In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization.

OBJECTIVE: To explore the use of ß-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized ß-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized ß-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to ß-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized ß-lactoglobulin. CONCLUSIONS: The polymerization of ß-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

Rosmarinus officinalis L. essential oil inhibits in vivo and in vitro leukocyte migration.

Rosmarinus officinalis L. (Lamiaceae), popularly known as rosemary, is used for food flavoring and in folk medicine as an antispasmodic, analgesic, antirheumatic, diuretic, and antiepileptic agent. Few studies have shown the anti-inflammatory effects of rosemary essential oil (REO). This study evaluated the effects of REO on leukocyte migration through in vivo leukocyte migration and in vitro chemotaxis assay. REO was analyzed by using gas chromatography-mass spectometry, and the main components identified were camphor (27.59%), 1,8-cineole (15.74%), α-pinene (16.58%), and ß-myrcene (10.02%). In rats, administration of REO reduced the number of leukocytes that rolled, adhered, and migrated to the scrotal chamber after carrageenan injection. All doses of REO tested significantly inhibited leukocyte chemotaxis induced by casein. The effects of REO on leukocyte migration highlight an important mechanism of the anti-inflammatory action of rosemary.

The comparison of methods for determination of humoral and cell-mediated immunity in patients with breast cancer.

We observed the immunological answer to antigens obtained from the human malignant breast tumor and from the blood of inbred mice strain C3H/H2K infected by LDH virus. We compared the modified ELISA method for humoral immunity with the leukocyte adherence inhibition (LAI) assay for cell-mediated immunity. The modified ELISA method is suitable for early diagnosing and monitoring antibodies in a malignant breast tumor simultaneously with senological examinations which include mammography and clinical examinations, because the antibodies are determined in a high number of samples by single application.

The basophil activation test in immediate drug allergy.

Diagnosis of drug allergy is not always straight forward for several reasons. First, a broad spectrum of drugs can elicit various immune-mediated diseases with distinct pathomechanism, secondly, although exact epitope identification is not mandatory for clinical diagnosis, the epitope that causes the reaction is frequently unknown, thirdly in vitro or in vivo test results might not be predictive of a clinical situation, and finally the gold standard or reference test for diagnosis, the drug challenge, is a complicated and sometimes dangerous endeavour. Upon challenge with specific allergens that cross-link membrane-bound IgE antibodies, basophils upregulate the expression of different activation markers such as CD63 and CD203c. These immunophenotypic alterations can be detected on a single-cell basis by multicolour flow cytometry using specific monoclonal antibodies in the basophil activation test (BAT). This review intends to summarise our current experience with the BAT in the diagnostic management of immediate-type allergy to drugs and related compounds that are generally (but not always) mediated by drug-specific IgE antibodies.

The direct thrombin inhibitor melagatran counteracts endotoxin-induced endothelial leukocyte adherence and microvascular leakage in the rat mesentery. Rationale for the treatment of inflammatory disorders beyond sepsis?

The activation of the coagulation system in the course of an inflammatory reaction impairs the function of the microcirculation. By means of intravital videomicroscopy the effect of the direct thrombin inhibitor melagatran on endotoxin-induced microvascular permeability and leukocyte adhesion to microvascular endothelium of rat mesentery was evaluated. Secondly, plasma concentrations of melagatran or interleukin-6 in response to endotoxin or after treatment with melagatran respectively, were determined. Male Sprague-Dawley rats (300-400 g bw) were infused with 0.5 mg/kg lipopolysaccharide (LPS) (E. coli O55:B5) over 80 minutes. Vascular leakage was detected with FITC-marked rat serum albumin by fluorescence microscopy and evaluated by grey value analysis with a computer assisted image processing system. Light microscopy was used to evaluate the adherence of leukocytes to the vessel wall. Two treated groups received either 0.3 or 0.6 mg/kg bw melagatran iv in addition to LPS-infusion. The observation period was 3 hours after the beginning of LPS infusion. Groups of animals not infused with LPS or solely treated with melagatran (0.3 or 0.6 mg/kg) served as controls. Infusion of LPS led to a significant increase of microvascular permeability, leukocyte adherence and thrombin-antithrombin complex plasma concentration compared to unstimulated controls. These effects were significantly reduced by melagatran at both dosage levels. Elevated plasma concentrations of melagatran were observed in animals infused with endotoxin and higher plasma levels of interleukin-6 were found in endotoxemic animals treated with melagatran. The results indicate that thrombin is one of the most important clotting enzymes involved in inflammatory microvascular disturbance. Moreover, it should be clarified whether direct thrombin inhibitors themselves play a role within the immune response to endotoxin.

