Serum proteins modified by neutrophil-derived oxidants as mediators of neutrophil stimulation.

Reactive oxygen intermediates (ROI) released during inflammation may act as important mediators of neutrophil effector functions. The objective of this investigation was to evaluate the influence of ROI generation on neutrophil adhesion molecule regulation and degranulation. Induction of the neutrophil oxidative burst via Fcgamma receptor cross-linking was accompanied by up-regulation of neutrophil surface CD11b, CD35, and CD66b only in the presence of selected serum proteins, such as purified human C4, C5, or human serum albumin (HSA). Scavenging of ROI attenuated protein-dependent receptor regulations. Moreover, exogenous hydrogen peroxide was effective to increase neutrophil CD11b expression in a protein-dependent way. HSA exposed to neutrophil-derived ROI displayed signs of oxidative modification in terms of carbonyl formation. Such modified HSA transferred to resting neutrophils bound readily to the cell surface and effected receptor modulation as well as cellular spreading. In contrast, neither native HSA nor HSA protected against oxidation by the tocopherol analog Trolox exhibited agonistic properties. In conclusion, we demonstrate that neutrophil-derived ROI modify selected serum proteins, which, in turn, act as proinflammatory mediators of neutrophil stimulation.     

LPS activation of bone marrow natural suppressor cells.

Lipopolysaccharide (LPS) from Salmonella typhosa was injected into C57B1/6 mice and the effect on bone marrow (BM) natural suppressor (NS) cell activity was examined. It was shown that injection of LPS, as low as 0.01 microgram/g body weight, could enhance BM NS activity. The enhanced activity was apparent 24 hr postinjection, and returned to normal by Day 5. It was necessary to show that the enhanced suppression displayed characteristics of NS cells. The suppressor cell is Thy negative and can be found in low density Percoll fractions. Suppression was dependent upon interferon-gamma and could be augmented by lymphokines that were contained in the supernatant of TH2 helper cell. The data suggest that BM NS activity may be influenced in vivo during gram-negative sepsis.     

Neutrophil-derived Oxidants and Proteinases as Immunomodulatory Mediators in Inflammation

Neutrophils generate potent microbicidal molecules via the oxygen-dependent pathway, leading to the generation of reactive oxygen intermediates (ROI), and via the non-oxygen dependent pathway, consisting in the release of serine proteinases and metalloproteinases stored in granules. Over the past years, the concept has emerged that both ROI and proteinases can be viewed as mediators able to modulate neutrophil responses as well as the whole inflammatory process. This is well illustrated by the oxidative regulation of proteinase activity showing that oxidants and proteinases acts is concert to optimize the microbicidal activity and to damage host tissues. ROI and proteinases can modify the activity of several proteins involved in the control of inflammatory process. Among them, tumour necrosis factor-alpha and interleukin-8, are elective targets for such a modulation. Moreover, ROI and proteinases are also able to modulate the adhesion process of neutrophils to endothelial cells, which is a critical step in the inflammatory process.     

Stem cells as bone marrow residents

In addition to being a source of hematopoietic and mesenchymal stromal cells, bone marrow is known to be the source of stem cell populations, which express pluripotent markers and are capable of differentiating into cells of three germinative layers (ectoderm, mesoderm, and endoderm). Recently, a new population of so-called “very small embryonic like cells” (VSELs) has been identified in the bone marrow in addition to well-known pluripotential cells, such as MIAMI, MSC, and MAPS. The presence of stem cells with a wide spectrum of differentiation capacities in the bone marrow allows an alternative interpretation of the phenomenon of plasticity and the possibility of their switch from a canonical to a nontrivial path of differentiation. The phenotypic features of VSELs, their differentiation capacities, ontogenetic origin, and relationships with other types of stem cells are studied. The role of bone marrow stem cells and induced pluripotential stem cells in regeneration processes and their possible therapeutic application are discussed.     

Complement activation in heart diseases. Role of oxidants

Increasing evidence demonstrated that atherosclerosis is an immunologically mediated disease. Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Three major components are recognized as the major contributing factors in reperfusion injury. These are: (1) molecular oxygen; (2) cellular blood elements (especially the neutrophils); and (3) components of the activated complement system. The latter two often act in concert. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue damage mediated by neutrophils can be initiated by complement fragments, notably C5a, which are potent stimulators of neutrophil superoxide production and adherence to coronary artery endothelium. The complement cascade, particularly the alternative pathway, is activated during myocardial ischemia/reperfusion. Complement fragments such as the anaphylatoxins C3a and C5a, are produced both locally and systematically, and the membrane attack complex is deposited on cell membranes and subsequent release of mediators such as histamine and platelet activating factor (PAF), thereby causing an increase in vascular permeability with concomitant manifestation of cellular edema. Complement increases the expression of CD18 on the neutrophils and increases P-selectin expression on the surface of the endothelium. Mitochondria may be a source of molecules that activate complements during ischemia/reperfusion injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexities of the complement cascade provide numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vitro models of ischemia/reperfusion injury. The mechanism(s) that initiates complement activation is not clearly known, although loss of CD59 (protectin) from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex. Therapeutic approaches to ischemia/reperfusion injury in general, and especially those involving complements, are at the very beginning and their potential benefits have still to be adequately evaluated. It may be noted that complement activation has both positive and negative effects and, therefore, might be modulated rather than abruptly blunted.     

