The effectiveness of the anti-CD11d treatment is reduced in rat models of spinal cord injury that produce significant levels of intraspinal hemorrhage.

We have previously reported that administration of a CD11d monoclonal antibody (mAb) improves recovery in a clip-compression model of SCI. In this model the CD11d mAb reduces the infiltration of activated leukocytes into the injured spinal cord (as indicated by reduced intraspinal MPO). However not all anti-inflammatory strategies have reported beneficial results, suggesting that success of the CD11d mAb treatment may depend on the type or severity of the injury. We therefore tested the CD11d mAb treatment in a rat hemi-contusion model of cervical SCI. In contrast to its effects in the clip-compression model, the CD11d mAb treatment did not improve forelimb function nor did it significantly reduce MPO levels in the hemi-contused cord. To determine if the disparate results using the CD11d mAb were due to the biomechanical nature of the cord injury (compression SCI versus contusion SCI) or to the spinal level of the injury (12th thoracic level versus cervical) we further evaluated the CD11d mAb treatment after a T12 contusion SCI. In contrast to the T12 clip compression SCI, the CD11d mAb treatment did not improve locomotor recovery or significantly reduce MPO levels after T12 contusion SCI. Lesion analyses revealed increased levels of hemorrhage after contusion SCI compared to clip-compression SCI. SCI that is accompanied by increased intraspinal hemorrhage would be predicted to be refractory to the CD11d mAb therapy as this approach targets leukocyte diapedesis through the intact vasculature. These results suggest that the disparate results of the anti-CD11d treatment in contusion and clip-compression models of SCI are due to the different pathophysiological mechanisms that dominate these two types of spinal cord injuries.

Effects of a triple antibiotic solution on pulpal dynamics after intentionally delayed tooth replantation in mice.

INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

Evaluation of the function of polymorphonuclear neutrophilic leukocytes in healthy dogs given a high dose of methylprednisolone sodium succinate.

OBJECTIVE: To evaluate effects of a high dose of methylprednisolone sodium succinate (MPSS) on function of polymorphonuclear neutrophilic leukocytes (PMNs) in dogs. ANIMALS: 7 healthy male Beagles (body weight, 10.5 to 15 kg; age, 2 to 4 years). PROCEDURES: All dogs were treated by IV administration of a high dose of MPSS (30 mg/kg). Additional doses of MPSS (15 mg/kg) were administered IV at 2 and 6 hours and then at 6-hour intervals until 48 hours after the initial dose. Blood samples were collected before and 1, 2, 4, 7, and 14 days after completion of the MPSS administrations and used for evaluation of PMN functions. Isolated PMNs were used for assessment of functions, such as adhesion, migration, phagocytosis, and oxidative burst. RESULTS: On days 1, 2, and 4 after completion of MPSS administration, there was a decrease in PMN expression of adhesion markers such as CD11b and CD18. There was a decrease in the phagocytotic ability of PMNs on days 1, 2, and 7 after completion of MPSS administration, with a reduction in the oxidative burst of PMNs detected on day 7. No significant changes were identified for migration. All functional changes returned to their pretreatment values by 14 days after completion of MPSS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with a high dose of MPSS suppressed PMN functions in dogs. Analysis of these results suggested that treatment with a high dose of MPSS can suppress some of the major functions of PMNs for at least 7 days.

Effects of memantine on soluble Alphabeta(25-35)-induced changes in peptidergic and glial cells in Alzheimer's disease model rat brain regions.

