Inhibition of retrovirus-induced syncytium formation by photoproducts of a brominated 1,8-naphthalimide compound.

A major disadvantage of conventional phototherapy is the requirement for the in situ delivery of stimulating photoenergy subsequent to the binding of photochemicals to target malignant cells, or virus-infected cells, or viruses. This drawback has resulted in considerable limitation in the use of photochemicals in photomedicine. To circumvent this problem, we have investigated the antiviral efficacy of a brominated 1,8-naphthalimide photocompound, termed LY66Br [3-bromo-4-(hexylamino)-N-hexyl-1,8-naphthalimide], which upon exposure to visible light at 420 nm generates independently of oxygen one or more stable antiviral molecular photoproducts (e.g., is 'preactivated'). Human cell lines infected with the human immunodeficiency virus type 1 (HIV-1), or with the human T-lymphotropic virus type-1 (HTLV-I) exposed to photochemical products of LY66Br (P-LY66Br) completely lost their ability to form syncytia in vitro. Photoproducts of P-LY66Br retain full antiviral activity for at least 3 and 6 weeks when stored at room temperature and at -80 degrees C, respectively. Concentrations of P-LY66Br, effective in inhibiting syncytium formation mediated by HIV-1 and HTLV-I, were nontoxic to normal red cell components of whole blood (red blood cell 2,3-diphosphoglyceric acid, adenosine triphosphate, osmotic fragility or blood type antigens). Additionally, no evidence of acute toxicity was demonstrated in mice following an intravenous bolus inoculation to achieve plasma concentration of 600 microM of P-LY66Br. These findings represent the first demonstration of inhibition of retrovirus-induced syncytium formation by a photochemical product, and justify further investigation of the preactivation process of photochemicals in the treatment of systemic viral infections such as the acquired immunodeficiency syndrome (AIDS), in cancer therapy, and in sterilization of banked blood products.

Neutralization of HIV-1 and inhibition of HIV-1-induced syncytia by 1,8-naphthalimide photoactive compound.

The antiviral property of a newly designed class of 1,8-naphthalimide photochemical compounds was investigated. One such photoactive compound, 1,14-bis-(N-hexyl-3'-bromo-1,8'-naphthalimide-4'-yl)-1,4,11,14- tetraazatetradecane-5,10-dione (diED66Br), when activated to an excited state by visible light (420 nm), effectively neutralized the in vitro infectivity of human immunodeficiency virus (HIV-1). Light-activated diED66Br also inhibited syncytium formation induced by cells infected with HIV-1. Nonactivated diED66Br was completely ineffective. The neutralizing and syncytium-inhibiting doses of activated diED66Br had no effect on normal human peripheral blood mononuclear cells. Radioimmunoprecipitation analysis indicated that diED66Br neutralizing activity resulted primarily from its ability to inhibit the binding of HIV-1 envelope glycoprotein gp120 to the CD4 cellular receptors. Although the exact molecular mechanism of viral neutralization by diED66Br has not been elucidated, its ability to neutralize HIV-1 infectivity and to inhibit syncytium formation supports further investigations of this photochemical as a potential therapeutic treatment of HIV-1 infection.

Inactivation of viruses with photoactive compounds.

The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.

Preliminary studies of photoinactivation of human immunodeficiency virus in blood.

The transmission of human immunodeficiency virus (HIV) by blood or blood components is a major concern in blood banking. A photodynamic flow cell system was designed to inactivate cell-free HIV mixed with blood from a healthy donor. Blood containing 4 x 10(3) infectious units of HIV was treated with 10 and 20 micrograms per mL of commercially available dihematoporphyrin ether (DHE) per mL. Aliquots of this mixture were then held in the dark or irradiated in a flow cell illuminated at a light energy density of 5 J per cm2 provided by a xenon light source equipped with a 630 +/- 5 nm band-pass interference filter; the aliquots were subsequently placed in A.301 cells. All infected cultures were assessed for reverse transcriptase (RT) activity for 17 days. RT activity for either concentration of dye was significantly reduced in irradiated samples as compared to that in samples held in the dark. Blood samples from volunteers also were assessed for the effects of the inactivation process on red cells at concentrations of DHE up to 200 micrograms per mL. No effects were observed on red cell 2,3 DPG or ATP, whole blood potassium concentrations, red cell osmotic fragility, or blood cell antigens.

Protective qualities of mitochondrial and cytosolic fluorescent dyes against in vitro and in vivo infection by the Tulahuen strain of Trypanosoma cruzi.

This study demonstrates the binding of various fluorescent dyes (3,3' dihexyloxacarbocyanine iodide [DiOC6I], doxycycline [DOTC], rhodamine 123, and merocyanine 540) to infectious and intracellular forms of the Tulahuen strain of Trypanosoma cruzi. These dyes predominantly localize in mitochondria. Following treatment with DiOC6I and DOTC, both irradiated and nonirradiated samples showed dark toxicity to T. cruzi, whereas the other dyes effected toxicity only following irradiation with light. Under in vitro conditions, 91% protection was obtained 96 hr postinfection under dark conditions through the use of 0.573 micrograms/ml of DiOC6I. During in vivo studies, the onset of parasitemia was delayed by 7 days through the use of DiOC6I in ng/ml levels. Host deaths occurred in the infected control group on day 11 postexposure, whereas in the 5.7-ng/ml dye-treated group, no death had occurred after 20 days postexposure. This study demonstrates delay of onset of T. cruzi infections with the use of DiOC6I at concentrations well below the levels toxic to the host.

