Preferential localization of varying forms of photoactive 1, 8-naphthalimide compounds within the atheromatous arterial wall.

BACKGROUND AND OBJECTIVE: We are currently working with a novel class of photoactivated 4-amino substituted 1,8-naphthalimide compounds for tissue bonding. With promising results in other tissues, we are pursuing potential vascular applications. This study focused on determining the appropriate compound formulation(s), concentration, and exposure times to optimize penetration of the heterogeneous arterial wall. STUDY DESIGN/MATERIALS AND METHODS: Segments of atheromatous rabbit carotid artery were immersed in hydrophilic or lipophilic forms of the compound, then frozen, cryosectioned, and examined by confocal microscopy. RESULTS: The hydrophilic compound exhibited preferential localization within the intima and media and limited presence in the adventitia. Conversely, the lipophilic compound concentrated in the intima and adventitia with virtual exclusion from the media. Exposure to both forms resulted in complete penetration of the arterial wall. CONCLUSION: These results extend our knowledge and permit a more practical approach to potential vascular applications using these photoactivated compounds for tissue bonding.

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.

Jet-assisted laser tools for tooth preparation.

Previous oral calcified-tissue laser ablations have yielded inadequate results because of the difficulty in producing a desired effect on a surface without concomitant pulp or osseous damage. The purpose of this study was to characterize a new modality of ablating teeth using argon and diode lasers (488.5 nm, 805 nm) in combination with the repetitive placement of specific photoabsorptive dyes. In this design, energy from laser light, that would otherwise be reflected, is coupled to the tooth-dye interface. Thirty-two specimens of recently extracted human enamel were sectioned and prepared into 3 x 2 x 2 rectangular blocks and smoothed with a polishing point. Two-microliter droplets of dye were placed on the external enamel surface and subsequently air-dried. Specimens were then ablated with the laser-dye combinations, producing craters approximately 100-200 mum in depth and devoid of visual carbonization. Similar irradiations were performed on enamel specimens without dye application, and displayed no cavitation or surface carbonization. SEM studies showed evidence of crater formation within the enamel surface. Optimization of laser parameters integrated with specific dispensing of dye is necessary before this technique can be studied further.

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.

Soft tissue studies with 805 nm diode laser radiation: thermal effects with contact tips and comparison with effects of 1064 nm Nd:YAG laser radiation.

This study compares the perioperative depths of thermal coagulation, charring, and incision in rabbit liver, internal anterior abdominal wall skeletal muscle, and abdominal skin and in swine liver and abdominal skin obtained with 805 nm diode laser and 1,064 nm Nd:YAG laser radiation using 300-microns-diameter conical-tip and 1,200-microns-diameter spherical-tip contact fibers by hand. Additionally, the total depth of tissue necrosis surrounding incisions made with both lasers and contact tips is determined 48 hours postoperatively in the three tissues, and healing of the liver and abdominal wall muscle 21 and 35 days postoperatively is assessed histologically. Perioperatively determined charring, coagulation, and incision depth obtained in all tissues with either 805 nm or 1,064 nm laser radiation were sensibly equivalent at equal laser power values for each of the two contact tip shapes tested. At equal laser power values, coagulation depths obtained in rabbit abdominal skin using the 300-microns-diameter conical tip differed significantly (P < or = 0.01) from those values obtained with the 1,200-microns-diameter spherical tip. Incision depths obtained with the two different contact tip shapes at equal laser power settings in the different tissues studied differed in a few instances with no apparent pattern relating to tissue type or laser power. Depth of incisions obtained with both laser and tip types increases in the range of 6-12 w, but plateaus in the range 12-18 w in the tissues studied. Incisions obtained with both diode and Nd:YAG laser contact were essentially hemostatic, with self-limiting oozing at most.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal effects in tissues from combined simultaneous coaxial CO2 and Nd:YAG laser beams.

This study examines the depth of thermal coagulation and charring in swine liver, kidney cortex, tongue (inferior surface), skeletal muscle, inflated lung, and skin resulting from in vivo incision with simultaneous coaxial CO2 and Nd:YAG (1.064 microM) laser beams. At values of 20 w and 40 w, respectively, and at values of 30 w and 60 w, respectively, of combined CO2 and Nd:YAG laser radiation, coagulation depths determined histologically in liver were significantly greater (P less than 0.01) than in the other tissues and were significantly less in inflated lung (P less than 0.05) than in other tissues for the larger laser power settings employed. Coagulation depths achieved at 10 w and 20 w, respectively, and at 20 w and 40 w, respectively, of CO2 and Nd:YAG laser power were comparable to those obtained by other workers in liver and other relatively vascular tissues using a contact Nd:YAG laser tip. Charring depths obtained at power settings of 30 w (CO2) and 60 w (Nd:YAG) were greater in liver (P less than 0.001) than in all other tissues examined. Hemostasis during incision was achieved only for values of the ratio of CO2 to Nd:YAG laser power in the range 2-3 in the more vascular tissues, liver and kidney cortex, whereas hemostasis was achieved also in the lesser vascular tissues at higher values. These results strongly suggest the usefulness of combined simultaneous CO2 and Nd:YAG laser beams in surgery of the more vascular organs and tissues.

