Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36.

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.

Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells.

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

Effects of corticosterone and microgravity on inflammatory cell production of superoxide.

In this investigation we studied the effects of corticosterone and microgravity on Propionibacterium acnes-induced inflammatory cells ability to produce superoxide (O2-). We found in vitro and in vivo exposure of murine peritoneal inflammatory cells to corticosterone did not inhibit the O2- response. We also found that in microgravity P. acnes-induced inflammatory cells were capable of producing four times as much O2- as at 1g. Therefore, neither corticosterone nor microgravity experienced during parabolic flight prevents an O2- response by inflammatory cells.

Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.

We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase.

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought-stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought-inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought-sensitive cultivar than in a drought-tolerant one. An involvement in drought-induced galactolipid degradation is proposed for VUPAT1.

Cytokines and rejection of mouse cardiac allografts.

Graft survival is prolonged by pretransplant transfusion of the graft recipient. It has been postulated that graft rejection is associated with Th1-like cytokines. We tested whether transfusion shifts cytokine production from a Th1-type (gamma-IFN production) to a Th2-type (IL-4 production). Transfusion prolonged cardiac allograft (C3H/HeN donor to a C57BL/6 recipient) survival (10.4+/-0.5 versus 7.2+/-0.2 days for controls, P<0.0001). Splenocyte cultures from nontransfused recipients produced supernatant IFN-gamma concentrations of 13.4+/-1.4 ng/ml upon anti-CD3 stimulation; the same cells produced 32.3+/-3.5 pg/ml IL-4 stimulated with Con A. Spleen cells from transfused animals did not produce gamma-IFN with or without stimulation; (P<0.0001) and produced 21.5+/-3.2 pg/ml IL-4 without stimulation (P<0.0001 compared with controls). C57BL/6 CD8+ lymphocytes isolated from rejected C3H grafts were adoptively transferred (6.7+/-1x10(6)/animal) to pretransfused, C57BL/6 recipients of a C3H graft. Graft survival for these recipients was 7.8+/-0.3 days compared with 10.4+/-0.5 days for recipients pretreated with transfusion only (P<0.005). Transcripts of the gamma-IFN gene were present in unmodified grafts but not in the grafts from transfused recipients given the CD8 cells. In conclusion, transfusion downregulated gamma-IFN production and up-regulated IL-4 production and slowed (but did not abrogate) rejection; CD8 graft-infiltrating cells given adoptively restored normal rejection but not IFN-gamma. Further studies are needed to elucidate the role of cytokines in cardiac allograft rejection.

Patterns of Epstein-Barr virus latent and replicative gene expression in Epstein-Barr virus B cell lymphoproliferative disorders after organ transplantation.

B cell lymphoproliferative disorders arising in organ transplant recipients (B cell posttransplant lymphoproliferative disorders [PTLD]) are generally associated with EBV. In previous reports, B cell PTLD were shown to express the full pattern of EBV latent genes, as in vitro-established lymphoblastoid cell lines. Although viral linear DNA was detected in 40% of lymphoproliferative disorders from immunocompromised hosts, immunophenotypic studies failed to detect late EBV replicative antigens. The aim of this study was to investigate the relationship of EBV latent gene expression in B cell PTLD to morphology, clonality, and immunophenotype, and to examine the replicative state of EBV in malignant cells. For this purpose, 9 cases of EBV-related B cell PTLD were analyzed. Immunoglobulin gene rearrangements were detected by Southern blot analysis. The presence of EBV was assessed by Southern blot and by in situ hybridization. B cell differentiation antigens, adhesion and activation molecules, and EBV latent and replicative gene expression were studied using immunohistochemistry techniques. We demonstrated that EBV-related B cell PTLD exhibited varying patterns of latent viral gene expression. Higher levels of adhesion molecules were detected in latent membrane protein 1 (LMP1) or LMP1 plus EBV nuclear antigen 2 (EBNA2)-positive tumors than in LMP1 and EBNA2-negative tumors. In contrast, there was no relationship between CD21 and CD23 expression and latent EBV phenotype. Activation of the EBV replicative cycle was highlighted by BamHI Z left frame 1 expression in 5 of 9 cases. Less frequent expression of late viral proteins suggested that the initiation of the EBV lytic cycle might not always lead to virions production.

