Marinesco bodies and substantia nigra neuron density in Parkinson's disease.

AIM: Marinesco bodies (MB) are intranuclear inclusions in pigmented neurons of the substantia nigra (SN). While rare in children, frequency increases with normal ageing and is high in Alzheimer's disease, dementia with Lewy bodies and other neurodegenerative disorders. Coinciding with the age-related rise in MB frequency is initiation of cell death among SN neurons. Whether MB have a role in this process is unknown. Our aim is to examine the association of MB with SN neuron density in Parkinson's disease (PD) in the Honolulu-Asia Aging Study. METHODS: Data on MB and neuron density were measured in SN transverse sections in 131 autopsied men aged 73-99 years at the time of death from 1992 to 2007. RESULTS: Marinesco body frequency was low in the presence vs. absence of PD (2.3% vs. 6.6%, P < 0.001). After PD onset, MB frequency declined as duration of PD increased (P = 0.006). Similar patterns were observed for SN neuron density. When MB frequency was low, neuron density was noticeably reduced in the SN ventrolateral quadrant, the region most vulnerable to PD neurodegeneration. Low MB frequency was unique to PD as its high frequency in non-PD cases was unrelated to parkinsonian signs and incidental Lewy bodies. Frequency was high in the presence of Alzheimer's disease and apolipoprotein ε4 alleles. CONCLUSIONS: While findings confirm that MB frequency is low in PD, declines in MB frequency continue with PD duration. The extent to which MB have a distinct relationship with PD warrants clarification. Further studies of MB could be important in understanding PD processes.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

BACKGROUND: Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. OBJECTIVES: To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. METHODS: Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. RESULTS: Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. CONCLUSIONS: PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133+ /CD166+ /EphB1+ /EfnB2+ , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures.

Topical axitinib suppresses angiogenesis pathways induced by pulsed dye laser.

BACKGROUND: The recurrence of port-wine stain (PWS) blood vessels by pulsed dye laser (PDL)-induced angiogenesis is a critical barrier that must be overcome to achieve a better therapeutic outcome. OBJECTIVES: To determine whether PDL-induced angiogenesis can be suppressed by topical axitinib. METHODS: The mRNA expression profiles of 86 angiogenic genes and phosphorylation levels of extracellular signal regulated kinases (ERKs), phosphorylated protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) in rodent skin were examined with or without topical axitinib administration after PDL exposure. RESULTS: The PDL-induced increased transcriptional levels of angiogenic genes peaked at days 3-7 post-PDL exposure. Topical application of 0·5% axitinib effectively suppressed the PDL-induced increase in mRNA levels of the examined angiogenic genes and activation of AKT, P70S6K and ERK from days 1 to 7 post-PDL exposure. After topical administration, axitinib penetrated into rodent skin to an approximate depth of 929·5 µm. CONCLUSIONS: Topical application of 0·5% axitinib can systematically suppress the PDL-induced early stages of angiogenesis via inhibition of the AKT/mammalian target of rapamycin/p70S6K and Src homology 2 domain containing transforming protein-1/mitogen-activated protein kinase kinase/ERK pathway cascades.

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo.

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.

Improving fertility in beef cow recipients.

In the 1970s, bovine embryo transfer (ET) shifted from research in a laboratory environment to commercialization of this technology for beef producers. With the quarantine requirements and expense of importing Continental breeds of cattle from Europe, embryo transfer became the logical means to reproduce greater numbers of these animals at a lower cost. The ET industry grew very rapidly and soon would become what it is today, a common practice utilized by select ranchers and breeders. Research over the years has primarily focused on methods to increase the number of ovulations and fertilized ova from the donor female, but the total number of transferable embryos has not changed markedly in the last 20 years. More recent advances have been in the area of in vitro production of embryos that allow for greater numbers of embryos to be produced and easier accessibility to incorporate technologies such as sexed sperm, sperm injection, or transgenics. This paper will focus on the second part of the equation, the recipient, and decisions that will enable both the customers and practitioners to most efficiently utilize embryos from superovulation, in vitro production, or nuclear transfer, so that the maximum number of pregnancies can be produced.