Soluble forms of amyloid-beta (Abeta) have been considered responsible for cognitive dysfunction prior to senile plaque formation in Alzheimer's disease (AD). As its mechanism is not well understood, we examined the effects of repeated i.c.v. infusion of soluble Alphabeta(25-35) on peptidergic system and glial cells in the pathogenesis of AD. The present study aims to investigate the protective effects of memantine on Abeta(25-35)-induced changes in peptidergic and glial systems. Infusion of Alphabeta(25-35) decreased the level of immunoreactive somatostatin (SS) and substance P (SP) in the hippocampus prior to neuronal loss or caspase activation, which is correlated with the loss of spine density and activation of inducible nitric-oxide synthase (iNOS). Biochemical experiment with peptide-degrading enzymes, prolyl oligopeptidase (POP) and endopeptidase 24.15 (EP 24.15) activities demonstrated a concomitant increase with the activation of glial marker proteins, glial fibrillary acidic protein (GFAP) and CD11b in the Abeta-treated hippocampus. Double immunostaining experiments of EP 24.15 and GFAP/CD11b antibodies clearly demonstrated the co-localization of neuro peptidases with astrocytes and microglia. Treatment with memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist significantly attenuated Abeta(25-35)-induced changes of neuropeptides, their metabolizing enzymes, glial marker proteins, and activation of iNOS. Taken together, the data implies that memantine exerts its protective effects by modulating the neuropeptide system as a consequence of suppressing the glial cells and oxidative stress in AD model rat brain regions.

Gene expression profiling in anti-CD11d mAb-treated spinal cord-injured rats.

Acute administration of a mononclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. We have profiled gene expression in anti-CD11d and isotyped-matched control mAb-treated rats after SCI. Microarray analysis demonstrated reduced expression of pro-inflammatory cytokines and increased expression of inflammatory mediators that promote wound healing and the expression of scar proteins predicted to improve nerve growth. These changes in gene expression may reflect changes in the types of macrophages that populate the lesions in anti-CD11d mAb-treated rats.

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells.

The clinical efficacy and safety of realgar (arsenic sulfide, As(4)S(4)) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL-60 cells. Treatment with RNPs at 6 microM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP-induced differentiation in HL-60 cells.

Efalizumab for the treatment of psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis. Efalizumab, a T cell-targeted, recombinant human monoclonal antibody, is approved for the treatment of adult patients with chronic moderate to severe plaque psoriasis. The effect of efalizumab therapy on PsA has not previously been investigated. OBJECTIVE: This phase II randomized, double-blind, placebo-controlled multicenter study evaluated the efficacy and safety of efalizumab for the treatment of PsA. METHODS: Patients were required to be on at least one of the following concomitant systemic therapies for PsA: nonsteroidal anti-inflammatory drugs, corticosteroids, and/or sulfasalazine or methotrexate. One hundred fifteen patients with active PsA were enrolled and randomized in the study. Of these, 107 were treated weekly with efalizumab 1 mg/kg or placebo for 12 weeks, followed by 12 additional weeks of open-label efalizumab. RESULTS: At week 12, 28% of efalizumab-treated patients achieved ACR-20 response (a 20% reduction from the baseline in the American College of Rheumatology response criteria), the primary end point, compared with 19% of placebo patients (p = .27). The safety profile was comparable between efalizumab- and placebo-treated patient groups, regardless of methotrexate background therapy, and no worsening of joint disease occurred with efalizumab therapy. CONCLUSIONS: Efalizumab was not effective in treating PsA; efalizumab therapy did not worsen PsA. The efalizumab safety profile does not appear to be altered with the concomitant use of methotrexate therapy.

Comparison of effects of methylprednisolone and anti-CD11d antibody treatments on autonomic dysreflexia after spinal cord injury.

Autonomic dysreflexia is a condition of episodic hypertension that develops after spinal cord injury (SCI). We previously showed that a two-day anti-inflammatory treatment with an anti-CD11d integrin monoclonal antibody (mAb), soon after SCI in rats, reduced the magnitude of dysreflexia for at least 6 weeks. Effects of methylprednisolone (MP), a commonly used neuroprotective treatment for SCI, on dysreflexia have never been examined. We compared the effects of a 2-day MP treatment and/or the anti-CD11d mAb on autonomic dysreflexia, elicited by colon distension, after clip-compression SCI at the 4th thoracic segment (T4) in rats. We assessed the effects of each treatment on the size of the calcitonin gene-related peptide (CGRP)-immunoreactive afferent arbour in the dorsal horn, as changes in this arbour can correlate with the development of dysreflexia. MP reduced autonomic dysreflexia by approximately 50% at 2 weeks after SCI, but this effect was lost by 6 weeks. At 2 weeks, the combined effects of MP and the mAb were not additive, reducing dysreflexia by approximately 50%. Neither MP nor the mAb treatment altered the area of CGRP-immunoreactive fibres in the lumbar cord, the crucial input region for dysreflexia initiated by colon distension. However, both treatments led to increased fibre areas in the T9 segment, correlated with greater tissue integrity and smaller lesions, delineated by inflammatory cells. In summary, MP only temporarily decreases autonomic dysreflexia after SCI. The early beneficial effects of both treatments on dysreflexia do not relate to changes in the CGRP-immunoreactive afferent arbour but may correlate with decreased lesion progression.