Polyethylene glycol and a novel developed polyethylene glycol-nitric oxide normalize arteriolar response and oxidative stress in ischemia-reperfusion.

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion (P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.

Homocysteine triggers mucosal microvascular activation in inflammatory bowel disease.

OBJECTIVES: Increased homocysteine contributes to the pathophysiology of several chronic inflammatory diseases. Whether homocysteine could participate in mucosal inflammation in inflammatory bowel disease (IBD) has not been explored yet. Our aims were to study the levels of plasma and mucosal homocysteine in IBD patients and to assess whether homocysteine can trigger an inflammatory reaction on human intestinal microvascular endothelial cells (HIMECs). METHODS: Homocysteine was measured in the plasma, mucosal biopsy, and lamina propria mononuclear cell (LPMC) supernatants from normal and IBD subjects. HIMEC were cultured in presence of homocysteine, TNF-alpha, or folic acid, alone or in combination. Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular cell adhesion molecule 1 was measured by flow cytometry and monocyte chemoattractant protein-1 (MCP-1) production by ELISA. Phosphorylation of p38 and p42/44 was assessed by immunoblot in HIMEC extracts. T-cell- and monocyte-HIMEC adhesion assays were used to evaluate the impact of homocysteine on leukocyte adhesion to intestinal endothelial cells. RESULTS: Patients with IBD displayed significantly higher homocysteine plasma and mucosal levels than control subjects. IBD-derived LPMC released higher homocysteine than control-derived LPMC. Treatment of HIMEC with homocysteine, and synergistically with the combination of TNF-alpha and homocysteine, triggered HIMEC inflammation, resulting in VCAM-1 up-regulation, MCP-1 production, and p38 phosphorylation. These events lead to an increased capacity of HIMEC to adhere T- and monocyte cells and were blocked by folic acid treatment. CONCLUSIONS: Homocysteine is increased in both the mucosa and plasma of patients with Crohn's disease and ulcerative colitis and contributes to the inflammatory state of the mucosal IBD endothelium. Therefore, homocysteine could play a proinflammatory role in IBD, which can be efficiently targeted by folic acid supplementation.

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia.

OBJECTIVE: To characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DESIGN: Animal study using intravital microscopy. SETTING: Animal research facility. SUBJECTS: Male Syrian golden hamsters, 6-8 wks old with a body weight of 60-80 g. INTERVENTIONS: In skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 microg/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p <.01 vs. baseline). Activated protein C treatment inhibited (p <.05) lipopolysaccharide-mediated leukocytic response and attenuated (p <.05) endotoxic perfusion failure in nutritive capillaries. CONCLUSIONS: Activated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.

Dehydroepiandrosterone protects the microcirculation of muscle flaps from ischemia-reperfusion injury by reducing the expression of adhesion molecules.

