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P J Bugelski

Publications and source records attributed to P J Bugelski.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy.

Macrophage-derived foam cells are a prominent component of developing atherosclerotic lesions. We describe an in vitro model of foam cell formation which mimics some aspects of the evolution of foam cells in mature atherosclerotic lesions. Thioglycollate-elicited mouse peritoneal macrophages were incubated with copper-oxidized LDL (ox-LDL) for periods up to 168 hr. Identifiable foam cells were present after incubation with ox-LDL at 24, 72, and 168 hr. Control cells incubated without ox-LDL did not form foam cells. Fluorescence microscopy after staining with Nile red exhibited progressive accumulation of lipids, and transmission electron microscopy (TEM) showed distinct ultrastructural changes over time. Macrophages at 24 hr had a few non-membrane-bound lipid droplets but were otherwise identical to control cells. These lipid droplets fluoresced yellow-gold after Nile red staining. After 72 hr of incubation with ox-LDL, in addition to increased numbers of non-membrane-bound lipid inclusions, macrophages contained membrane-bound multilamellar lipoid structures. These multilamellar structures corresponded to areas of reddish-orange fluorescence after Nile red staining. In macrophages incubated with ox-LDL for 168 hr, the amount of cellular lipid was further increased and cholesterol crystal profiles were apparent within some multilamellar lipoid structures. Biochemical analysis showed that the total cholesterol content steadily increased over 168 hr. The increase in total cholesterol was accompanied by a dramatic increase in free cholesterol between 72 and 168 hr. These results demonstrate that long-term incubation of macrophages with ox-LDL increased lipid deposition in cultured cells and that, under the conditions studied, cholesterol crystals formed in macrophage foam cells. Moreover, this system allows investigation of the evolution of foam cells showing some characteristics of those found in atherosclerotic lesions.

Animals↗
PubMed1995

Separation of immunomodulatory and cholesterol-lowering activities of heterocyclic azaspiranes.

Azaspiranes are novel immunomodulators which are effective in a variety of autoimmune diseases. One azaspirane analog, SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine dihydrochloride), caused a decrease in total serum cholesterol in dogs after oral administration. To determine whether an effect on cholesterol was common to this class of compounds, the immunomodulatory activity was compared with the cholesterol-lowering activity of six azaspirane analogs. The compounds were given to beagles at a dose of 1 mg/kg p.o. for 28 days, and the effect on serum cholesterol was determined. The results from this study showed a clear dissociation between the immunomodulatory and hypocholesterolemic activities of these compounds. Studies performed to determine the mechanism of the decrease in serum cholesterol caused by SK&F 105685 indicated that it was not due to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase or acyl-CoA:cholesterol acyltransferase activities, or to a potentiation of cholesterol-7 alpha-hydroxylase activity. In addition, analysis by gas chromatography of the nonsaponifiable sterol fraction in dog plasma after treatment with SK&F 105685 or SK&F 106333 showed a decrease in cholesterol and an accumulation of lathosterol and an unknown sterol, indicating that the conversion of these sterols is inhibited and cholesterol synthesis is blocked at these steps. SK&F 105685 affected the sterol profile in human hepatoblastoma cells (Hep G2) in a similar way. Characterization of the unknown sterol by gas chromatography and mass spectrometry indicated that the unknown sterol is very similar to cholesterol and lathosterol, but its identity has yet to be established. These results show that the hypocholesterolemic effects of azaspiranes are related to inhibition of one or more of the final steps in the biosynthetic pathway of cholesterol.

Animals↗
PubMed1995

Renal catabolism of recombinant human soluble CD4 after intravenous administration to male Sprague-Dawley rats.

