Neutralization of heparin activity by neutrophil lactoferrin

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin- induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.     

Methotrexate reduces inflammatory cell numbers, expression of monokines and of adhesion molecules in synovial tissue of patients with rheumatoid arthritis

Methotrexate (MTX) is one of the most widely prescribed drugs in the treatment of rheumatoid arthritis (RA). The mechanism by which MTX exerts its anti-rheumatic effect has not yet been defined. The aim of the present study was to investigate the effect of MTX treatment (7.5- 15 mg/week) on synovial tissue in RA. For this purpose, synovial biopsies were taken from 11 RA patients before and 16 weeks after initiation of MTX therapy. Immunohistochemistry was performed using monoclonal antibodies (MAb) specific for CD3, CD4, CD8, CD22, CD25, CD38, CD68, MAb67, Ki67, interferon gamma (IFN-gamma), interleukin (IL)- 1alpha, IL-1beta, tumour necrosis factor alpha (TNF-alpha), E-selectin, ICAM-1 and VCAM-1. All parameters for disease activity improved during the period of treatment. Immunohistochemical analysis revealed a statistically significant decrease in scores for CD3, CD8, CD38, CD68, Ki67, IL-1beta, TNF-alpha and the adhesion molecules E-selectin and VCAM-1. The observed decrease in synovial scores for inflammatory cells, monokines and adhesion molecules suggests that the anti- inflammatory effect of MTX is, in part, dependent on a reduction in monokine-inducible vascular adhesion molecules and subsequent reduction of cell traffic into joints.      

Heparin cofactor II-proteinase reaction products exhibit neutrophil chemoattractant activity

The physiologic function of the plasma glycoprotein heparin cofactor II (HCII) is not well understood. An in vivo role for thrombin (IIa) inhibition by HCII in the presence of certain glycosaminoglycans (dermatan sulfate and heparin) can be proposed. Many proteins, such as complement components, can be proteolyzed to generate secondary bioactive molecules. HCII is a substrate for the human neutrophil (PMN) proteinases cathepsin G (CG) and elastase (LE). We found that degradation of HCII by CG or LE generated products with potent PMN chemotactic activity, which did not stimulate the PMN oxidative burst. Our results suggest that HCII may be a physiologic regulator of the acute inflammatory response.     

In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells

Recently, the ligand for c-mpl has been identified and cloned. Initial studies of this molecule indicate that it is the platelet regulatory factor, thrombopoietin (TPO). Previous work has indicated that c-mpl is expressed in very immature hematopoietic precursors and thus raised the possibility that TPO may act directly on the hematopoietic stem cell. Therefore, in these studies, we investigate the effects of TPO on hematopoietic stem cell populations isolated from the murine fetal liver and bone marrow. Cocultivation of stem cells with fetal liver stroma give rise to multilineage expansion of the stem cells but with little or no megakaryocytopoiesis. Addition of TPO to these cocultures gives significant megakaryocyte production. This production is enhanced in combination with Kit ligand or interleukin-3. The addition of TPO to stem cell suspension cultures produces a dynamic thrombopoietic system in which stem cells undergo differentiation to produce megakaryocytes and proplatelets. These experiments show that the megakaryocytopoietic and thrombopoietic activities of TPO are initiated at the level of an early progenitor cell or upon the hematopoietic stem cell.     

Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4-5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.     

A fatal case of AIDS-defining meningoencephalitis by C. Neoformans,sensitive to antifungal therapy

Cryptococcus neoformans is the most common cause of life threatening meningoencephalitis in HIV-infected patients. Diagnosis is based on tests for cryptoccocal antigen in serum and cerebrospinal fluid, and on culture of the organism. We present a case of AIDS-related cryptococcal meningoencephalitis unresponsive to antifungal combination therapy, despite of evidence of fungal susceptibility in vitro. Significant decreases in cryptococcal antigen titers in serum and cerebrospinal fluid did not correlate with progress in disease and fatal outcome.     

Nutrition as a Vehicle for Cardiovascular Translational Research

It is becoming increasingly evident that poor nutrition plays an important role in inducing cardiovascular disease. Just as importantly, data now support the contention that appropriate nutritional interventions may have just as important an effect in preventing or delaying the appearance of cardiovascular disease. If this is indeed true, then it is critical that these advances in our knowledge of the effects of nutritional interventions be translated into effective strategies to combat cardiovascular disease. It is argued in this paper, with a few specific examples, that the translation of nutritional interventions can provide powerful approaches to alleviating the clinical challenges currently facing us today in the cardiovascular field. Furthermore, the value-added economic advantages of translating nutritional strategies on a wide scale into the public become another intriguing argument to further support investigations in this growing field.     

Tricortical Calcaneal Bone Graft and Management of the Donor Site

The clinical outcomes of 19 patients requiring autogenous grafts for foot surgery were followed up until healing at the donor site occurred. In all cases, tricortical bone was extracted from the calcaneus for use at another pedal site. The first cohort of 9 patients had the calcaneal deficit replaced with allogenic cubes. The second cohort received no tissue replacement. Patients were reviewed postoperatively with a questionnaire and clinical examination to evaluate the outcome of the operations. Radiographic outcomes were observed at the donor and recipient sites in both groups until healing was confirmed as bridging trabeculation. Incorporation of the graft at the donor site was also reviewed. Clinical outcomes, namely pain, local sensory function, and return to footwear, were satisfactory in all patients and were not significantly different between groups. One patient from each group sustained a heel fracture. The donated autogenous grafts at the recipient sites were all incorporated uneventfully at 6 months. In the first cohort, allogenic graft incorporation in the calcaneus was complete in only 2 patients at the 12-month stage. The remaining 7 cases showed reduction of the deficit by new bone formation arising from the calcaneus. Donor sites with allogenic bone replacement healed at a median of 18 months (interquartile range, 18-18 months). In the group without replacement, healing occurred at a median of 6 months (interquartile range, 6-12 months), a highly statistically significant difference (P < .001). In the second cohort without allogenic graft replacement, radiographic filling at the donor site was complete within a 12-month period. Tricortical bone can be successfully harvested from the calcaneus, but there may an associated risk of heel fracture. The role of replacement allogenic bone in assisting healing at the donor site is unclear.