Reduction of the multiple organ injury and dysfunction caused by endotoxemia in 5-lipoxygenase knockout mice and by the 5-lipoxygenase inhibitor zileuton.

The role of 5-lipoxygenase (5-LOX) in the pathophysiology of the organ injury/dysfunction caused by endotoxin is not known. Here, we investigate the effects of treatment with 5-LOX inhibitor zileuton in rats and targeted disruption of the 5-LOX gene in mice (5-LOX(-/-)) on multiple organ injury/dysfunction caused by severe endotoxemia. We also investigate the expression of beta2-integrins CD11a/CD18 and CD11b/CD18 on rat leukocytes by flow cytometry. Zileuton [3 mg/kg intravenously (i.v.)] or vehicle (10% dimethyl sulfoxide) was administered to rats 15 min prior to lipopolysaccharide (LPS; Escherichia coli, 6 mg/kg i.v.) or vehicle (saline). 5-LOX(-/-) mice and wild-type littermate controls were treated with LPS (E. coli, 20 mg/kg intraperitoneally) or vehicle (saline). Endotoxemia for 6 h in rats or 16 h in mice resulted in liver injury/dysfunction (increase in the serum levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, bilirubin), renal dysfunction (creatinine), and pancreatic injury (lipase, amylase). Absence of functional 5-LOX (zileuton treatment or targeted disruption of the 5-LOX gene) reduced the multiple organ injury/dysfunction caused by endotoxemia. Polymorphonuclear leukocyte infiltration (myeloperoxidase activity) in the lung and ileum as well as pulmonary injury (histology) were markedly reduced in 5-LOX(-/-) mice. Zileuton also reduced the LPS-induced expression of CD11b/CD18 on rat leukocytes. We propose that endogenous 5-LOX metabolites enhance the degree of multiple organ injury/dysfunction caused by severe endotoxemia by promoting the expression of the adhesion molecule CD11b/CD18 and that inhibitors of 5-LOX may be useful in the therapy of the organ injury/dysfunction associated with endotoxic shock.

Pharmacological hyperprolactinemia attenuates hydrocortisone-induced expression of CD11b on human CD8+ cells in vivo.

OBJECTIVE: To study the short-term influences of pharmacologic hyperprolactinemia on hydrocortisone (HC)-induced effects on selected immune parameters. METHODS: A single dose of HC (40 mg per os) was administered to eleven healthy female volunteers 1 h after domperidone (10 mg per os) or placebo administration. Immune cell subsets and expression of adhesion molecules was assessed by flow cytometry at baseline and 4 and 6 h after HC administration. Intracellular staining of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) production in CD4+ lymphocytes after phorbol myristate acetate and ionomycin stimulation was performed at the same time points. RESULTS: HC administration was followed by a significant increase in cortisol levels, numbers of leukocytes and granulocytes and the percentage of CD16+, CD19+, CD11a+, CD11a+CD8+, CD11b+ and CD11b+CD8+ cells. The number of lymphocytes and monocytes and the percentage of CD3+, CD4+, CD4+/CD8+ ratio, CD62L+, CD54+ and CD54+CD16+ cells decreased, while the percentage of CD8+ cells was unaffected. Domperidone administration resulted in a significant increase in prolactin (PRL) concentrations. During hyperprolactinemia, the HC-induced increase in CD11b+CD8+ cells was significantly (p < 0.05) attenuated at 4 h. HC-induced changes in other immune parameters remained unaffected. No significant changes in the intracellular production of IL-4 and IFN-gamma in CD4+ lymphocytes were observed after a single dose of HC alone or during hyperprolactinemia. CONCLUSIONS: This study shows an attenuated HC-induced increase in CD11b+CD8+ cells in the peripheral blood of healthy females during hyperprolactinemia. Our in vivo observations suggest that short-term interactions occur between PRL and glucocorticoids, affecting selected immune functions. Further studies are needed for confirmation of these results.

