia; and 20%, chronic myeloid leukemia. Over the 35-year period, the incidence of acute leukemia increased threefold in the age groups 0-9 and 50-70+ years; whereas the increase in the age group 0-9 years climaxed in the 5-year period 1968-72, and the increase in the age group 50-70+ years was sustained. The incidence of chronic lymphocytic and chronic myeloid leukemia remained unchanged. No geographic pattern was discernible.

Short-term liquid marrow cultures are supported by a mixture of haematopoietic cytokines but do not purge for acute myeloid or lymphoid leukemic marrow cells.

Short-term liquid marrow culture (STLMC) is a potential source for autografting in leukemia. In a preclinical setting, including candidates for autologous marrow transplantation, we have studied STLMC supported by a selected mixture of clinical available recombinant human haematopoietic growth factors. STLMC of leukemic marrow cells were prospectively performed to evaluate the purging effect. Bone marrow cells cultured and supported by the selected mixture of rhIL-3/rhGM-CSF/rhEpo revealed an increased number of day 10-12 cultured cells, parallelled by an increased proliferation rate when compared to unstimulated cultures. The median number of myeloid progenitors recognized as day 7 and day 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) was significantly increased in the supported STLMC to 145/305 from 105/115 per ml culture (n = 7, p < 0.01). Further addition of rhKL did not enhance the numbers of day 7 or day 14 GM-CFUs per ml culture. In no instance was the number of clonogenic cells at the end of culture greater than the input day 0, except in cultures of purified CD34-positive marrow progenitors which resulted in an expansion of late myeloid progenitors. Cytokine-supported cultures of leukemic marrow cells from acute myeloid (n = 14) and lymphoblastic (n = 7) leukemia patients were established at the time of diagnosis. In the supported cultures, the cell number increased for myeloblast but was unchanged for lymphoblast leukemic marrow cells compared to non-supported cultures. Immunophenotypic and cytogenetic studies of selected leukemic cell samples identified unchanged myeloid or slightly reduced frequencies of lymphoblastic leukemic cells at the end of culture. This preclinical study supports the idea that the addition of a mixture of clinical available haemopoietic cytokines to STLMC increases the recovery of detectable myeloid progenitors which may enhance myeloid regeneration after autografting. No substantial selective loss of myeloid leukemic cells was found in the cytokine-supported short-term culture system.

Priming with recombinant human hematopoietic cytokines before bone marrow harvest expands in vivo and enhances ex vivo recovery of myeloid progenitors in short-term liquid cultures.

BACKGROUND AND AIM: Short-term liquid marrow cultures (STLMC) are a potential source for autografting. We have previously shown that the quality of such grafts from transplantation candidates may be improved by hematopoietic cytokine support, especially if purified CD34-positive progenitors are cultured. The aim of this preclinical work was to quantitate ex vivo recovery of myeloid progenitors (colony-forming units-granulocyte/macrophage [CFU-GM]) in STLMC before and after short-term, in vivo treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF), granulocyte-macrophage colony-stimulating factor (rhGM-CSF), or interleukin-3 (rhIL-3). EXPERIMENTAL SETUP: Twenty-two sequential patients in marrow remission for hematological malignancies and eligible for autologous marrow transplantation received rhG-CSF or rhGM-CSF for 5 days or rhIL-3 for 10 days before marrow harvest. Marrow samples before and after in vivo priming were studied for CFU-GM in pre- and post-STLMC. RESULTS: After priming with rhG-CSF, rhGM-CSF, or rhIL-3, the number of isolated light-density cells increased nine-, six-, and two-fold, respectively. The total number of sampled (18 mL marrow) myeloid progenitors preculture (day 7/14 CFU-GM x 10(4) increased significantly from median 0.7/1.1 before to 37.3/26.7 after priming with rhG-CSF (n = 8) and from 5.6/3.4 before to 46.6/44.9 after priming with rhGM-CSF (n = 8) but remained unchanged (3.7/1.5 to 3.6/5.7) after priming with rhIL-3 (n = 6). The number of myeloid progenitors postculture (day 7/14 CFU-GM x 10(4) per 18 mL marrow) in cytokine-supported STLMC significantly increased from median 0.3/0.6 before to 7.0/5.3 after priming with rhG-CSF and from 1.9/1.6 before to 24.4/14.4 after priming with rhGM-CSF but remained unchanged (0.4/0.6 to 0.4/0.2) after priming with rhIL-3. Cytokine-primed and purified CD34+ marrow cells may be expanded in STLMC by a cytokine-driven differentiation into late myeloid progenitors and endstage cells. CONCLUSION: In vivo priming of bone marrow cells by hematopoietic cytokines significantly increases the recovery of harvested pre- and postculture myeloid progenitors. During cytokine-supported STLMC, early myeloid progenitors may differentiate into a "very late" progenitor pool with a potential for fast marrow regeneration. The number of such progenitors in cytokine-supported short-term liquid cultures may be sufficient for fast myeloid engraftment and complete peripheral blood or marrow stem-cell support after high-dose chemotherapy.

Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation.

In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10-5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.

Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue.

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.

