Biomedical subjects

M W Berns

Publications and source records attributed to M W Berns.

At least 145 records · Page 8Linked to original sources

PubMed1989

Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle.

A laser-induced optical force trap was used to alter the movement of chromosomes in mitotic cells in vitro. The trap was produced by using a 1.06-microns neodymium YAG (yttrium/aluminum garnet) laser focused through a phase-contrast microscope. The trap was applied to one side of centrophilic chromosomes off the mitotic spindle and to late-moving chromosomes on the mitotic spindle. In both situations, chromosome movement was initiated in the direction opposite to that of the applied force. When the force was applied, chromosomes moved at velocities 10-20 times normal. These studies verify and extend the feasibility of using this new technique to study factors that influence organelle motility.

Animals↗

PubMed1989

Singlet oxygen generation of porphyrins, chlorins, and phthalocyanines.

The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin II, TPPS4), chlorins (MACE, DACE), and a phthalocyanine (CASPc). Buffered solutions of sensitizers and singlet oxygen acceptors were irradiated with a CW dye laser and the oxygen depletion was monitored electrochemically with a Clark-type microelectrode. A comparison of oxygen-depletion rate constants and quantum efficiencies yields the order of efficiency of the sensitizers: TPPS4 greater than MACE greater than PII greater than DACE greater than CASPc. For singlet oxygen acceptors the order was: furfuryl alcohol greater than imidazole greater than tryptophan. CHO cell suspensions were also used as acceptors. Here the order of efficiency (per absorbed photon) was PII greater than MACE approximately CASPc. Expressed in terms of oxygen depletion per cell the order was CASPc approximately PII greater than MACE. When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc greater than PII greater than MACE approximately DACE. The discrepancy between the efficiencies of sensitizers to generate singlet oxygen and their cytotoxicity was explained in terms of photodegradation (for the chlorins), intracellular localization (for PII), and contributions from a Type I mechanism (for CASPc).

Animals↗

PubMed1989

Mutation and sister chromatid exchange induction in Chinese hamster ovary (CHO) cells by pulsed excimer laser radiation at 193 nm and 308 nm and continuous UV radiation at 254 nm.

We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose-related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.

Animals↗

PubMed1989

Skin photosensitivity and photodestruction of several potential photodynamic sensitizers.

The major side effect associated with porphyrins (Photofrin II) in clinical photodynamic therapy is skin photosensitivity. In order to avoid this deleterious reaction, patients must remain out of the sunlight for approximately 1 month. A possible procedure to reduce the amount of skin photosensitivity is to photodegrade (photobleach) the compound in the skin. In this study, we report a series of experiments describing the photodegradation rates of two photosensitizers currently receiving attention due to their potential for use in PDT (mono L-aspartyl chlorin e6 and chloroaluminum sulfonated phthalocyanine). These compounds are compared to Photofrin II (PfII). Experiments consisted of determining photodegradation rates and efficiencies of the sensitizers in (i) phosphate buffered saline (PBS), (ii) PBS with fetal calf serum (to enhance absorption and simulate cellular binding or deaggregation), (iii) Chinese Hamster Ovary cells, and (iv) Balb/c mice. We performed two standardized skin sensitivity assays using the Hartely albino guinea pig irradiated with a UV blue point lamp and Balb/c mice irradiated with the therapeutic wavelength of each sensitizer. In addition, we performed a cell clonogenicity assay comparing photodegraded and fresh PfII on CHO cells. The photodegraded PfII exhibited significant phototoxicity, although the fluorescence was bleached by more than 70%. The results show that PfII causes major skin photosensitization and that the other compounds produce no substantial skin sensitivity. Our studies suggest that photodegradation of PfII with 630 nm light has little influence on the phototoxicity of the compound. In addition, skin sensitivity was not alleviated with prior photobleaching with 405 nm light.

Animals↗

PubMed1989

Micromanipulation of sperm by a laser generated optical trap.

The force generated by the radiation pressure of a low power laser beam induces an optical trap which may be used to manipulate sperm. We studied the effect of the optical trap on sperm motility. A Nd:YAG laser beam was coupled to a conventional microscope and focused into the viewing plane by the objective lens. Sperm were caught in the trap and manipulated by a joy stick controlled motorized stage. After different exposure periods, the velocity and patterns were analysed by a computerized image processor. There were minor changes in sperm velocity when exposed to the trap for 30 seconds or less. A gradual decrease in the mean linear velocity was observed after 45 seconds of exposure. This optical micromanipulator may also be useful for studying the force generated by a single spermatozoa and evaluating the influence of drugs on motility.