Sulfonated human immunoglobulin enhances CD16-linked CD11b expression on human neutrophils.

Intravenous human immunoglobulin therapy infrequently results in excessive inflammatory responses in vivo; these effects are not fully understood. We assessed whether sulfonated human immunoglobulin (SHIG) or polyethylene glycol-treated human immunoglobulin (PHIG) enhanced expression of inflammatory receptors on peripheral blood neutrophils in vitro, such as alphaMbeta2 (CD11b/CD18) and Fc gamma receptor type III (FcgammaRIII). CD11b and CD16 expression on neutrophils was measured by fluorescence flow cytometry. Various cytokines were assessed using a highly sensitive fluorescence microsphere system. SHIG enhanced/induced CD11b expression and partial aggregations on neutrophils, but PHIG did not. No detection of aggregation IgG was observed in SHIG and PHIG. SHIG-induced CD11b expression was inhibited by treatment of corticosteroid (dexamethasone) and by anti-CD16 monoclonal antibody. Concentrations of various cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, RANTES, tumor necrosis factor (TNF)-alpha, and interferon (INF)-gamma in culture supernatant were not significantly changed by SHIG or PHIG. SHIG and PHIG did not enhance CD16 on neutrophils. SHIG enhanced CD16-linked CD11b expression on neutrophils in vitro. CD11b induction was inhibited by dexamethasone and by anti-CD16 antibody. These in vitro results suggest that aggregations and enhancement of CD11b on neutrophils by SHIG may induce excessive inflammatory responses in vivo.

Efaluzimab in the treatment of psoriasis.

We present a review of the literature concerning treatment of psoriasis with humanized monoclonal antibody (hu 1124, efaluzimab, Xanelin) against the CD11a component of lymphocyte-function-associated antigen-1 (LFA-1). Efaluzimab inhibits the interaction of CD11a (LFA-1) with various ICAM molecules. Because ICAM-1 (CD54) is expressed on activated endothelial cells and antigen presenting cells (APCs), the antibody inhibits both the APC-T cell interaction and the T- cell adhesion to endothelial cells, their subsequent activation, which results in decreasing of transendothelial migration. Treatment with Efaluzimab was well tolerated and the majority adverse events were dose-related. Adverse events were described as mild at doses of 0.3 mg/kg or less and included mild chills, abdominal discomfort, headache, and fever (flu-like complaints), apart from this white blood cell counts and lymphocyte counts transient increase were observed. Headache was the most common dose-limiting toxicity observed at a single dose of 0.6 mg/kg or higher.

Psoriasis as a model for T-cell-mediated disease: immunobiologic and clinical effects of treatment with multiple doses of efalizumab, an anti-CD11a antibody.