Adhesion molecules contribute to ischemia-reperfusion injury by increasing the endothelial adhesion and extravasation of leukocytes. Scientific evidence suggests that presurgical treatment with dehydroepiandrosterone may protect the microvasculature against this damage, but the exact mechanism is not known. The purpose of this study was to investigate the effects of presurgical dehydroepiandrosterone treatment on microcirculatory hemodynamic parameters and the expression of adhesion molecules in a rat cremaster muscle flap model. Twenty male rats were randomly assigned to three experimental groups. In group I (n = 5), the muscle flaps did not receive presurgical treatment. In group II (n = 6), propylene glycol (30 mg/kg), the vehicle for dehydroepiandrosterone, was injected intravenously before ischemia was induced. In group III (n = 9), dehydroepiandrosterone (30 mg/kg) was injected intravenously before ischemia was induced. All flaps were subjected to 6 hours of ischemia and 90 minutes of reperfusion. Microcirculatory variables (functional capillary density, red blood cell velocity in the main flap arteriole, and numbers of rolling, sticking, and transmigrating leukocytes), blood levels of three adhesion molecules (L-selectin, Mac-1 integrin, and CD44), and the numbers of leukocytes expressing those molecules were analyzed. Analysis of the microcirculatory parameters revealed that dehydroepiandrosterone treatment before ischemia had significant preservative effects on the red blood cell velocity and functional capillary density 30 and 90 minutes after reperfusion, compared with the control and vehicle-treated groups. Leukocyte-endothelial cell interactions were also affected by dehydroepiandrosterone treatment, as reflected by significant decreases in the numbers of sticking and transmigrating leukocytes 30 and 90 minutes after reperfusion. In dehydroepiandrosterone-treated animals, leukocytes exhibited lower levels of expression of adhesion molecules after the onset of ischemia, compared with the control groups. In this study, intravenous dehydroepiandrosterone administration reduced the activation of leukocytes and improved red blood cell velocity and capillary perfusion in the muscle flap microcirculation during ischemia-reperfusion injury. This protective effect was most likely the result of delayed expression of Mac-1 integrin, L-selectin, and CD44 molecules on leukocytes.

SK&F 95654-induced acute cardiovascular toxicity in Sprague-Dawley rats--histopathologic, electron microscopic, and immunohistochemical studies.

The characteristics and pathogenesis of the cardiovascular toxicity induced by the type III selective phosphodiesterase inhibitor SK&F 95654 were examined in 2 studies. Sprague-Dawley rats received either a single sc injection of 50, 100, or 200 mg/kg SK&F 95654 and were euthanized at 24 hours after administration of the drug (Study 1), or were given a single subcutaneous (sc) injection of 100 mg/kg SK&F 95654 and euthanized at 1, 2, 4, 6, 8,12, 24 hours, or 2 weeks after treatment (Study 2). Control rats received either DMSO or saline. Myocardial lesions and vascular lesions of the mesentery, spleen, and pancreas were seen 24 hours after dosing with either 50,100, or 200 mg/kg SK&F 95654. The frequency and severity of these lesions (evaluated after the 100 mg/kg dose) increased with time over a period of 1 to 24 hours. By 2 weeks, the lesions subsided. Cardiac lesions consisted of myocyte necrosis with hypercontraction bands, inflammatory cell infiltration, interstitial hemorrhage, and interstitial edema. Vascular lesions of the mesentery were most prominent and consisted of vasodilatation and inflammation in the small-sized vessels, arterial medial necrosis and hemorrhage, and venous thrombosis. The vascular lesions included: leukocyte adhesion to endothelial cells, transendothelial migration of leukocytes, and inflammatory cell infiltration into vessel walls. Affected vessels included arteries, terminal arterioles, capillaries, postcapillary venules, and veins. Apoptosis of endothelial and smooth muscle cells was detected in the mesenteric vasculature by both TUNEL assay and electron microscopy. Evidence of endothelial cell activation in the mesenteric arteries and veins was also observed by electron microscopy. Immunohistochemical staining detected enhanced endothelial cell expression of intercellular adhesion molecule- 1 (ICAM- 1) and von Willebrand factor (vWF) in the mesenteric arteries and veins. Mast cells were noted to be more prevalent in affected mesenteric tissue from drug-treated animals. The present findings suggest that apoptosis of endothelial and smooth muscle cells, activation of endothelial cells, recruitment of mast cells, and increased expression of adhesion molecules are important factors to the overall pathogenesis of SK&F 95654-induced vasculitis.

Método citoquímico para evaluar adherencia en leucocitos polimorfonucleares neutrófilos / Cytochemical method for evaluating adherence in polymorphonuclear neutrophil leucocytes

Se desarrolló un método citoquímico cuantitativo y sencillo para evaluar adherencia en leucocitos polimorfonucleares neutrófilos. Las células fueron aisladas por gradiente de densidad a partir de sangre periférica de 47 donantes voluntarios que acudieron al Servicio de Transfusiones del Instituto Superior de Medicina Militar "Dr. Luis Díaz Soto". La cualidad adhesiva se amplificó mediante la determinación de proteínas totales por el método de Lowry, luego de incubar los fagocitos en placas plásticas de cultivo de 24 pozos. El mayor número de valores se encontró entre 6 y 10 mg/dL, lo que representó el 70 porciento de los sujetos evaluados. La combinación de la placa de cultivo como superficie de adhesión y la determinación de proteínas como forma de amplificar esta cualidad, constituye una novedad del método propuesto

Effect of the calcium phosphate-mediated RNA uptake on the transfer of cellular immunity of a synthetic peptide of HIV-1 to human lymphocytes by exogenous RNA.