Quantitation of platelet glycoprotein IV (CD36) in healthy subjects and in patients with essential thrombocythemia using an immunocapture assay.

Glycoprotein IV (GPIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV. Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 micrograms/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 +/- 0.05 (mean +/- SD, n = 15) micrograms per 100 micrograms of platelet proteins and a mean value of 27,440 +/- 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean +/- SD = 81,850 +/- 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean +/- SD = 26,810 +/- 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.

Molecular characterization of a monoclonal murine anti-blood group A antibody.

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.

Spectrum of advanced upper airway obstruction due to goiters.

Five patients with advanced upper airway obstruction due to goiter were identified in our institution. All had symptoms of respiratory insufficiency to such a degree that surgery was clearly indicated. Functional characteristics of this group were compared with prior series of goiter patients who had less severe respiratory symptoms. A peak inspiratory flow less than 1.5 L/sec characterized this group who required surgery.

Mapping of 5-HT-moduline binding sites in guinea-pig brain by film and digital autoradiography.

5-HT-moduline is a cerebral tetrapeptide [Leu-Ser-Ala-Leu] that was recently isolated from bovine brain tissue and shown to interact specifically with 5-HT1B receptors, particularly in rodents. The pharmacological properties of 5-HT1B receptors in rodents are different from those in other species. In order to better understand the role of this peptide in non-rodent species, we determined the distribution of 5-HT-moduline binding sites in guinea-pig brain using both the film autoradiography and digital autoradiography with a newly developed high resolution beta-imaging techniques. We found that 5-HT-moduline binding sites were expressed in various brain regions. Quantitative analysis showed that densities of binding sites were similar to those observed previously in rat brain. Regions with the highest labelling included cortex, septum, hippocampus and some regions of basal ganglia. Our results extend previous data and show that 5-HT-moduline interacts with the two forms of 5-HT1B receptors that are distinct pharmacologically. By this interaction, 5-HT-moduline may play an important role in regulating the functional activity of 5-HT1B receptors, thereby contributes to the pathophysiology of serotonergic transmission.

Serum interleukin-10 in acquired immunodeficiency syndrome lymphoma patients. Seroco-Hemoco Study Group.

Interleukin-10 (IL-10) has multiple effects on lymphoid development, particularly as a stimulant of activated B-cell proliferation and differentiation. It is thought that IL-10 might play a role in the development of B lymphoid malignancies based on the observation that lymphomatous tissues from HIV+ patients contain numerous cells containing IL-10 mRNA as well as IL-10 protein. The aim of this study using an Elisa test was to analyze IL-10 in the serum of 18 HIV+ patients with non Hodgkin's B lymphoma (NHL) and compared the presence of this cytokine in the serum of 18 HIV+ patients without NHL. In this comparative study we also considered the different parameters such as the mode of contamination, sex, age and number of CD4 cells. 44% of the patients with HIV-related NHL had significant levels of IL-10 (> or = 12 pg/ml) in their serum, in comparison to the patients without NHL who did not show detectable serum IL-10.

Burn center management of necrotizing soft-tissue surgical infections in unburned patients.

BACKGROUND: Patients with necrotizing soft-tissue infections present great challenges in management from initial presentation through definitive care. Because burn centers concentrate expertise in critical care, wound management, and rehabilitation, we examined the effectiveness of burn center care for patients with necrotizing infections. METHODS: We reviewed our burn center's experience with all patients admitted from 1990 through 1999 with a primary diagnosis of necrotizing fasciitis (NF) or Fournier's gangrene (FG). RESULTS: Fifty-seven patients were identified, 18 with FG and 39 with NF. Patients had a high incidence of preexisting medical problems, including diabetes (37%), obesity defined as greater than 20% above ideal body weight (33%), and hypertension (33%). Seven of 57 (12%) patients died. Patients required a mean of 4.1 operative procedures (range 1 to 15) for definitive wound closure. The mean length of stay (survivors only) was 28.5 days, (range 3 to 70). Although costs increased throughout this period, a formal program of cost-containment resulted in no increase in actual charges per day, from a mean of $4,735 in 1991 to $5,202 in 1999. CONCLUSIONS: Burn centers can provide successful and cost-effective acute care, definitive wound closure, and rehabilitation for patients with NF and FG.