Histiocytic sarcomas and monoblastic leukemias. A clinical, histologic, and immunophenotypical study.

Eight histiocytic sarcomas, identified by examination of more than 2000 malignant lymphomas, are described. For comparison, tumor infiltrates from five monoblastic leukemias were also analyzed. The histiocytic sarcomas were all high-grade malignancies consisting of markedly pleomorphic large cells with many mitotic figures. At presentation, six of the patients had systemic symptoms (fever, fatigue, loss of weight), skin infiltrates, and lymphadenopathy. Despite aggressive chemotherapy, clinical remissions were short, and six patients died of disease .5-48 months (mean, 6.5 months) after diagnosis. The remaining two patients are alive and in partial or complete remission 7 and 12 months after diagnosis. Immunotypic examination showed that all the histiocytic sarcomas were positive for macrophage-related antigens and negative for antigens on B cells, T cells, myeloid cells, epithelial cells, and melanocytes. T-cell receptor and immunoglobulin genes were studied in three cases and were present in a germline configuration. One of the histiocytic sarcomas resembled Langerhans' cells in phenotype and morphology; it was classified as a Langerhans' cell sarcoma. The remaining histiocytic sarcomas did not express accessory cell-associated antigens, but more closely resembled "ordinary" tissue macrophages; they were positive for lysozyme and/or CD68, followed in frequency by CD11c, CD4, CD11b, CDw32, peanut agglutinin receptor, and CD13. Similar features were seen in the monoblastic leukemias. These conditions could only be distinguished from histiocytic sarcoma by clinical and morphologic, rather than immunophenotypic, criteria. Expression of oncoprotein p53 was studied in nine cases and was positive in six of six histiocytic sarcomas and one of three monoblastic leukemias. Rare malignancies show features consistent with the derivation from macrophages. Two entities may be distinguished: those that resemble antigen-presenting accessory cells and those that more closely resemble ordinary tissue macrophages. Recognition of these tumors is important clinically and requires assessment of clinical, morphologic, and immunophenotypic features, supplemented by analysis of T-cell receptor and immunoglobulin genes. Whether (or how) p53 gene mutations are implicated in their pathogenesis will be an important topic for future investigation.

Short-term rhG-CSF priming before chemotherapy does mobilize blood progenitors but does not prevent chemotherapy induced myelotoxicity: a randomized study of patients with non-Hodgkin's lymphomas.

The aim of this study was to evaluate the efficacy, safety and toxicity of short-term priming with recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately after diagnosis but before combination chemotherapy (CHOP) for non-Hodgkin's lymphomas. Of fourteen patients entering the study, seven received five days subcutaneous injection of rhG-CSF (5 micrograms/kg/day) before CHOP (CSF-group), and seven were treated with CHOP alone (control group). Blood samples were studied before and on days 1-5 during rhG-CSF priming as well as twice weekly after treatment. The number of blood and bone marrow progenitors was identified by clonogenic growth day 7, 14 and 21 of GM-CFU in semisolid medium. Blood absolute neutrophil counts increased in all rhG-CSF primed patients. The expansion of marrow myelopoiesis resulted in increased myeloid:erythroid ratios, increased bone marrow cellularity and increased numbers of myeloid progenitors both in the blood as well as the marrow. Chemotherapy induced neutropenia developed on day 9-12 in all patients independent of myeloid growth factor priming. However, neutropenia appeared earlier in the cytokine primed group (P = .0038). Five patients in the CSF-group and three patients in the control group were hospitalized with neutropenic fever, and septicemia was documented in three patients in the CSF-group. RhG-CSF induced expansion of myelopoiesis immediately before combination chemotherapy mobilized sufficient number of blood progenitors for apheresis but did not result in reduction of duration and degree of neutropenia in patients with newly diagnosed non-Hodgkin's lymphoma. Although the small number of patients prevents drawing definite conclusions, this time schedule for priming should be used with caution in the future due to an increased risk of hematologic toxicity.

Stimulation tests for the bone marrow neutrophil pool in malignancies.

It has been known for decades that blood neutrophilia occurs after the administration of etiocholanolone, adrenocortical steroids, and endotoxins. Neutrophil leukocytosis in general may be due to several mechanisms such as increased stimulation of the myelopoiesis, increased release from the marrow, a shift from the marginated to the circulating pool (demargination), prolongation in the peripheral half-life, and decreased migration of neutrophils from the blood to the tissue. However, the principal cause of the neutrocytosis for each of the above mentioned agents is increased release of neutrophils from the bone marrow reserves. Since a sufficient reserve capacity is a prerequisite for optimal defenses against infections, the marrow response has been used to estimate the dose of chemotherapy expected to be tolerated without life-threatening neutropenia. However, none of the above "test substances" have gained widespread use due to adverse reactions or undesirable effects on neutrophil function. Recent progress in biotechnology has developed recombinant human (rh) hematopoietic growth factors ready for clinical use. Marrow myelopoiesis is stimulated by granulocyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage CSF (rhGM-CSF). The immediate effect, however, is mobilization of mature neutrophil granulocytes to the blood. Bone marrow cellularity seems to influence the neutrophil number mobilized during 24 hours by one subcutaneous injection of either rhG-CSF or rhGM-CSF. A recent pilot study has suggested such a "24 hour stimulation test" to predict severe neutropenia following cyclic chemotherapy. This concept is illustrated by two case reports. The "stimulation test" suggests that we may devise strategies to define patient subsets which may benefit from prophylactic growth factor administration during cyclic chemotherapy.