BACKGROUND: Leukocyte function-associated antigen 1 (LFA-1), consisting of CD11a and CD18 subunits, plays an important role in T-cell activation and leukocyte extravasation. OBJECTIVE: To test whether blocking CD11a decreases immunobiologic and clinical activity in psoriatic plaques. DESIGN: Open-label, multicenter, dose escalation study. PATIENTS: Thirty-nine patients with moderate-to-severe psoriasis. INTERVENTION: Intravenous infusions of efalizumab, a humanized anti-CD11a monoclonal antibody, for 7 weeks at doses of 0.1 mg/kg every other week or 0.1 mg/kg weekly (category 1), 0.3 mg/kg weekly (category 2), and 0.3 increasing to 0.6 or 1.0 mg/kg weekly (category 3). Skin biopsies were performed on days 0, 28, and 56. MAIN OUTCOME MEASURES: Serum efalizumab levels, levels of total and unoccupied T-cell CD11a, T cell counts, epidermal thickness, cutaneous intercellular adhesion molecule 1 (ICAM-1) and keratin 16 (K16) expression, Psoriasis Area and Severity Index (PASI) scores. RESULTS: Dose-response relationships were observed for pharmacokinetics and pharmacodynamic measures. Category 1 failed to maintain detectable serum efalizumab or T cell CD11a down-modulation between doses. Category 2 achieved both. Category 3 achieved both and additionally maintained sustained T-cell CD11a saturation between doses. A dose-response relationship was also observed clinically and histologically. The mean decrease in the PASI score was 47% in category 3, 45% in category 2, and 10% in category 1 (P<.001). Epidermal and dermal T-cell counts, epidermal thickness, and ICAM-1 and K16 expression decreased in categories 2 and 3 but not in category 1. Circulating lymphocyte counts increased in categories 2 and 3. CONCLUSIONS: At doses of 0.3 mg/kg or more per week, intravenous efalizumab produced significant clinical and histologic improvement in psoriasis, which correlated with sustained serum efalizumab levels and T-cell CD11a saturation and down-modulation.

Impaired sensitivity to beta 2 integrin-blocking in ICAM-1-mediated neutrophil migration in ulcerative colitis.

BACKGROUND: Factors influencing the directed migration of neutrophils into colonic tissue in ulcerative colitis (UC) are poorly described. ICAM-1 has recently been shown to possess chemotactic properties, and the aim of this study was to evaluate the involvement of beta 2 integrins in this ICAM-1-mediated migration. METHODS: The chemotactic effect of ICAM-1 on neutrophils isolated from 13 UC patients and 17 healthy volunteers was studied in microchemotaxis chambers. Physiological concentrations of ICAM-1 (0.05-500 pM) were separated from neutrophils by nitrocellulose filters, and cell migration was evaluated using the leading front technique. beta 2 integrins on neutrophils were blocked with antibodies to CD11a, CD11b, CD11c and CD18, and migration towards ICAM-1 was examined. RESULTS: Migration towards ICAM-1 was equal for UC and control neutrophils, showing a bell-shaped ICAM-1 dosemigratory response curve with peak migration at 5 pM ICAM-1 (30.0 microns; interquartile range 22.9-35.7; P < 0.001). Blockade of the CD11 subunits on control cells inhibited the chemoattractant effect of ICAM-1 by 43.6%-58.0%, whereas the migration was decreased by only 20% in UC under similar blocking conditions (P < 0.01). Anti-CD18 mAbs had no effect. Inhibition of protein kinases with staurosporin only slightly decreased the ICAM-1-mediated migration, whereas incubation with staurosporin and CD11 antibodies showed additive effects on UC neutrophils and synergistic effects on control cells. No quantitative differences in beta 2 integrin expression were detected between control and UC neutrophils. CONCLUSIONS: The chemotactic property of ICAM-1 was shown to be CD11-dependent and UC neutrophils were found to be less dependent on CD11/ICAM-1-mediated migration than were control neutrophils.

Abrogation of surgery-induced decline in circulating dendritic cells by subcutaneous preoperative administration of IL-2 in operable cancer patients.