Recombinant soluble CD4 (sT4; mol. wt. 45,000) has been studied extensively in Sprague-Dawley rats, and substantial renal processing has been indicated. In rats and monkeys, renal filtration and precipitation of sT4 in the distal nephron caused tubular cast nephropathy. Intravenous pharmacokinetics in the rat demonstrated that sT4 plasma clearance exceeded the glomerular filtration rate. In an effort to determine quantitatively the extent to which kidney and other tissues were responsible for sT4 catabolism, sT4 was labeled with trace amounts of dilactitol-[125I]tyramine and administered intravenously to Sprague-Dawley rats (1 mg/kg). Dilactitol-tyramine accumulates in lysosomes at the site of protein degradation. It has been used primarily to demonstrate hepatic catabolism of endogenous proteins. Blood samples were drawn for pharmacokinetic analysis, and selected tissues were removed to assess radiolabel distribution. Comparison of pharmacokinetic parameters derived from total plasma radiolabel and functional ELISA were not significantly different. Thus, covalent modification of sT4 with dilactitol-tyramine did not appreciably change the rate of clearance. From 3 to 24 hr after intravenous administration, 81.5 +/- 0.1% of the total administered radioactivity was found in the kidney. Approximately 8-13% of the administered dose was recovered in the liver. Macroscopic autoradiography of the kidney demonstrated accumulation of radiolabel in the cortex. Light microscopic autoradiography of the kidney following intravenous administration of directly radioiodinated sT4 confirmed cortical processing, because radiolabel was located primarily in epithelial cells of P1 and P2 segments of the proximal tubule after low intravenous doses (0.4-4 mg/kg). At 40 mg/kg, distal tubules and cortical collecting ducts were labeled as well. Thus, sT4 was filtered by the glomerulus, reabsorbed in the proximal tubule, and degraded in the lysosomal compartment.

Animals↗
PubMed1994

HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages.

Human immunodeficiency virus (HIV), the retrovirus believed to be the cause of acquired immunodeficiency syndrome (AIDS), infects a variety of CD4+ cells, including lymphocytes and cells of the monocyte/macrophage lineage. Encoded in the HIV genome are several precursor proteins that must undergo proteolytic cleavage to yield functional proteins. The gag precursor protein of HIV (p55) is cleaved by a virally encoded aspartate protease (HIV protease). Because cleavage of p55 is required for viral maturation and infectivity, inhibition of HIV protease is an attractive target for therapy designed to block the progression of HIV infection. Inhibitors of HIV protease from a variety of chemical classes have been synthesized and antiviral activity has been demonstrated in lymphocytes and cells from the monocyte/macrophage lineage. A few HIV protease inhibitors have progressed to the clinic and some have shown promise in early trials. There are, however, several important issues that will affect the development of a successful HIV protease inhibitor, including cell and tissue distribution and immunotoxicity. These issues are tissue distribution and immunotoxicity. These issues are discussed, with an emphasis on the complexities posed by cells of the monocyte/macrophage lineage.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus.

Some isolates of simian immunodeficiency virus (SIV) have been shown to infect Sup-T1 cells with slow kinetics and in the absence of cytopathic effects, including cell fusion or CD4 down-modulation (J. A. Hoxie, B. S. Haggarty, S. Bonser, J. Rackowski, H. Shan, and P. Kanki, J. Virol. 62:2557-2568, 1988). In the present study, we describe the isolation and characterization of a SIVmac variant, derived from the BK28 infectious molecular clone, that became highly cytopathic for Sup-T1 cells. This variant, termed CP-MAC, exhibited a number of differences from BK28, including (i) an altered tropism which largely restricted its host range to Sup-T1 cells, (ii) the ability to induce cell fusion and CD4 down-modulation, and (iii) a highly stable interaction of its external (SU) and transmembrane (TM) envelope glycoproteins. In addition, a marked increase in the level of surface envelope glycoproteins was observed both on CP-MAC-infected cells and on virions. The CP-MAC env gene was PCR amplified from infected cells, and sequence analysis identified five amino acid changes in SU and six in TM compared with BK28. The introduction of these changes into BK28 was shown to fully reconstitute the biological and morphological properties of CP-MAC. The limited number of mutations in CP-MAC should enable the molecular determinants to be more precisely defined and help to identify the underlying mechanisms responsible for the striking biological and structural alterations exhibited by this virus.