Human bone marrow lymphocytes. III. Polyclonal activation of B lymphocytes in human bone marrow measured by a direct plaque-forming cell assay.

Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.     

No narcosis for bone marrow harvest in autologous bone marrow transplantation

A prospective study with mild general analgesia and sedation together with local anesthesia during bone marrow harvest was performed. Thirty-one patients underwent 33 bone marrow collections. Pretreatment consisted of 100 mg meperidine i.m. and 20 mg diazepam i.m. 1 h before start of procedure. Eight patients got additional meperidine and diazepam during the procedure, all patients got lidocaine 1% locally. A mean volume of 1.321 was obtained with 42.5 punctures. Twenty-two patients had no complications, 4 vomited, 4 had easily correctable hypotension of short duration, one got oxygen for cyanosis of short duration. Acceptance was good in 23 patients, in 6 reasonably well, in two bad. Only one patient experienced pain problems, due to suction. Anxiety was no major problem due to good information before the procedure and mild sedation. This form of anesthesia for bone marrow collection is a safe procedure, it is generally well accepted by the patient and it can be performed on an out-patient basis.     

Iron in bone marrow

In the bone-marrow, non-haemoglobin iron can predominantly be found in the reticulum. Slight granules containing iron can also be observed in parts of erythroblasts by means of the Berlin blue reaction. These cells are called sideroblasts. In chemical respect, non-haemoglobin iron consists of ferritin soluble in water and haemosiderin insoluble in water. Erythroblasts will only take their iron from plasma transferrin. For the most part, this iron uptake is being regulated by erythropoietin adapting erythropoiesis to the oxygen requirements of the tissue. The iron contained in erythroblasts is predominantly utilized for haemoglobin synthesis in these cells. A slight part is being taken up by ferritin. The bone-marrow reticulum will phagocytise aged erythrocytes and store liberated iron as ferritin and haemosiderin. Part of the iron is being delivered again to plasma transferrin. With constant serum iron level the liberation of iron from the reticulo-endothelial tissue must correspond to the iron uptake by erythropoiesis. The absence of iron capable of being coloured in the bone-marrow reticulum is considered to be a reliable parameter of iron deficiency. It enables the diagnosis of iron deficiency anaemia to be made even in those patients with serum iron level and a total iron binding capacity lying within the normal range and no hypochromia of erythrocytes being present. It enables iron deficiency anaemia to be separated from sideropenic anaemia with reticulo-endothelial siderosis in differential-diagnostic manner. Even in patients with sideroblastic anaemia, iron colouring of bone-marrow smears is required for ensuring the diagnosis. Recently, a separation has also been made for idiopathic anaemia with abnormal sideroblasts. In these patients there is an increased risk for acute leukemia to develop.     

In vitro assay of cytogenetic damage induced in bone marrow cells in vivo by chemical carcinogens

Bone marrow cells explanted after the host animal has been exposed to chemicals can be assayed in vitro for cytogenetic damage. Cells were grown in medium for two cell cycles in the presence of 5-bromodeoxyuridine and then analyzed for the frequency of sister chromatid exchanges and cell replication kinetics. The patterns for induction of these endpoints as assayed in vitro were very similar to those observed if the assay was performed totally in vivo. Several chemicals were tested in this system and shown to be positive; hycanthone, ethidium bromide, 4-fluoro-3-nitro-phenyl azide, mitomycin-C, 2-aminoanthracene, benzo(a)pyrene, and cyclophosphamide. Dehydroemetine, known as a nonmutagenic drug, was negative in this assay. Since this assay incorporates host metabolism, is rapid, and has a wide dynamic range, it may be useful to perform to determine the potential of chemicals to induce genetic damage.     

Elimination of T lymphocytes from the bone marrow in HLA-incompatible bone marrow transplantation

The method presented is very well suited to eliminate T-lymphocytes from great amounts of bone-marrow. The stem cells required to reconstitute the bone-marrow are enriched in this way. It can be completely performed in a closed system. Any contamination with germs is excluded. It can be reproduced well and learnt quickly. It takes 10 hours for two trained co-workers to process 1,500 ml of bone-marrow. The vitality of cells is very good (100%). Its suitability for transplantation has still to be checked.