Humans↗

PubMed1988

Mechanism of tumor destruction following photodynamic therapy with hematoporphyrin derivative, chlorin, and phthalocyanine.

The effect of photodynamic therapy on the tumor microvasculature in the first few hours after treatment was studied at the light and electron microscopy levels. BALB/c mice with EMT-6 tumor received ip injections of hematoporphyrin derivative, chlorin, or phthalocyanine, and 24 hours later, the tumors were treated with light at 100 J/cm2 at the appropriate therapeutic wavelength for each photosensitizer. Animals were killed and their tumors removed at time 0, 30 minutes, 1 hour, and 2, 4, 6, 8, 12, 16, and 24 hours after treatment. The results indicate that for all three sensitizers the effects of photodynamic therapy leading to rapid necrosis of tumor tissue are not the result of direct tumor cell kill but are secondary to destruction of the tumor microvasculature. The first observable signs of destruction occur in the subendothelial zone of the tumor capillary wall. This zone, composed of dense collagen fibers and other connective tissue elements, is destroyed in the first few hours after phototherapy. However, the ultrastructural changes seen in this zone are different for the hematoporphyrin derivative, compared with chlorin and phthalocyanine. Binding of photosensitizers to the elements in this zone as well as altered permeability and transport through the endothelial cell layer because of the increased intraluminal pressure may be key features of tumor destruction.

Animals↗

PubMed1988

Combination cytotoxic chemotherapy with cisplatin or doxorubicin and photodynamic therapy in murine tumors.

This study was designed to evaluate the interaction of photodynamic therapy (PDT) and chemotherapy in an animal model. PDT is based on the interaction of hematoporphyrin derivative and red light of the appropriate wavelength (630 nm) and intensity. Two tumor models were utilized: C3H/Km mice bearing the RIF-1 tumor and BALB/c mice bearing the EMT-6 tumor. Tumor-bearing mice were treated with either cisplatin (DDP), doxorubicin (ADM), PDT, or a combination of drug and PDT. It was demonstrated that the RIF-1 tumor was sensitive to DDP and insensitive to both PDT and ADM. There was no additional antitumor effect when either drug was combined with PDT. The EMT-6 tumor was moderately sensitive to PDT and mildly sensitive to both DDP and ADM. Although the addition of DDP did not potentiate tumor destruction, the addition of ADM significantly enhanced the effect of PDT (P = .01). The enhanced activity of the combination of PDT and ADM appeared to be the result of increased activity of ADM alone, when illuminated with red (630 nm) light. This potentiation may be due to a photochemical process or may be secondary to the mild hyperthermia generated by illumination with the laser. This study demonstrates that PDT combined with cytotoxic chemotherapy is well tolerated in these animals and that certain combinations of PDT and chemotherapy may result in an enhanced tumoricidal effect.

Animals↗

PubMed1988

In vitro characterization of monoaspartyl chlorin e6 and diaspartyl chlorin e6 for photodynamic therapy.

The characteristics of two new chlorin photosensitizers were studied in cell culture by determining phototoxicity, subcellular localization, and photophysical properties. Monoaspartyl chlorin e6 (MACE) and diaspartyl chlorin e6 (DACE) are new photosensitizers that show promise for use in photodynamic therapy. These chlorins are pure, monomeric compounds as determined by high-pressure liquid chromatography. Both compounds absorb substantially at a longer wavelength (664 nm) than does dihematoporphyrin ether-ester (DHE). Tumor diagnosis with the use of fluorescence should be facilitated due to the purity of the compounds and the single fluorescence emission peak. Phototoxicity dose-response curves of the sensitizers were completed using a standard clonogenic assay to determine cell viability. The chlorins showed good sensitizing capabilities with light. In addition, subcellular localization of MACE, DACE, and DHE was studied using fluorescence microscopy. Whereas DHE was located throughout the cytoplasm, the primary site of localization of the chlorins appeared to be in the lysosome. The results demonstrate that MACE and DACE are effective photosensitizing agents in vitro and compare favorably to DHE.

Cell Line↗

PubMed1988

Photodynamic therapy of human malignant melanoma xenografts in athymic nude mice.