Surgery-induced immunosuppression is characterized by a decline in lymphocyte count, particularly T lymphocyte number. In addition, preliminary studies have shown that the postoperative period is also characterized by a decline in the number of circulating dendritic cells (DC), whose fundamental anticancer role has been recently demonstrated. Previous studies had already shown that the preoperative injection of IL-2 may completely abrogate surgery-induced lymphocytopenia, whereas its eventual influence on DC system during the perioperative period is still unknown. The present study was performed to evaluate the influence of IL-2 preoperative immunotherapy on the perioperative changes in circulating DC number in patients affected by colorectal cancer. The study included 14 consecutive patients, who were randomized to be treated with or without IL-2 presurgical immunotherapy (12 million IU/day for 3 days subcutaneously). Circulating immature and mature cells were evaluated before surgery and at days 3 and 7 of the postoperative period. The detection was made by FACS using monoclonal antibodies against CD123 and CD11c to recognize immature and mature DC, respectively. Surgery induced a significant decline in the mean number of both immature and mature DC. The pre-surgical administration of IL-2 completely abrogated surgery-induced decline in immature DC cell amount. Moreover, mature DC mean number was diminished only at day 3 of the postoperative period, since the value observed at day 7 was not significantly lower than that found before surgery. This preliminary study shows that surgery-induced immunosuppression is characterized also by a significant decline in the mean number of both immature and mature DC. Moreover, this study would suggest that the preoperative immunotherapy with IL-2 may counteract surgery-induced failure of DC system. Because of the fundamental antitumor role of DC, this evidence could have a prognostic impact on the clinical course of the neoplastic disease.

Interleukin-8 and leukotriene-B(4), but not formylmethionyl leucylphenylalanine, stimulate CD18-independent migration of neutrophils across human pulmonary endothelial cells in vitro.

Although neutrophil migration from the systemic circulation involves the beta2- (or CD18) integrin family, the existence of an alternative, CD18-independent route of neutrophil extravasation to tissues has been demonstrated in animal models. The molecular interactions involved in this alternative migratory route have not yet been characterized. The objective of this study was to assess the CD18-dependency of neutrophil migration across human endothelial cells from an organ known to support CD18-independent migration, the lung, with a view to establishing an in vitro model to facilitate study of CD18-independent migration. Neutrophil migration across human pulmonary artery endothelial cells (HPAECs) in response to three different chemoattractants, formylmethionyl leucylphenyl-alanine (FMLP), interleukin (IL)-8, and leukotriene (LT) B(4), was examined. Results demonstrated that a function-blocking antibody to CD18 decreased FMLP-stimulated migration by 71.7 +/- 4.4% (P < 0.001). In contrast, migration in response to LTB(4) was decreased by only 20.5 +/- 10.2% (P < 0.01), and no significant decrease was observed with migration to IL-8. Neutrophils that migrated to FMLP had 1.7-fold more surface CD11b/CD18 compared with nonmigrated neutrophils (P < 0.01), whereas this integrin complex was not significantly upregulated on neutrophils that had migrated to IL-8 or LTB(4). Further investigation of this migratory route indicated that it did not involve the beta1 integrins (CD29) or the endothelial selectins, E- or P-selectin, nor did it require the activity of either metalloproteinases or neutrophil elastase. These results indicate that neutrophil migration across HPAECs in vitro to IL-8 and LTB(4) is predominantly CD18-independent and provides a much-needed in vitro system for examination of the neutrophil-endothelial interactions involved in this alternative migratory route.

Comparison of the distribution of progenitor cells in G-CSF-mobilized peripheral blood and steady-state bone marrow after counterflow centrifugal elutriation.

Blood-derived progenitor cells obtained following mobilization with granulocyte colony-stimulating factor (MoPBSC) are increasingly being used as an alternative to bone marrow (BM) in allogeneic stem cell transplantation. The higher numbers of mature T lymphocytes in MoPBSC grafts may increase the risk of (chronic) graft-vs.-host disease. Counterflow centrifugal elutriation (CCE) is an effective method for T-cell depletion of BM grafts. The elutriation characteristics of steady-state BM and MoPBSC were compared using a CCE procedure in which fractions were obtained after small incremental increases in flow rate with constant centrifugal force. Counterflow centrifugal elutriation experiments with MoPBSC from six healthy volunteers showed that 54% of all cells collected were recovered in the < or = 15 mL/minute fractions, whereas experiments with mononuclear BM cells from five healthy volunteers resulted in recovery of 52% of collected cells from the > or = 19 mL/minute fractions. The peak concentrations of CD34+ cells were found in the same fraction (18 mL/minute), but more CD34+ cells from MoPBSC were recovered from the small (< or = 16 mL/minute) fractions (54% for MoPBSC, 26% for BM; p = 0.08). The small CD34+ cells from BM were more frequently lacking CD38 and human leucocyte antigen-DR expression than the small CD34+ cells from MoPBSC. Mature T-cells (CD3+) in BM and MoPBSC samples had similar CCE features, as did early (long-term culture initiating cells, high-proliferative potential colony-forming cells) and more mature (colony-forming units granulocyte/macrophage, BFU-e) hematopoietic progenitor cells. The results of this study suggest that T-cell depletion by CCE of MoPBSC as compared to BM products, may lead to a greater loss of CD34+ cells, but not of immature hematopoietic progenitor cells.