Amino Acid Sequence↗
PubMed1994

Effects of a novel azaspirane (SK&F 105685) on arthritic lesions in the adjuvant Lewis rat: attenuation of the inflammatory process and preservation of skeletal integrity.

OBJECTIVE: To determine the effects of SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5] decane-2-propanamine dihydrochloride) on the arthritic lesions in the tibiotarsal joint of adjuvant arthritic (AA) rats. METHODS: Inhibition of hindpaw inflammation was measured by water displacement. The protective effects on joint integrity were determined by measuring radiographic and histological changes and by scanning electron microscopy. RESULTS: Compared to AA control rats, SK&F 105685 suppressed hindpaw edema 64% or 41-54% in AA rats receiving 30 or 20 mg/kg/day, respectively. Radiographic evaluation showed marked decreases in soft tissue swelling and in the severity of skeletal tissue loss at the tibiotarsal joint in both dose groups. Histologically SK&F 105685 markedly attenuated the extent and severity of the inflammatory lesion and preserved the basic integrity of bone and cartilaginous tissues, including the content and distribution of proteoglycans of the articular cartilages. Cellular changes included decreases in the inflammatory infiltrate and in the number of osteoclasts and chondroclasts. Whereas AA control rats exhibited marked to severe loss (41-70%) of skeletal tissue mass, the loss in rats given 30 mg/kg SK&F 105685 was mild (< 20%). Scanning electron microscopy of the talus revealed only slight erosion of the articular cartilage and general preservation of the underlying bone. In contrast, in AA controls, there was marked erosion of the talar articular cartilage and severe loss of subchondral bone. Spleen cells from SK&F 105685 treated rats had a reduced capacity to respond to concanavalin A and contained suppressor cell activity as measured in a coculture assay. CONCLUSION: Our studies show that SK&F 105685 has remarkable protective effects on the joints of AA rats and suggests that it may attenuate the overall inflammatory process and retard the degenerative loss of skeletal tissue in rheumatoid arthritis in humans.

Administration, Oral↗
PubMed1993

Beneficial effects of SK&F 105685 in rat adjuvant arthritis: prophylactic and therapeutic effects on disease parameter progression.

Prophylactic administration of SK&F 105685 (N, N-dimethyl-8,8,-dipropyl-2-azaspiro[4.5]decane-2-propanamine dihydrochloride) at 30 mg/kg/day inhibited hindpaw lesions in adjuvant arthritic (AA) rats following 16 (84% inhibition) and 24 (70% inhibition) days of treatment. In a therapeutic protocol, where dosing was initiated on day 10 after disease induction, SK&F 105685 (20 mg/kg/day) effectively halted disease progression, and the inflammatory lesion was suppressed by 70% following treatment for 20 days. Histological evaluation of the joint periarticular soft tissue, bone and articulation (joint space and joint surface) from therapeutically treated rats showed a clear beneficial effect. Most rats presented moderate lesions rather than the severe lesions seen in the AA animals. AA control rats and AA rats treated prophylactically or therapeutically with SK&F 105685 had significant and similar increases in their total white blood cell (WBC), neutrophil, monocyte and platelet counts. Although increases were seen in the absolute number of neutrophils and platelets, there were no differences in the levels of leukotriene B4 (LTB4) and hydroxyheptadecatrienoic acid (HHT), per cell when these cells were stimulated with the calcium ionophore A23187. These results, describing the beneficial effects of SK&F 105685 administered therapeutically to the AA rat, indicate that this compound possesses properties desirable of an anti-arthritic agent and may potentially modify the disease outcome.