While photodynamic therapy (PDT) for cutaneous malignancies including dermal recurrences of breast cancer and basal cell carcinomas has shown great promise, PDT of malignant melanoma has remained incompletely understood. A comparison study of the effects of PDT on human xenograft amelanotic and melanotic malignant melanoma in the athymic nude mouse model was performed. Twenty-four hours after ip administration of Photofrin II, the responses to total laser light doses of 25-300 J/cm2 were evaluated by histologic examination. Animals were also sacrificed 24 hours after administration of Photofrin II without light, and their uptake and localization of hematoporphyrin derivative (HpD) for each tumor were measured and compared. The results indicate that human xenograft melanotic melanoma, despite the fact that it contains more HpD than does amelanotic melanoma, is far less responsive to PDT. This result appears to be due to the competing chromophore melanin, which may inhibit the photochemical reaction at several key points.

Animals↗

PubMed1988

Ablation of bone and methacrylate by a prototype mid-infrared erbium:YAG laser.

An erbium:YAG laser was used to generate 200-microseconds pulses of mid-infrared 2.94-microns light in both the single and multimode configurations. Laser pulses were focused on the surfaces of both rabbit long bones and methacrylate blocks, and the tissue response was examined histologically. The depth of thermal injury was determined by ocular micrometry. Over all energy levels tested, the erbium:YAG laser produced ablation of bone and methacrylate with minimal thermal damage to adjacent tissue. Increasing the laser energy per pulse produced increasingly wider and deeper grooves in both bone and methacrylate. However, such increase in laser energy produced a proportionately greater increase in the zone of thermal injury in methacrylate as compared with bone. These studies suggest the feasibility of a surgical erbium:YAG laser in orthopaedics and other forms of ablative surgery.

Animals↗

PubMed1988

An acute light and electron microscopic study of ultraviolet 193-nm excimer laser corneal incisions.

The 193-nm ultraviolet beam from an argon fluoride excimer laser was focused on the corneas of rabbits to produce incisions of the type necessary for radial keratotomy. The energy densities used were in two ranges, 1.0 to 2.1 J/cm2 per pulse and 200 to 700 mJ/cm2 per pulse. The eyes were enucleated and fixed for histologic and electron microscopic examination immediately after exposure. Structural analysis of the higher energy density exposures showed ridging on the surface of the cornea, micro-pitting on the stromal surface inside the cut, and denudation of the endothelium under the ablation zone. The lower energy density incisions did not exhibit significant surface ridging or endothelial cell loss but did exhibit significant stromal swelling during the laser exposure thus making it difficult to produce incisions of a precisely controlled depth. Beam profile measurements and infrared thermal measurements of the cornea surface during laser exposure were made.

Animals↗

PubMed1988

Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd/YAG laser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by staining the treated cells with antitubulin mAb at various time points after laser irradiation. The results demonstrate that microtubules grow back into the transected zones individually; neither simultaneous growth nor shrinkage of all microtubules has been observed. The half-time of replacement of laser-dissociated microtubules is observed to be approximately 10 min. On the other hand, exposure of the core of the microtubule, which is expected to consist almost completely of GDP-tubulin, by transecting the internal regions of the microtubule does not render the remaining polymer catastrophically disassembled, and most transected microtubules with free minus ends do not quickly disappear. Taken together, these results suggest that most microtubules in cultured interphase cells exhibit some properties of dynamic instability (individual regrowth or shrinkage); however, other factors in addition to the hydrolysis of GTP-tubulin need to be involved in modulating the dynamics and the stability of these cytoplasmic microtubules.

Animals↗

PubMed1987

In vivo studies on the utilization of mono-L-aspartyl chlorin (NPe6) for photodynamic therapy.

The in vivo photosensitizing efficacy of mono-L-aspartyl chlorin has been studied by determining the percentage of BALB/c mice cured at varying doses of drug. Using an EMT-6 tumor model, animals received i.p. injections of mono-L-aspartyl chlorin (0.5-100 mg/kg) and then were subsequently exposed to light at 664 nm. Tumor biopsies were taken from selected animals sacrificed at 24 h after treatment and routine histopathological sections made. The other animals remained in the dark for a period of 6 weeks to determine the cure rate. Our results show that mono-L-aspartyl chlorin is an effective tumor localizer that brings about the selective degradation of tumor tissue following light exposure.

Animals↗

PubMed1987

Biological studies on the main fractions of hematoporphyrin derivative.

