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.

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry.

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developed specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.

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.

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.

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.

Assessment of tissue viability in complex extremity injuries: utility of the pyrophosphate nuclear scan.

BACKGROUND: Extensive extremity injuries often require difficult decisions regarding the necessity for amputation or radical debridement. During the past decade, we have used technetium-99 pyrophosphate (PyP) scanning as an adjunct in this setting. This study was performed to assess the accuracy of PyP scan in predicting the need for amputation in relation to clinical, operative, and pathologic findings. METHODS: Review of our computerized registry identified 11 patients (10 men, age 36.1 +/- 14.9 years) admitted from 1990 to 1999 who underwent PyP scan. Using operative and pathologic findings, accuracy of the PyP scan was graded as supporting or refuting the clinical assessment of the need for amputation. RESULTS: Eight patients suffered high-voltage electrical injuries, one had severe frostbite, and two suffered soft-tissue infections. In most cases, PyP scan showed clear demarcation of viable and nonviable tissue, verifying the need for amputation (positive); those that demonstrated viable distal tissues confirmed at operation were considered negative. PyP scan had a sensitivity of 94%, a specificity of 100%, and an accuracy of 96% in this setting. CONCLUSION: Technetium-99 PyP scanning is a useful adjunct in predicting the need for amputation in extremities damaged by electrical injury, frostbite, or invasive infection. In addition, by providing an objective "picture" of extremity perfusion, PyP scans can be helpful in convincing patients of the need for amputation.

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.

An unusual manifestation of acquired syphilis.

Bone involvement is an unusual manifestation of acquired syphilis. We report a case of clinically apparent osteitis of the skull, secondary to acquired syphilis, which was the patient's presentation of human immunodeficiency virus infection.

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.

Combined use of radioimagers and radioactive 3'OH DNA nick end labelling to quantify apoptosis in cell lines and tissue sections: applications to virus-induced apoptosis.

DNA fragmentation is a key feature of the degradation phase of apoptosis. In this work we have developed an assay, based on radioimager (beta-IMAGER and micro-IMAGER) quantification of radioactive nick end labelling (RANEL), which is quantitative, rapid and sensitive to study in vitro and in vivo induced apoptosis. To establish the technique, in vitro apoptosis of T cell lines was induced by stimulation of the Fas receptor; cells were labelled using TdT-mediated [alpha-33P] dCTP nick end labelling, after which then radioactivity was quantified using a beta-IMAGER. We have also shown that the RANEL method can be applied to the quantification and visualisation, by micro-IMAGER analysis, of liver tissue sections from mouse Fas-induced fulminant hepatitis or from Dengue-1 virus infected individuals. Finally, this system has also been used to detect apoptosis induced by rabies virus in Jurkat T cells. These data have established a large field of application for the RANEL assay.