Protection of NIH 3T3 cells from infection by trypomastigotes and sphaeromastigotes of Trypanosoma cruzi, Telahuen strain, by porphyrins in the presence and absence of light (630 and 690 nm).

The light and dark toxicities of naturally occurring and synthesized porphyrins were investigated for their potential to protect NIH 3T3 cells from infection with mammalian tissue culture-derived trypomastigotes of Trypanosoma cruzi. The fluorescent nucleic acid dye Hoechst 33342 was employed to trace T. cruzi intracellular development following porphyrin exposure with and without irradiation. The synthetic porphyrins investigated included isomers A and B of mono- and diacid benzoporphyrin (BPD-MA, BPD-DA, BPD-MB, and BPD-DB). derivatives of naturally occurring hematoporphyrin dihematoporphyrin ether (DHE), and hydroxyethylvinyl deuteroporphyrin (HEVD). These porphyrins provided positive protection following irradiation with light Protection also was obtained due to dark toxicity phenomena. HEVD at a concentration of 1.0 micrograms/ml followed by irradiation of 20 J/cm2 of light at 630 nm provided 90% protection, whereas BPD-MA and BPD-MB provided greater than 85% protection using irradiation at 690 nm. Dark protection was greatest at 1.0 micrograms/ml with the BPD (55-70%), but HEVD provided the greatest toxicity (89%) at a concentration of 10 micrograms/ml. Further investigations are in progress to find methods to increase the protection obtained and to determine the mechanism(s) involved in protection.

Photodynamic inactivation of simian immunodeficiency virus.

A photodynamic flow system employing a dihematoporphyrin ether (DHE) was tested for its ability to inactivate the in vitro infectivity of simian immunodeficiency virus (SICMac) at 630 +/- 5 nm with a light fluence of 5 J/cm2. Cell-free SIVMac was inactivated by photoactivated hematoporphyrin derivative in a dose-dependent fashion. Since SIVMac is closely related to human immunodeficiency virus type 2 (HIV-2) and we have previously reported the successful photodynamic inactivation of HIV-1 in cell-free medium as well as in whole human blood, this technology has the potential for the eradication of transfusion-associated acquired immunodeficiency diseases caused by the above-mentioned retroviruses.

Photodynamic therapy of viral contaminants with potential for blood banking applications.

A photodynamic method has been evaluated as a means of eradicating viral contaminants with the potential for rendering blood safe for transfusion. Herpes simplex virus type 1 (HSV-1) was tested under flowing conditions in culture media or in blood supplemented with the virus. Hematoporphyrin derivative was used as the sensitizer and was photoactivated with visible light at 630 nm and 5 J/cm2. HSV-1 in suspension both in culture medium as well as in blood was shown to be killed. The human immunodeficiency virus was also found to be photoinactivated in flowing cell culture medium and, thus, potentially may be inactivated in blood. These findings extend our previous studies which demonstrated that enveloped viruses can be photoinactivated with hematoporphyrin derivative in a static fluid system. Analysis of blood cell number, red cell lysis, plasma proteins, and other standard hematological tests showed no significant change. The possibility that transfusion-associated acquired immunodeficiency syndrome (AIDS) may result from a blood unit infected with human immunodeficiency virus that tested negative makes it imperative that a safe and effective means of viral killing be developed. The system reported here offers promise as an effective approach to this problem.

The selection of antigens for the diagnosis, prognosis and evolutive study of parasitic diseases.

The hypothesis is set forth that schizodeme (kDNA) typing of Trypanosoma cruzi, and possible other parasites, may be used to resolve the problem associated with the protean clinical manifestations of the disease. kDNA typing of T. cruzi clones may turn out to be useful in: (1) diagnosis; (2) prognosis; (3) production of species (generic) vaccines; (4) the study of autoimmunity. We recommend that an international culture bank of schizodeme-type T. cruzi be established.

Secretory/excretory antigens produced by tetrathyridia of Mesocestoides corti defined by SDS--PAGE and HPLC.

Antigens derived from culture medium in which metacestodes of Mesocestoides corti Hoeppli, 1925, had been maintained were studied by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and high performance liquid chromatography (HPLC). By SDS--PAGE a minimum of 18 Coomassie-staining bands were discerned, of which 4 major bands and 4 major peaks by HPLC of similar molecular weights were observed. The HPLC eluate peaks were analyzed for antigenic activity in vitro by double diffusion in two dimensions, and in vivo in a rabbit. The rabbit had been artificially sensitized with a dialyzable leukocyte extract, showing transfer-factor-like activity, from peritoneal exudate cells removed from mice infected with tetrathyridia. All of the HPLC fractions reacted with rabbit antibody prepared against secretory/excretory antigens, but the sensitized rabbit responded only to two fractions. It is now possible by HPLC to fractionate complex antigens without denaturing them and to elucidate further the role they play during infection.

