Proteinuria without albuminuria: urinary protein excretion by a subset of patients with burn injuries.

BACKGROUND: There is disagreement regarding the utility of urinary albumin excretion as a marker for capillary injury in patients with severe burn injuries. We examined protein components in urine specimens from patients with burn injury. METHODS: Detailed analysis was performed for a set of 5 urine specimens selected based on a high ratio of albumin-sized molecules by size-exclusion HPLC (Accumin) versus albumin by immunoassay methods. Specimens were analyzed for total protein, alpha(1)-microglobulin, alpha(1)-acid glycoprotein, cystatin C, and retinol-binding protein. Urine components were analyzed by chromatographic and electrophoretic methods. Major components were identified by mass spectrometry of tryptic peptides. RESULTS: A subset of urine specimens had increased total protein with slight increases in albumin by immunoassay or by polyacrylamide gel electrophoresis. Albumin values by size-exclusion HPLC were more than 10-fold higher. Immunoassays for alpha(1)-microglobulin and alpha(1)-acid glycoprotein yielded concentrations 5-10 fold higher than for albumin. Other major components identified included zinc-alpha(2)-glycoprotein and leucine-rich-alpha(2)-glycoprotein. CONCLUSIONS: A subset of patients with burn injury had increased total urinary protein resulting primarily from increased excretion of proteins such as alpha(1)-microglobulin and alpha(1)-acid glycoprotein with little increase in albumin excretion. The unusual composition of urinary proteins in these patients may relate to decreased filtered load of albumin and increased filtered load of acute phase reactants or to alterations in renal tubular protein processing. Thus, measurement of urinary albumin may have decreased sensitivity for detecting kidney injury in burn patients.

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.

A novel blocking monoclonal antibody recognizing a distinct epitope of human CD40 molecule.

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.

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.

The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up in vivo by lymphoid organs where it forms a complex with nucleolin.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using beta-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16-37% could be accounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19-nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.

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.

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.

Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector.

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.

Remote tumor cells in the case of breast cancers: significance of their presence for prognosis [corrected].

Through two clinical studies, tumor cells were searched for in the bone biopsies and cytapherisis of patients suffering from inflammatory tumors and who had undergone intensive therapy and autografts (Pegase 2 program). In these studies we used immunocytochemical test with two monoclonal antibodies. The results have shown the presence of tumor cells in 14% of the patients (respectively 18%), with no correlation to the appearance of metastases after 4 years in the first study. Nevertheless, the presence of these tumor cells seems to be an important factor in the number of relapses. It seems important to develop research into tumor contamination especially in the selection of grafts over the next few years.

Cytomegalovirus induction of interleukin-6 in lung fibroblasts occurs independently of active infection and involves a G protein and the transcription factor, NF-kappaB.

Cytomegalovirus (CMV) infection induces the proinflammatory cytokine, interleukin (IL)-6, which may contribute to the pathology of the infection. In vitro CMV induction of IL-6 by human lung fibroblasts was studied. The quantity of cytokine in culture supernatants was maximal 20 h after infection and decreased thereafter. Transcription of the IL-6 gene and IL-6 protein expression were equally stimulated by infectious and UV-inactivated virus (CMV-UV). CMV-UV-stimulated IL-6 was inhibited by pyrrolidinedithiocarbamate (an inhibitor of the transcription factor, NF-kappaB) and by pertussis toxin (suggesting the involvement of a G protein) and occurred in the absence of CMV immediate-early antigen transcription. Neutralizing antibodies to IL-1beta or tumor necrosis factor-alpha did not affect CMV-UV-induced IL-6, but expression was inhibited by antibody to the CMV attachment glycoprotein. IL-6 production by fibroblasts occurs independently from productive infection but has characteristics that suggest a ligand receptor-mediated pathway. This function may be important in pathology or disease resolution.

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.

Distinct effects of allogeneic blood transfusion on splenocyte cytokine production after hemorrhagic shock.