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.

Enamel surface roughness and dental pulp response to coaxial carbon dioxide-neodymium: YAG laser irradiation.

The purpose of this study was to evaluate the effect of a coaxial carbon dioxide/neodymium:yttrium aluminium garnet laser beam on enamel surface roughness and the dental pulps of mongrel dogs. In four dogs, four maxillary left posterior teeth were irradiated at 16 cm source-tooth distances. Two teeth were irradiated with 16 W CO2/16 W Nd:YAG and the remaining two with 16 W CO2/40 W Nd:YAG. Two maxillary right teeth were untreated controls. In addition, mandibular premolars were irradiated at the same distance and power levels, extracted, and analysed for surface roughness. Significant differences in surface roughness were found between control samples and either power level, but not between enamel surfaces at the two power levels. Maxillary teeth were removed at 10 days postoperatively, sectioned and stained (H & E). The reaction of pulpal cells to irradiation was scored. Data analysis revealed statistically significant differences between the control and lower power Nd:YAG groups and between the control and higher power Nd:YAG groups. The difference in pulpal response between both laser groups approached significance.

Choice of lasers for minimally invasive spinal surgery.

Recently, there has been increased interest in less invasive spinal surgery techniques. This has led to the development of procedures such as automated percutaneous lumbar discectomy and arthroscopic microdiscectomy. Lasers are now used in many areas of medicine and may have applications in minimally invasive spinal surgery. A number of different laser systems have been evaluated for their effectiveness in removing disc tissue in the laboratory, but technical problems have limited their clinical use. Only the Nd:YAG (1,064 nm and 1,320 nm) and KTP (532 nm) systems have been used clinically. Unsuccessful clinical results were obtained with the 1,064 nm Nd:YAG, whereas the other two systems appeared to produce results similar to the present mechanical systems but required less time for disc removal. This paper discusses considerations for choosing a laser system for spinal applications and reviews the work performed in this area.

In vitro photodynamic inactivation of herpes simplex virus with sapphyrins: 22 pi-electron porphyrin-like macrocycles.

The photodynamic inactivation of HSV-1, a virus having a membranous envelope, with both a decaalkyl sapphyrin and its dicarboxy-substituted analog was studied. The decaalkyl sapphyrin was as efficient in the inactivation of HSV-1 on a per macrocycle basis as DHE, whereas the efficiency of the dicarboxy-substituted sapphyrin was approximately two orders of magnitude less. Fluorescence studies of sapphyrin's binding to liposomes and VSV suggested that the decaalkylsapphyrin bound monomerically to cholesterol-rich regions of the viral envelope, whereas its charged analog localized in a more polar environment.

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.

Photooxidative changes of lysozyme with 337.1 nm laser radiation.

Initial photoinduced oxidative changes in the protein lysozyme were studied using the 337.1 nm radiation from a pulsed nitrogen laser without exogenous sensitizing compounds. Irradiation of lysozyme and tryptophan in aerated solution results in the temperature and solvent dependent loss of tryptophan absorption and fluorescence, and the appearance of fluorescent "daughter products," primarily N-formyl-kynurenine and kynurenine. Exposures that resulted in 15% loss of tryptophan fluorescence produced no measurable loss in enzymatic activity. Fluorescence quenching experiments on irradiated lysozyme at low conversion percentage suggest that an exposed residue (Trp-62) is favored as an initial target of attack.

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.

Muscle diffraction theory. Relationship between diffraction subpeaks and discrete sarcomere length distributions.

A theoretical discussion is presented that describes the diffraction on monochromatic light by a three-dimensional sarcomere array having the following properties. The basic repetitive diffracting unit is the sarcomere. The contiguous arrangement of physically attached serial sarcomeres in the myofibril is contained within the model so that relative position of sarcomeres depend upon the lengths of intervening ones. Sarcomere length is described by a distribution function. This function may be discrete or continuous and contain one or more subpopulations. Two arrangements of sarcomeres are considered: (a) when sarcomeres of different lengths are arranged randomly in myofibrils the amplitude and width of mth order (m greater than or equal to 1) peaks and associated secondary diffraction maxima decrease and increase monotonically, respectively, as the standard deviation of the length distribution increases. No subpeaks are present regardless of the number of subpopulations within the distribution function. This behavior is shown to follow from the dependence of sarcomere position on the length of intervening sarcomeres. (b) When sarcomeres belonging to the same length subpopulation are arranged in serial contiguous fashion to form domains and more than one length subpopulation is present, then mth order diffraction peaks split to form subpeaks. The theoretical basis for this behavior is developed for the first time and may explain the subpeaks evident in diffraction patterns from cardiac and skeletal muscle.