Adjuvants, Immunologic↗
PubMed1993

Effects of SK&F 105685, a novel anti-arthritic agent, on immune function in the dog.

SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The activity of SK&F 105685 in these models is associated with the induction of non-specific suppressor cell (SC) activity as defined by the ability of cells from drug-treated animals to inhibit the proliferative response of lymphocytes from control animals to concanavalin A. To evaluate the immunotoxicologic potential of SK&F 105685, the effect on immune function of one month of dosing with 1 mg/kg/day of SK&F 105685 was examined in the dog. Differential blood cell counts and ex vivo immune function assays were performed using blood collected before dosing on days 1 (baseline), 15 and 29, of the study. Immune function assays were performed on spleen cells on day 30. Under the conditions of the study, SK&F 105685 displayed pharmacological activity as demonstrated by the induction of splenic SC activity. The drug did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood and did not cause generalized immunosuppression. The ability of peripheral blood lymphocytes or spleen cells to produce IL-2 or proliferate in response to mitogenic stimulation was not affected by drug treatment. SK&F 105685 also failed to affect the candidacidal activity of polymorphonuclear leucocytes and spleen cells indicating that it is unlikely to compromise nonspecific resistance to infection. SK&F 105685 however, was able to inhibit the generation of a specific in vitro antibody response to sheep red blood cells (SRBC) by splenocytes from treated animals. Inhibition of the anti-SRBC antibody response was also observed upon addition of the drug to normal spleen cells. Addition of the drug at different time points during the culture period indicated that SK&F 105685 was interfering with an event(s) occurring during the first 72 h of culture. Taken together, these results suggest that, in a therapeutic setting, SK&F 105685 is unlikely to compromise the immune status of the host as it can down-regulate a specific immune response without causing generalized immunosuppression.

Animals↗
PubMed1993

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.

CD4 Antigens↗
PubMed1993

ARDS-like lung injury produced by endotoxin in platelet-activating factor-primed rats.

We recently reported that the combined administration of lipopolysaccharide (LPS) and platelet-activating factor (PAF) in rats, at doses that are completely devoid of any effect when given alone, caused lung injury characterized by neutrophil adhesion to lung capillaries and postcapillary venules, neutrophil accumulation in the lung parenchyma, platelet-fibrin deposits in postcapillary venules, and pulmonary edema. A marked increase in lung myeloperoxidase activity and an elevation of serum tumor necrosis factor-alpha and thromboxane B2, along with leukopenia and thrombocytopenia, were also noticed. The present study aimed to examine whether repeated LPS-PAF stimulus can cause progressive lung injury reminiscent of adult respiratory distress syndrome (ARDS). A second LPS-PAF challenge, 4 h (n = 11) after the original challenge, induced mortality (69% at 24 h, P < 0.01) and some of the pathological changes seen in clinical ARDS, including severe pulmonary edema, alveolar proteinaceous exudates, monocytic infiltration, and a further increase in lung myeloperoxidase activity (700%, P < 0.01). Repeated LPS-PAF dosing also resulted in sustained increased serum tumor necrosis factor-alpha levels (1,610 +/- 470 pg/ml, P < 0.01) and further exacerbation of the leukopenia (-68 +/- 6%, P < 0.01) and thrombocytopenia (-65 +/- 8%, P < 0.01). These data suggest that repeated LPS-PAF actions are sufficient to elicit pathophysiology of ARDS-like lung injury.

Animals↗
PubMed1993

Morphometric analysis of envelope glycoprotein gp120 distribution on HIV-1 virions.

