Mucosal injury and disruption of intestinal barrier function in HIV-infected individuals with and without diarrhea and cryptosporidiosis in northeast Brazil

The effects of AIDS-related diarrhea--with and without cryptosporidiosis and microsporidiosis--on intestinal function and injury were studied in 40 AIDS patients and 13 healthy volunteers from Fortaleza, Brazil. The differential urinary excretion of ingested lactulose and mannitol was used as a marker of barrier disruption and overall villous surface area. HIV-infected patients with diarrhea had a 2.8-fold higher lactulose to mannitol excretion ratio than HIV-positive patients without diarrhea and a 10.4-fold higher ratio than healthy volunteers. Moreover, those with crypotosporidial infection had a lactulose to mannitol ratio almost 6-fold greater than those without diarrhea and nearly 3-fold higher than those with non-cryptosporidial diarrhea. This effect involved both decreased mannitol excretion (decreased intestinal absorptive area) and increased lactulose excretion (mucosal barrier disruption). The single patient with microsporidial infection had a nearly 3-fold higher ratio than healthy volunteers. Alpha1-antitrypsin tests were positive in two of five (40%) HIV-positive patients with cryptosporidial infections compared with none of 12 HIV-infected patients with non-cryptosporidial diarrhea. These findings confirm that HIV infection is associated with profound intestinal dysfunction and injury, even in those without diarrhea. Disruption of the intestinal barrier is even greater, however, in HIV-infected patients with cryptosporidial diarrhea, with potential nutritional consequences.     

Cytolytic activity of peripheral blood blast cells from patients with acute myeloid leukemia

The results of the present study demonstrate that cells with the morphologic and phenotypic characteristics of blast cells that are obtained from the peripheral blood of patients with newly-diagnosed or recurrent acute myeloid leukemia (AML) can be stimulated by gamma interferon + lipopolysaccharide (IFN/LPS) to mediate in vitro cytolysis of an NK-insensitive hepatoma cell line. The conditions of IFN/LPS induction and subsequent assessment of cytotoxicity that were employed were identical to those used conventionally to test macrophage-mediated tumor cell cytotoxicity. What was totally unexpected was that these same blast cells, in the absence of stimulation with IFN/LPS, were also found to mediate high levels of spontaneous cytotoxicity against autologous bone marrow cells and against the U937 human promonocytic leukemia cell line in vitro. This high level of spontaneous cytotoxicity against autologous bone marrow or U937 promonocytic leukemia cells was not enhanced by IFN/LPS or MCSF under conditions that stimulated cytotoxic function in normal blood monocytes and was markedly reduced by pretreatment of the blast cells with IL2 under conditions that induced potent NK/LAK-mediated cytotoxicity. Neutralizing antibodies against TNFalpha and/or IL1alpha/beta eliminated the cytolytic function of blast cells against autologous bone marrow or U937 promonocytic leukemia targets. These findings demonstrate the existence of a population of cells with the morphologic characteristics of blast cells in the peripheral blood of AML patients which has the capacity to mediate spontaneous cytolysis of autologous bone marrow cells or a promonocytic leukemia cell line. These cells may be an immature variant of normal precursors produced as a consequence of the disordered hematopoietic environment in the marrow of AML patients. Alternatively, this function may be mediated by a subset of the leukemic blasts themselves.     

Evaluation of diagnostic peritoneal lavage in stable patients with gunshot wounds to the abdomen

BACKGROUND: Diagnostic peritoneal lavage (DPL) is used to diagnose intra-abdominal injury in patients with stab wounds and blunt trauma. Because exploratory celiotomy is routinely performed on patients with gunshot wounds to the abdomen, DPL is rarely employed. However, several studies have questioned routine exploration and have drawn attention to the associated morbidity of negative celiotomy. Diagnostic peritoneal lavage is an easily performed and inexpensive test that may be useful in this situation. OBJECTIVE: To evaluate the performance of DPL in the diagnosis of intra-abdominal injury in hemodynamically stable patients with gunshot wounds to the abdomen. DESIGN: A prospective clinical trial. SETTING: Two urban trauma centers. PATIENTS: Patients with gunshot wounds to the abdomen and a systolic blood pressure of at least 90 mm Hg. INTERVENTIONS: Clinical predication of intra-abdominal injury in the emergency department and DPL performed in the operating room before the initiation of celiotomy. Injuries found during the celiotomy were recorded. MAIN OUTCOME MEASURES: The results of the clinical evaluation and DPL were compared with the findings of the celiotomy. RESULTS: Forty-four patients were enrolled into the study. Intra-abdominal injury was present in 32 (73%) of these patients. The senior surgery resident correctly predicted the presence of intra-abdominal injury in 36 (82%) of the patients (sensitivity = 90.0%, specificity = 58.3%, positive predictive value = 85.3%, negative predictive value = 63.6%, phi = 0.52, P < .01) in the emergency department before DPL and celiotomy were performed. Diagnostic peritoneal lavage correctly identified the presence or absence of intra-abdominal injury in 40 (91%) of the patients (positive predictive value = 96.7%, negative predictive value = 78.6%, phi = 0.79, P < .01). CONCLUSIONS: Clinical judgment is highly accurate in separating patients with tangential gunshot wounds to the abdomen from those with intra-abdominal injury but may miss patients with intra-abdominal hemorrhage. Diagnostic peritoneal lavage is highly predictive of the presence of intra-abdominal injury. The return of gross blood on aspiration or a lavage red blood cell count greater than 10 x 10(9)/L should prompt an urgent celiotomy. Missed injuries are rare and most likely to be bowel perforations. Diagnostic peritoneal lavage is an objective test that may augment clinical judgment in selecting hemodynamically stable patients with potential tangential gunshot wounds for observation and is especially useful in identifying intra-abdominal hemorrhage.     

Molecular cloning of the murine IL-1 beta converting enzyme cDNA

IL-1 beta is a potent modulator of immune and inflammatory responses. Murine IL-1 beta is initially synthesized as an inactive 33-kDa pro-molecule that is activated by proteolytic cleavage between Asp-117 and Val-118 to generate the 17-kDa mature IL-1 beta protein. This cleavage is catalyzed by a specific protease that has been designated the IL-1 beta converting enzyme (or IL-1 beta convertase). We have used a human IL-1 beta convertase cDNA to isolate murine convertase cDNA from a WEHI-3 library. These cDNA predicted that the murine convertase is a 402-residue protein. Overall, the murine convertase showed 71% nucleotide and 62% predicted amino acid sequence identity with the human convertase. Southern blot analysis of interspecific backcross mice indicated that the murine IL-1 beta convertase is encoded by a single copy gene located on murine chromosome 9. The murine convertase showed broad constitutive expression, being detected in mononuclear phagocyte and T lymphocyte cell lines as well as in spleen, heart, brain, and adrenal glands. The expression of the murine convertase in mononuclear phagocytes was up-regulated by treatment with LPS or rIFN-gamma. These studies establish that the IL-1 beta convertase is an evolutionarily conserved, widely expressed enzyme that can be regulated at a pretranslational level.     

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.     

UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.