It is known that exogenous RNA molecules can be taken up by eukaryotic cells and can exert a variety of biological effects both in vitro and in vivo. The modulation of human lymphocytes by exogenous RNAs has medical implications. The exogenous RNA used in this study was obtained from lymphoid organs of animals immunized with the synthetic peptide p12 of HIV-1 and was referred to as p12-RNA. Human lymphocytes were transfected with the p12-RNA and the transfer of immunoreactivity of p12 was assessed by the lymphocyte proliferation and the leukocyte adherence inhibition assays. Our results indicate that the transfer of cellular immune response to the p12 occurred in 9 donors (60%) who were named responsive individuals whereas 6 donors (40%) were non-responsives. We also found that the calcium phosphate-mediated RNA uptake method is effective in converting non-responsive into responsive donors. The calcium phosphate-mediated RNA uptake may also be used to increase the efficiency of RNA transfection in other models with medical implications and to contribute to a better understanding of the molecular events involved in the uptake of RNA. Our findings give support for the use of exogenous RNAs obtained from lymphoid organs of immunized animals with synthetic peptides of HIV-1 in the immune reconstitution of individuals infected with HIV-1.

Leukocyte adhesiveness/aggregation test (LAAT) to discriminate between viral and bacterial infections in children.

OBJECTIVES: We previously noted that white blood cells (WBC) have increased adhesive properties during bacterial infections. Here, we aim to explore the possibility of using the different adhesive properties of WBC as a means of differentiating between viral and bacterial infections, a common problem in paediatrics. METHODS: The adhesive properties of WBC in the peripheral blood of 25 children with documented bacterial infections, 15 with documented viral infections and 36 with probable viral infections, were studied by means of a leukocyte adhesiveness/aggregation slide test (LAAT). The results of the LAAT were compared with those of the other acute phase reactants, namely WBC, differential count and erythrocyte sedimentation rate (ESR), which were taken in the same blood sample in each patient. RESULTS: The sensitivity, specificity and positive predictive value were 92%, 96%, and 92%, respectively for the LAAT; 83%, 87% and 80% for the ESR; 56%, 78% and 56% for the white blood cell count; and 54%, 74% and 50% for the differential count. CONCLUSIONS: The presence of bacterial infections in children can be tested using a simple slide test to reveal the increased state of leukocyte adhesiveness/aggregation in the peripheral blood. The LAAT is a reliable, rapid and inexpensive test, and it can be a useful laboratory tool for the paediatrician treating a child with acute febrile illness.

Cutting edge: a small molecule antagonist of LFA-1-mediated cell adhesion.

LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3, 5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.

Acquired increase in leucocyte binding by intestinal microvascular endothelium in inflammatory bowel disease.

BACKGROUND: Endothelial cells that line microvascular blood vessels have an important role in inflammation through their ability to bind and recruit circulating leucocytes. Endothelial cells from the intestines of patients with chronically inflamed Crohn's disease and ulcerative colitis--the two forms of inflammatory bowel disease--display an increased leucocyte-binding capacity in vitro. We investigated whether this enhanced leucocyte binding is a primary or an acquired defect. METHODS: We cultured human intestinal microvascular endothelial cells (HIMEC) from the uninvolved intestine and chronically inflamed bowel of three patients with inflammatory bowel disease (two Crohn's disease, one ulcerative colitis). We assessed HIMEC binding to polymorphonuclear leucocytes and U937 cells by means of an adhesion assay. FINDINGS: After activation with interleukin-1beta or lipopolysaccharide, HIMEC from the chronically inflamed tissue in all three patients with inflammatory bowel disease bound twice as many polymorphonuclear leucocytes and U937 cells as endothelial cells from uninvolved tissue. INTERPRETATION: Enhanced leucocyte binding by HIMEC from chronically inflamed tissue in patients with inflammatory bowel disease is an acquired defect since it is not found in the uninvolved intestinal segments from the same individuals. Because interaction between endothelial cells and leucocytes is a key regulatory step in the inflammatory process, this enhanced binding may contribute to the pathophysiology of chronic intestinal inflammation.

Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium.

A clinically available mixture of hydroxyethylrutosides (HR) was examined as a protector against endothelial cell activation by hypoxia in perfused human umbilical vein. The results showed that 500 micrograms/mL HR totally inhibited the adherence of human unstimulated neutrophils to the endothelium of umbilical vein incubated in hypoxic conditions. This inhibition was confirmed by a morphological study performed by scanning electron microscopy. In addition, neutrophils adherent to the hypoxic umbilical vein endothelium became activated, as evidence by the increased release of superoxide anions and synthesis of leukotriene B4. These processes could also be inhibited by HR. In conclusion, the results of this study suggest that the improvement in venous insufficiency observed clinically with HR could, in part, be the result of their ability to inhibit the recruitment and activation of neutrophils by endothelium activated during blood stasis.

Role of selectin-dependent adhesion in cardiac allograft rejection.

BACKGROUND: Selectins play important roles in the inflammatory responses by eliciting leukocyte rolling. The roles of E- and P-selectins in the acute rejection of cardiac allografts remain unclear. This study was designed to evaluate whether E- and P-selectins participate in the pathophysiology of heart rejection. METHODS: Heterotopic heart transplantation was performed in both mice and rats in full histoincompatibility combinations. Immunohistochemistry, flow-cytometry, and reverse transcriptase- polymerase chain reaction were performed to evaluate E-, P-selectin and sialyl Lewis X (SLeX) expression in rejecting cardiac allografts. The effects of short-term administration of monoclonal antibodies (mAbs) to E- and P-selectins on cardiac allograft survival were also evaluated. RESULTS: Significant prolongation of graft survival was observed in mice treated with either anti-E- or P-selectin mAbs, or both. The enhanced endothelial and mRNA expression of E- and P-selectins was observed in the rejecting cardiac allografts. Some graft- infiltrating mononuclear cells were double-stained with both anti-SLeX and anti-alphabetaT cell receptor mAbs. Flow-cytometric analysis of graft-infiltrating cells also showed enhanced SLeX expression. CONCLUSION: These results suggest that both P- and E-selectins are critically involved in the early development of acute heart rejection.

Nitric oxide inhibits neutrophil adhesion during experimental extracorporeal circulation.

BACKGROUND: Myocardial and pulmonary injuries often occur after cardiopulmonary bypass, mediated in part by neutrophil activation and adhesion to endothelial cells. The effects of nitric oxide (NO) administration on neutrophil adhesion to endothelial cells after simulated extracorporeal circulation were investigated. METHODS: Two identical extracorporeal circulation circuits were primed with fresh human blood and circulated for 2 h at 37 degrees C. Nitric oxide at a 40-ppm concentration was added to one of the oxygenators in each pair. Neutrophil CD11b/CD18 expression and their adhesion to human umbilical vein endothelial cell monolayers were assayed in leukocytes isolated from samples drawn from the circuit 30, 60, 90, and 120 min after circulation began. In another series of experiments, blocking monoclonal antibodies to both neutrophil CD11b and CD18 were incubated with polymorphonuclear leukocytes after removal from the circuit before the adhesion assay. RESULTS: After 60 min of circulation, the neutrophils from NO-treated circuits showed significantly reduced CD11b/CD18 surface expression compared with the control group. There was also a significant reduction in neutrophil-endothelial adhesion in the NO group after 120 min of circulation. Monoclonal antibodies to both CD11b and CD18 significantly inhibited the adhesion of polymorphonuclear leukocytes at endothelial cells after 120 min of circulation. CONCLUSIONS: These results confirm that neutrophil activation occurs during cardiopulmonary bypass. The addition of NO to the circuits of extracorporeal circulation significantly affects neutrophil adhesion to endothelial cells.