Role of C3 cleavage in monocyte activation during extracorporeal circulation.

BACKGROUND: We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation. METHODS AND RESULTS: SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 micrograms/mL and 4 experiments at 30 micrograms/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation-dependent monocyte-platelet conjugates. CONCLUSIONS: Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

Pharmacokinetics and pharmacodynamics of the leukotriene B4 receptor antagonist CP-105,696 in man following single oral administration.

AIMS: CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxy-chroman-7-yl] cyclopropane carboxylic acid is a potent, novel LTB4 receptor antagonist advanced to clinical trials to determine its efficacy in inflammatory diseases. The pharmacokinetics and pharmacodynamics of CP-105,696 were investigated in healthy male volunteers following oral administration of single doses of 5 to 640 mg. METHODS: Forty-eight subjects participated in a randomized, double-blind, parallel group study. Plasma and urine concentrations of CP-105,696 were determined at intervals after drug administration. As an indication of LTB4 receptor antagonism following oral administration of CP-105,696, the inhibiton of LTB4-induced upregulation of the neutrophil cell surface complement receptor (CR3), CD11b/CD18, was monitored at 4 h following drug administration using an ex vivo whole blood flow cytometry assay. RESULTS: Cmax and AUC(0, infinity) increased in a dose-related manner. Respective mean Cmax values were 0.54 to 30.41 microg ml(-1) following doses of 5 to 640 mg. Respective mean AUC(0, infinity) values were 1337 to 16819 microg ml(-1) h for the 40 to 640 mg dose groups. Plasma concentrations declined in a monoexponential manner, with terminal elimination half-lives ranging from 289 to 495 h. Group mean terminal elimination half-lives were dose-independent. Urinary excretion of unchanged drug accounted for < 1% of the administered dose. A linear relationship was observed between CP-105,696 plasma concentrations and inhibition of LTB4-mediated CD11b upregulation on human neutrophils in whole blood. CP-105,696 plasma concentrations of 5-6 microg ml(-1) were necessary to elicit a two-fold shift to the right of the LTB4 concentration response curve for CD11b upregulation. CONCLUSIONS: These studies demonstrate pharmacologically significant LTB4-receptor antagonism following a single dose of CP-105,696 and pharmacokinetics consistent with once-daily dosing.

The effect of the bacterial product, succinic acid, on neutrophil bactericidal activity.

We investigated the effect of succinic acid on neutrophil bactericidal activity in a model of intra-abdominal abscess induced in mice by the peritoneal inoculation of 5 x 10(6) cfu ml-1 E. coli and 5 x 10(8) cfu ml-1 B. fragilis plus 1 mg of bran as faecal fibre analogue. The mean pH of the induced abscesses at week 1 was 6.7, higher than the pH associated with succinic acid inhibitory activity. We therefore determined the effect of succinic acid (0-100 mM) at pH 6.7 on the bactericidal activity of mouse bone marrow-derived neutrophils. Phagocytic killing of Proteus mirabilis by neutrophils was significantly inhibited by 30-100 mM succinic acid at pH 6.7 but there was no significant effect of succinic acid on engulfment of bacteria at this pH. However, significant inhibition of intracellular killing (assayed by adding succinic acid to suspensions of neutrophils which had engulfed bacteria in low serum concentrations but in the absence of succinic acid) was noted at 70 and 100 mM. These results indicate that succinic acid inhibits neutrophil bactericidal activity at a physiological pH, principally through inhibition of intracellular killing mechanisms and therefore contributing to bacterial persistence in this model of abscess formation.