Early infections after autologous transplantation for haematological malignancies.

The purpose of this study was to evaluate the early infectious complications following autologous transplantation in haematological patients. Sixty-one patients who underwent either autologous bone marrow (BM; 28 patients) or peripheral blood stem cell (PBSC; 33 patients) transplantation for haematological malignancies were reviewed retrospectively. Engraftment happened significantly faster and the length of hospital stay was shorter in the PBSC group compared with the BM group. All patients in the study developed fever and all but two experienced temperatures > or = 38.5 degrees C. Overall, 57 patients had signs of oral mucositis, 23 with ulceration. Twenty patients had bacteraemia, 12 developed pneumonia, 6 systemic fungal infection. No major differences were found between the two groups in distribution or incidence of infections. This study indicates that the use of peripheral blood stem cells results in faster engraftment and shorter hospital stay, whereas the effect on the incidence of early infections seems to be unaffected.

[Autologous bone marrow transplantation in malignant hematologic diseases]. / Autolog knoglemarvstransplantation ved maligne haematologiske sygdomme.

Autologous bone marrow transplantation (Auto-KMT) involves harvesting of a portion of a patient's bone marrow for subsequent reinfusion and restoration of marrow function following ablative doses of cytotoxic therapy, used in the treatment of various malignancies. The use of autologous rather than allogeneic marrow stem cells reduces the probability of acute graft-versus-host disease and reduces the need for obtaining HLA-matched marrow from limited donor pools. The greatest problem in Auto-KMT involves efficacy of the cytotoxic therapy and the obvious lack of graft-versus-leukemia effect. In addition, a theoretical limitation is that the marrow may contain clonogenic malignant cells, which may be the source of reestablished disease. In absence of phase III clinical trials directly comparing Auto-KMT with conventional therapies in the treatment of most malignancies, its role continues to be poorly defined. In an attempt to identify subsets of patients with leukemia or lymphoma who might benefit from transplantation, we performed this study of recent reports from the literature. It is concluded that the associated mortality is acceptable. At present the indications for Auto-KMT are lymphoma in relapse after conventional therapy and acute myeloblastic leukemia in second remission. It is probable that Auto-BMT will be used in earlier disease stages in the future (first remission).

[A mini epidemic of varicella zoster virus reinfection at a department of hematology]. / Miniepidemi af varicel zoster virus reinfektion på en haematologisk afdeling.

Whereas primary infection with varicella zoster virus (VZV) (chickenpox) and reactivation of VZV (shingles) are common and recognized, clinical reinfection with VZV is rare. A little epidemic of presumed reinfection with VZV in six immune-compromised adults is presented here. The epidemic lasted for three months, during which a healthy young woman also developed a primary VZV infection in the form of chickenpox. In the immune-compromised patients, the clinical picture was dominated by disseminated, prolonged and frequently haemorrhagic and necrotic eruptions which may cause diagnostic difficulties. Skin biopsy proved helpful in the diagnosis while demonstration of the VZV antigen in the skin elements was specific and sensitive. All of the patients, with one exception, were treated with acyclovir and dissemination of the infection to the inner organs did not occur. One patient may have died on account of the VZV infection. In conclusion, immune-incompetent patients must be warned against infection from chickenpox or disseminated herpes zoster. In cases of proved exposure, prophylactic treatment with acyclovir should be considered and, in cases of clinical disease, immediate treatment with 10 mg acyclovir per kg body weight should be administered intravenously thrice daily.

[Malignant hematologic diseases. Diagnosis and treatment. A clearing report]. / De maligne haematologiske sygdomme. Diagnostik og behandling. En klaringsrapport.

The malignant haematological disorders comprise the main groups leukemia, malignant lymphoma and multiple myeloma and the potentially malignant disorders: myelodysplastic syndrome, polycythaemia vera, myelofibrosis and M-component of uncertain significance. The common feature of all these disorders is monoclonality, i.e. they originate from one single cell. Around 2,000 new cases are diagnosed per year in Denmark. Because of the relative small number of patients, complex diagnosis and treatment (especially the possibility of cure on intensive treatment) a high degree of centralization is warranted to secure an evenly distributed high level of patient care and research. The present rules for referral of patients are unsatisfactory. A new referral system is proposed based on a common set of rules, agreed upon by five haematological centers in Denmark and the surrounding region, comprising diagnostic procedures, treatment, research and development for all haematological patients in the area. Based on these common rules (functional centralization) it is decided whether the individual patient can be treated in the primary hospital or should be referred to a center (geographical centralization). Recommendations about diagnosis, treatment and referral are made in this report. Detailed suggestions are given for diseases which may be treated locally whereas no detailed regimens are given for diseases and disease stages which should be centralized. In the latter cases, the main emphasis is placed on a presentation of treatment results.