The four main fractions of hematoporphyrin derivative were separated by high-pressure liquid chromatography. Each fraction was studied with respect to photosensitizing capabilities, fluorescence, and tumor tissue uptake in mice bearing EMT-6 tumors. Animals received i.p. injections of 10 mg/kg of each fraction, and 24 h later tumors either were treated with 100 J/cm2 of light (630 nm) to evaluate photosensitizing capabilities, or the animals were sacrificed and tumors removed for fluorescence and fraction uptake determination. The results indicate that the fraction responsible for photosensitization has the highest tumor tissue uptake and retention. Furthermore, this fraction demonstrates the highest overall fluorescence localization in neoplastic tissue. The other poorly photosensitizing fractions have a lower overall fluorescence in vivo due to their poor tumor tissue localization.

Animals↗

PubMed1987

Spectroscopic, morphologic, and cytotoxic studies on major fractions of hematoporphyrin derivative and Photofrin II.

This study characterizes the special properties of hematoporphyrin derivative (HpD) (Photofrin I) and Photofrin II with respect to the absorption and fluorescence spectra of the subfractions of the compounds in both solution and cells. In addition, data are presented demonstrating variation in the spectral amplitudes with respect to the age (shelf-life) of the commercially available compounds. Data are also presented confirming that fraction III is the active ingredient-containing fraction with respect to photosensitivity. The morphologic and electron microscopic analyses demonstrate that the first visible effect on cells following porphyrin sensitization can be detected within 1 minute of low level light exposure (10 J/cm2, 630 nm). The initial sites of morphologic damage are the mitochondria.

Animals↗

PubMed1987

The thrombogenic potential of argon ion laser endarterectomy.

The surface thrombogenicity of atheromas, conventional endarterectomy (CE), laser endarterectomy (LE), and laser angioplasty (LA) were compared in the rabbit arteriosclerosis model. Normal (N = 6) and arteriosclerotic (N = 15) rabbits underwent thoracoabdominal exploration. Multiple CEs and LEs were performed in 12 arteriosclerotic rabbits leaving a segment of intact atheroma between each endarterectomy. Multiple LAs were performed in three arteriosclerotic rabbits. Argon ion laser radiation was used for all laser procedures. Blood (0.05 ml) from normal rabbits was placed on the CE surface, LE surface, LA surface, atheroma, and normal intima and clotting times were determined. Surface thrombogenicity was calculated as the ratio of the clotting time of the CE, LE, LA, or atheroma to normal intima. Surface thrombogenicity was 1.0 +/- 0.03 for normal intima (control), 0.58 +/- 0.06 for atheromas (P less than 0.001), 0.46 +/- 0.08 for CE (P less than 0.001 from atheromas), 0.46 +/- 0.08 for LE (P = NS from CE), and 0.27 +/- 0.09 for LA (P less than 0.001 from CE and LE). The thrombogenicity of LE is the same as the thrombogenicity of CE. Both forms of endarterectomy are less thrombogenic than LA in the rabbit model.

Angioplasty, Balloon↗

PubMed1987

Photosensitization of experimental atheromas by porphyrins.

Arteriosclerotic arteries have been shown to fluoresce when treated with hematoporphyrin derivative. This study investigates the incorporation and distribution of a partially purified form of hematoporphyrin derivative (Photofrin II) in normal and arteriosclerotic rabbit aortas. A thoracoabdominal exploration was performed in 15 rabbits. Group I comprised normal rabbits, Group II normal rabbits given 5 mg/kg Photofrin II 48 hours before surgery, Group III arteriosclerotic rabbits and Group IV arteriosclerotic rabbits given 5 mg/kg Photofrin II 48 hours before surgery. Multiple aortic biopsy specimens for frozen section were taken from all rabbits. In addition, open laser endarterectomy (with an argon ion laser) was performed on Group III and Group IV rabbits. Frozen sections were studied by digital video fluorescence microscopy to determine the distribution of Photofrin II within the layers of the aortic wall. The fluorescence of the intima of Group IV rabbits was found to be significantly greater than that of the intima, internal elastic lamina, media or adventitia of the other groups (p less than 0.01) and significantly greater than that of the internal elastic lamina, media or adventitia of Group IV rabbits (p less than 0.01). When open laser endarterectomy was performed, Group III rabbits required 103 +/- 14 J/cm2 and Group IV required 33 +/- 3 J/cm2 (p less than 0.01). It is concluded that porphyrins are selectively localized within the intima of arteriosclerotic arteries. This localization sensitizes atheromas to argon ion laser light and facilitates laser endarterectomy.

Animals↗

PubMed1987

Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane.

The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 X 10(-4)-3 X 10(-3). This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

Animals↗