Long-term cytokine alterations following allogeneic blood transfusion.

BACKGROUND: Allogeneic blood transfusion is associated with an increased risk of infection and higher cancer recurrence rates. Previous research has shown that blood transfusion results in multiple immune effects, including cytokine alterations. The purpose of this study was to measure the long term kinetics of splenocyte cytokine production in transfused mice. METHODS: Balb/c mice received either syngeneic transfusion (Syn-BT) or allogeneic transfusion (Allo-BT) from C3H-HeN mice. Splenocyte production of IL-2, IL-6, IL-10, and IFN-gamma was quantitated by ELISA on post-transfusion days 5, 10, 21, and 30. RESULTS: Both Allo-BT and Syn-BT produced significant alterations in cytokine production, but Allo-BT produced the most dramatic and enduring effects as summarized: IL-2: Production of IL-2 was suppressed at day 5, (p < 0.0001), but then rose, peaking at day 21, 30% greater than control values (p < 0.05). IL-6: Allo-BT mice showed suppression of IL-6 throughout the study period (p < 0.005 vs controls, each time point). IL-10: A 5-fold increase in IL-10 production was seen at day 5 after Allo-BT (p < 0.0001 vs control). Production of IL-10 was suppressed at days 10 and 21 (p < 0.001), but returned to control levels by day 30, gamma-IFN: At day 5 post Allo-BT, gamma-IFN was 4 x greater than controls (p < 0.0001). Gamma-IFN production was suppressed at day 10, but then rose at days 21 and 30 to nearly 3 x control levels (p < 0.0001). CONCLUSION: Allo-BT produced multiple cytokine alterations that were of prolonged duration. These results provide a theoretic explanation for the multiple, long-term immunomodulating effects seen in patients who have received transfusions.

The 1997 Lindberg Award. Effects of burn injury on bone and growth in a mouse model.

Bone growth and remodeling are inhibited by severe burns in adult and pediatric patients, resulting in alterations in linear growth, bone mass, osteoporosis, and increased risk for pathologic fractures. This study of a mouse model of burn injury showed skeletal changes similar to those reported in patients with burn injuries. Baseline, control, sham, and burned mice were injected with fluorescent markers calcein and tetracycline for histomorphometric analysis. Total femur dry and ash weights and total calcium content were significantly lower 10 days after burn injury compared with sham and control animals. There also were decreases in the percentage of fluorochrome-labeled bone surfaces and bone formation rates in the burn-injured mice compared with control and sham mice; however, there were no differences in the mineral apposition rates. This model now provides an opportunity to examine cellular and molecular mechanisms contributing to skeletal pathology in a well-defined burn injury model.

Current management of purpura fulminans: a multicenter study.

Seven burn centers performed a 10-yr retrospective chart review of patients diagnosed with purpura fulminans. Patient demographics, etiology, presentation, medical and surgical treatment, and outcome were reviewed. A total of 70 patients were identified. Mean patient age was 13 yr. Neisseria meningitidis was the most common etiologic agent in infants and adolescents whereas Streptococcus commonly afflicted the adult population. Acute management consisted of antibiotic administration, volume resuscitation, ventilatory and inotropic support, with occasional use of corticosteroids (38%) and protein C replacement (9%). Full-thickness skin and soft-tissue necrosis was extensive, requiring skin grafting and amputations in 90% of the patients. One fourth of the patients required amputations of all extremities. Fasciotomies when performed early appeared to limit the level of amputation in 6 of 14 patients. Therefore, fasciotomies during the initial management of these patients may reduce the depth of soft-tissue involvement and the extent of amputations.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.