UNLABELLED: Allogeneic blood transfusion is known to be immunosuppressive in the settings of cancer and transplantation, but the contribution of blood transfusion to immunomodulation after hemorrhage is unknown. Our purpose was to determine the influence of allogeneic blood transfusion upon cytokine profiles following hemorrhagic shock, using a model which approximates the clinical setting. METHODS: Male C3H/HeN mice were hemorrhaged via femoral arterial catheters to a mean arterial pressure (MAP) of 35 +/- 5 mm Hg, which was maintained for 1 h. Mice were resuscitated with autologous blood (auto BT) or allogeneic blood (allo BT) from Balb/c mice (both equivalent to volume of shed blood), and crystalloid (2X the volume of shed blood)-infused at 0.05 ml/min. Animals were sacrificed at 1, 2, and 5 days postshock, and splenocytes were cultured for 24 h with anti-CD3 antibody. Supernatants were assayed for IL-2, IL-6, IL-10, and gamma-IFN by ELISA. RESULTS: Regardless of transfusion status, hemorrhagic shock resulted in increased IL-6 and gamma-IFN by Day 2 postshock. Distinct cytokine alterations after allogeneic transfusion were as follows. IL-2: transient elevation of splenocyte IL-2 production in the shock + allo BT group (P < 0.005 vs. shock + auto BT) at Postshock Day 2. IL-6: suppression in IL-6 production in the shock + allo BT group by Postshock Day 5 (P < 0.05 vs. shock + auto BT). IL-10: persistently elevated IL-10 production following shock + allo BT (Day 1, P < 0.001 vs. shock + auto BT; Day 5; P < 0.05 vs. shock + auto BT). gamma-IFN: elevation in gamma-IFN production by Day 5 in the shock + allo BT group (P < 0.0005 vs. shock + auto BT). CONCLUSIONS: Allogeneic blood transfusion results in distinct alterations in splenocyte production of IL-2, IL-6, IL-10, and gamma-IFN after hemorrhagic shock. This model reflects the clinical usage of blood products and demonstrates some of the immune alterations after transfusion.

Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death.

The severe outcome of the dengue (DEN) virus infection known as DEN hemorrhagic fever-DEN shock syndrome (DHF-DSS) is, in some cases, accompanied by liver injury. Councilman bodies observed in liver biopsies of DHF-DSS cases may correspond to hepatocytes in apoptosis. We show here that infection of the hepatoma cell line HepG2 with DEN type 1 virus induced cell death typical of apoptosis late in the virus cycle. The transcription factor NF-kappaB was activated concomitantly with viral protein synthesis and thus before the appearance of apoptotic cells. Inhibition of apoptosis was observed when DEN virus-infected cells were treated with NF-kappaB decoys, indicating the involvement of this transcription factor in induction of cell death. Thus, infected hepatocytes appear to be subject to apoptosis in vitro, and this may be a key element in the pathophysiology of hepatic failure associated with DHF-DSS.

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.

Radioimagers as an alternative to film autoradiography for in situ quantitative analysis of 125I-ligand receptor binding and pharmacological studies.

Three radioimagers, the mu-imager, the beta-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacological in situ quantitative analysis. Two iodinated ligands 125I-interleukin-1 alpha and 125I-gonadotropin releasing hormone agonist were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical gamma detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the mu-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and beta-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion for in situ quantitative studies on tissue sections. They combine precise imaging for in situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, brought in situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.

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.

Epstein-Barr virus latent and replicative gene expression in post-transplant lymphoproliferative disorders and AIDS-related non-Hodgkin's lymphomas. French Study Group of Pathology for HIV-associated Tumors.

In acquired immunodeficiency, B-cell proliferation is usually associated with Epstein-Barr virus (EBV), implying the impairment of the normal control of EBV and EBV-infected cells. It has been assumed that EBV infection is latent in lymphoproliferative disorders. In order to determine the type of latency and to investigate whether any lymphoproliferative disorders enter into the lytic cycle, we analyzed the expression of latent and replicative EBV genes in 9 post-transplant lymphoproliferative disorders (PTLD) and in 23 EBV-positive AIDS-related non-Hodgkin's lymphomas (AR-NHL). The PTLD cases were categorized into polyclonal or monoclonal polymorphic tumors and monoclonal monomorphic tumors. The AR-NHL cases included large-cell/immunoblastic (LC/IB) and Burkitt's lymphoma (BL) groups. We demonstrated that varying patterns of latent-viral-gene expression are exhibited showing the 3 forms of latency. Polymorphic PTLD and LC/IB AR-NHL frequently expressed type II or III latency, whereas monomorphic tumors and BL AR-NHL showed type I latency. It is noteworthy that 3 cases of BL AR-NHL expressed latency II form. Induction of lytic cycle highlighted by the expression of BZLF1 occurred in 55.5% of PTLD, 36% of LC/IB and 22% of BL AR-NHL. In contrast, late viral proteins indicating productive cycle were present in 22% of PTLD, 14% of LC/IB, and were absent in BL cases. These data suggest that the impairment of EBV control permits disruption of latency, but the initiation of the lytic cycle may not always lead to viral production.