The surface of HIV-1, like that of other retroviruses, is studied with virally encoded glycoproteins which appear ultrastructurally as electron-dense spikes or knobs. The glycoprotein that forms the spike structure, gp120, is non-covalently bound to the transmembrane glycoprotein gp41. Mature HIV-1 virions do not have as many spikes as the genetically related retroviruses HIV-2 and SIV. gp120 is lost from HIV-1 during viral morphogenesis and after incubation of the virus with the soluble form of cellular receptor CD4. In this study we used ultrastructural cytochemistry and morphometry to quantitate the distribution of envelope glycoprotein spikes on budding and mature HIV-1 virions and to look for alternatives to the laborious and somewhat subjective spike-counting technique for envelope spike analysis on HIV-1. HIV-1, strain HTLV-IIIB, was examined after staining of envelope glycoproteins with either tannic acid, immunogold staining for gp120 (gp120-immunogold), or lectin-gold staining with concanavalin A for mannose residues (ConA-HRP-gold) and frequency distributions of spikes or gold particles per micron HIV-1 membrane generated. Envelope spikes were normally distributed on membranes of budding and mature HIV-1. However, the density of spikes per micron viral membrane on mature HIV-1 virions was approximately 50% of that observed on budding virions. ConA-HRP-gold and gp120-immunogold did not efficiently label budding virions. The shape of the frequency distribution for ConA-HRP-gold particles on mature virions was similar to that for envelope spikes and could be used to quantitate envelope glycoproteins on HIV-1. In addition, ConA-HRP-gold staining was able to detect the loss of envelope proteins after treatment of virus with soluble CD4. gp120-immunogold labeling was patchy and many virions were unlabeled. ConA-HRP-gold staining proved to be a rapid, reliable, and easily quantifiable method for estimation of envelope glycoprotein density on mature HIV-1. However, the loss of spike structures throughout the life cycle of HIV-1 can effectively be determined only by direct spike counting.

Concanavalin A↗
PubMed1993

Tumor necrosis factor-alpha mediates endotoxin-induced lung injury in platelet activating factor-primed rats.

We have reported recently that lipopolysaccharide endotoxin and platelet activating factor cooperate in priming relationships to elicit lung microvascular injury. Lung injury was associated with elevated serum levels of tumor necrosis factor-alpha (TNF alpha) and histological findings highly reminiscent of the adult respiratory distress syndrome. The present study was designed to examine the role of TNF alpha in lipopolysaccharide/platelet activating factor-induced lung injury by utilizing a highly specific monoclonal antibody which block TNF alpha actions (anti-TNF alpha monoclonal antibody). Pretreatment with anti-TNF alpha monoclonal antibody (2.5-25 mg/kg i.v., n = 5-9) dose-dependently prevented the lipopolysaccharide/platelet activating factor-induced histopathological changes, lung edema (P < .01), lung myeloperoxidase activity (P < .01), elevation of neutrophil count in bronchoalveolar lavage fluid (P < .01) and increased serum thromboxane B2 (P < .01). Indomethacin (6 mg/kg i.v., n = 5) failed to modify the lung injury despite complete inhibition of thromboxane B2 formation (P < .05). These data suggest that TNF alpha might play a key role in initiation of the early inflammatory changes which lead to adult respiratory distress syndrome.

Animals↗
PubMed1992

Effect of a human immunodeficiency virus protease inhibitor on human monocyte function.

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Encoded by the HIV genome are several precursor proteins that undergo proteolytic cleavage to yield functional proteins. The env precursor protein is cleaved by a cellular protease. The gag precursor protein of HIV (p55), however, is cleaved by a virally encoded aspartate protease (HIV Protease). Cleavage of p55 is required for viral maturation and infectivity. There are also several host cell aspartate proteases that serve important homeostatic functions. Cathepsins D and E are lysosomal aspartate proteases which are believed to play an important role in macrophage function, and it has been suggested that inhibition of these enzymes by an HIV protease inhibitor may exacerbate immunosuppression in AIDS patients. We have studied the effect of SK&F 107461 (a hydroxyethylene dipeptide isostere inhibitor of HIV protease), on various host defense functions of human monocytes. Pepstatin A (an inhibitor of most aspartate proteases) and leupeptin (an inhibitor of serine and cysteine proteases) were included as controls. Although less potent than the prototypic aspartate protease inhibitor pepstatin, SK&F 107461 inhibited partially purified cathepsin D in vitro. However, in cell-based assays, SK&F 107461 had no effect on the degradation of hemoglobin, antigen processing of the protein antigen streptokinase, or secretion of 17-kD IL-1 beta by monocytes at concentrations which inhibit maturation of intracellular virus in HIV infected monocytes. Furthermore, SK&F 107461 had no effect on constitutive candidacidal activity. In contrast, leupeptin and pepstatin A partially inhibited accessory cell function of monocytes in the proliferative response to the recall antigen streptokinase. In addition, leupeptin partially inhibited degradation of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Presenting Cells↗
PubMed1992