Circulating antigens in infections of mice by tetrathyridia of Mesocestoides corti Hoeppli, 1925.

Two circulating antigens were detected in the serum of ICR/Timco female mice infected intraperitonealy with tetrathyridia of the cestode Mesocestoides corti Hoeppli, 1925. One circulating antigen appeared by day 2 postinfection (p.i.) and remained in all mice until at least 90 days p.i. A second antigen appeared in the serum on day 14 p.i. and disappeared from all mice by day 28 p.i. Infected mouse serum also contained antibodies against one secretory/excretory antigen and two antigens in crude homogenate, as judged by double diffusion in two dimensions (Ouchterlony). Immune deposits were observed in the kidney tissue of Rockland mice by transmission electron microscopy, and their identity as products of tetrathyridia was confirmed by immunofluorescence. Further studies showed that the main antibody subclass associated with the mesangial immune deposits was 7S gamma l, and that other subclasses of IgG and IgM were not involved. Antigen was found in the proximal renal tubules of infected mice, as demonstrated by fluorescein-labeled IgG fraction of rabbit antitetrathyridia secretory/excretory antigen antisera. The presence of tetrathyridia antigen in the urine of infected mice was confirmed using the Ouchterlony technique.

Fine structure of the tegument of Mesocestoides tetrathyridia by scanning and transmission electron microscopy.

The tegumental surface of tetrathyridial stages of the tapeworm, Mesocestoides corti, was studied by scanning and transmission electron microscopy. Two structural types of microvilli were observed--blade-shaped or conical ones, and elongated, slender microvilli. The distribution and relative frequency of the two types of microvilli differs in different areas of the body surface. Host peritoneal cells were observed attached to the blade-shaped microvilli by slender cytoplasmic projections.

Immunoglobulins on the surfaces of tetrathyridia of Mesocestoides corti Hoeppli 1925 (Cestoda), from laboratory infections of ICR mice.

7Sgamma2b antibody was detected, by fluorescein labeled antibodies, on the body surfaces and wall of excretory bladder of tetrathyridia of M. corti removed from the peritoneums of 5--24-week laboratory infections of ICR/TIMCO mice. 7Sgamma1, 7Sgamma2a, 7Sgamma3, IgM, IgA, and C3 were not found by use of the same techniques. Tetrathyridia maintained in culture medium to which pooled normal mouse, infected mouse, and horse (control) serums containing all known classes and subclasses of immunoglobulins had been added showed only mouse 7Sgamma2b on their surfaces and wall of the excretory bladder, and then only when incubated in pooled serum from infected mice. No other mouse immunoglobulin classes or subclasses were found attached to the worms, nor was any reaction obtained from tetrathyridia incubated in culture medium alone, normal mouse serum, or horse serum. This suggests a highly selective nonspecific absorption of 7Sgamma2b, or more likely demonstrates the specificity of the antibody for the parasite surfaces and wall of the excretory bladder. The possibility that 7Sgamma2b may be acting in an enhanceing mode is discussed.

Antibodies sequestered in the liver granulomata of 8-week infections of CF1 mice by Schistosoma mansoni Sambon, 1907.

With our methods IgM, 7Sgamma1 and C3 were detected in the egg-induced liver granulomata of 8-week infections of CF1 mice by Schistosoma mansoni. It is speculated that, in addition to sequestration of antigens within these lesions by these antibodies, 7Sgamma1 may be associated with the Hoeppli phenomenon, and IgM in combination with C3 may be responsible for the ultimate death of the embryo or miracidium within the egg shell.

Immunoglobulins attached to and in the integument of adult Schistosoma mansoni Sambon 1907, from first infection of CF mice.

7Sgamma2b antibody was found attached to and in the integument of adult Schistosoma mansoni removed from CF1 white mice. With the techniques used, IgA, IgM, 7Sgamma1, 7Sgamma2a, 7Sgamma3, and C3 complement were not found to be attached to the integumental surfaces. 7Sgamma2b does not seem to fix C3, and it is suggested this antibody may be acting in enhancing and blocking roles which protect the worms from the host.

Delayed type hypersensitivity in guinea pig infected subcutaneously with Naegleria fowleri Carter.

A soluble fraction, derived from Naegleria fowleri trophozoites disrupted by freeze-thawing, was tested for antigenic properties. Intradermal injections of this preparation were administered to guinea pigs previously infected subcutaneously with viable N. fowleri. Delayed hypersensitivity to the antigen and loss of weight, the diagnostic symptom of visceral naegleriasis, were observed in the surviving animals. Fifty percent of the guinea pigs, however, did not lose weight and had a reduced reaction to the antigen. The apparent differences in the immunocompetence of guinea pigs inoculated subcutaneously and intranasally with N. fowleri are compared.