Strong association of simian immunodeficiency virus (SIVagm) envelope glycoprotein heterodimers: possible role in receptor-mediated activation.

Soluble forms of a human cell-surface molecule expressed on T lymphocytes (CD4) neutralize diverse strains of both human (HIV) and simian (SIV) immunodeficiency viruses through the induction of envelope shedding and direct competition with cellular CD4 for virus binding. However, we have previously shown that sCD4 enhances infection of simian immunodeficiency viruses from African green monkeys (SIVagm) and have theorized that this enhancement is due to the induction of conformational changes leading to viral fusion (receptor-mediated activation). In this report, we compared the relative association of the envelope glycoproteins of SIVagm with HIV type 1 (HIV-1) in order to determine if a more stable association of SIVagm envelope glycoproteins might account for the differential effects of sCD4 on the infectious process. Monospecific antisera to each of the SIVagm glycoproteins were generated and used to detect stable heterodimers by radioimmunoprecipitation. Standard solubilization buffers containing both ionic and nonionic detergents or saturating concentrations of sCD4 failed to disrupt SIVagm gp120 interactions with the transmembrane protein, gp36, whereas HIV-1 heterodimers were easily dissociated. Higher concentrations of SDS (1%) were necessary to disrupt the SIVagm envelope complexes demonstrating the existence of strong noncovalent interactions between these membrane glycoproteins. In addition, morphometric analysis by electron microscopy revealed that the linear density of SIVagm spikes was stable and resisted shedding when virus was incubated with sCD4 whereas a significant decrease in linear spike density was noted for HIV-1. Based on our original hypothesis, the strong association of SIVagm glycoprotein spikes during soluble receptor binding may allow for highly stable conformational intermediates important for viral fusion, while neutralization of HIV-1 by sCD4 results from less stable envelope associations.

Animals↗
PubMed1992

Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells.

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

Blotting, Western↗
PubMed1992

Myeloma-like cast nephropathy caused by human recombinant soluble CD4 (sCD4) in monkeys.

CD4 is the receptor for human immunodeficiency virus (HIV) on lymphocytes and macrophages. Soluble CD4 (sCD4), a recombinant truncated form of CD4, has been shown to inhibit HIV-1 in vitro and is being tested as a therapy for AIDS. Preclinical studies in cynomolgus monkeys revealed a protein cast nephropathy after four daily intravenous doses of 100 mg/kg/day. Renal lesions were not found in monkeys that received 10 mg/kg/day. The renal lesions consisted of proteinaceous tubular casts associated with multinucleate giant cells and neutrophils located in the tubules of the distal nephron. The affected tubules were surrounded by an interstitial mixed inflammatory cell infiltrate. By electron microscopy, the casts were composed of moderately electron dense, paracrystalline material. Immunostaining demonstrated that the casts contained sCD4-derived material and Tamm-Horsfall protein. Moreover, biochemical analysis of urine showed that a portion of sCD4 was excreted as intact protein. Because infection with HIV-1 can be associated with clinically significant nephropathy, these data suggest that renal function should be closely monitored in patients receiving soluble forms of CD4.

Animals↗
PubMed1991

Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.

Blotting, Western↗