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.

Comparison of fluorescence and photodynamic activities of whole hematoporphyrin derivative and its enriched active components.

The in vivo biologic activities of the hematoporphyrin derivative (Photofrin) and the enriched, so-called "active fraction" (Photofrin II) were determined by measuring the necrosis produced in implanted tumors in DBA/2Ha mice exposed to various total doses of light (20-100 J/cm2) after ip administration of 10 mg/kg standard doses of either Photofrin or Photofrin II. Total relative percentage increase in fluorescence in tumor tissue, as compared to fluorescence in control tissue, also was measured for both Photofrin and Photofrin II. In response to total light doses (630 nm) of 40-100 J/cm2, mice that received Photofrin had comparable amounts of tumor necrosis to those mice that received Photofrin II. At doses of 40-60 J/cm2, 80% tumor destruction resulted, and at 80-100 J/cm2, tumor destruction was 100%. However, at a total light dose of 20 J/cm2, the tumors that received Photofrin II exhibited 60-80% tumor necrosis, whereas those animals that received Photofrin had only small areas of patchy necrosis associated with signs of vascular thrombosis and hemorrhage into the surrounding perivascular stroma. A 25.2% total increase in maximal tissue fluorescence over that in controls was observed for animals that received Photofrin II, as compared to 13.9% for those animals that received Photofrin. It is concluded that the greater demonstrable efficacy of treatment with Photofrin II, as compared to treatment with Photofrin, is due to enrichment of those nonpolar hydrophobic components of the hematoporphyrin derivative mixture that are thought to be primarily responsible for the in vivo biologic activities.

Use of multiple photosensitizers and wavelengths during photodynamic therapy: a new approach to enhance tumor eradication.

Several studies have examined the synergism of hyperthermia or chemotherapy agents in combination with photodynamic therapy (PDT) to enhance tumor eradication. In our unique approach to treatment, multiple photosensitizers and wavelengths were used: two photosensitizers, Photofrin II and meso-tetra-(4-sulfonatophenyl)-porphine (TPPS4), irradiated at the appropriate therapeutic wavelength for each photosensitizer. EMT-6 mammary tumors were induced in the flanks of BALB/c mice. The mice were assigned to a control group (50 mice) or treatment group (150 mice). All treatment animals and some control animals received photosensitizing drug (5 mg/kg of TPPS4, 5 mg/kg of Photofrin II, or 2.5 mg/kg of both TPPS4 and Photofrin II). All treatment animals and some control animals also received light treatment (630 nm for TPPS4 and/or 658 nm for Photofrin II). The results show that the approach using both drugs and the corresponding therapeutic wavelengths enhanced the effectiveness of PDT. This approach achieved a cure rate of up to 100%, which was, depending on the light intensity used, as much as 40% greater than the rate achieved by the approach using one drug and one wavelength. The results also show that lesser amounts of drug and/or light may be required if both drugs and wavelengths are used, thus lowering the chances of side effects common to PDT. Furthermore, the results indicate that the increased tumor kill is due to a synergistic effect of the two photosensitizers that was tested on the tumor microvasculature in the first few hours after PDT.

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.

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.

Recursive partitioning analysis of prognostic factors in three Radiation Therapy Oncology Group malignant glioma trials.

BACKGROUND: Despite notable technical advances in therapy for malignant gliomas during the past decade, improved patient survival has not been clearly documented, suggesting that pretreatment prognostic factors influence outcome more than minor modifications in therapy. Age, performance status, and tumor histopathology have been identified as the pretreatment variables most predictive of survival outcome. However, an analysis of the association of survival with both pretreatment characteristics and treatment-related variables is necessary to assure reliable evaluation of new approaches for treatment of malignant glioma. PURPOSE: This study of malignant glioma patients used a non-parametric statistical technique to examine the associations of both pretreatment patient and tumor characteristics and treatment-related variables with survival duration. This technique was used to identify subgroups with survival rates sufficiently different to create improvements in the design and stratification of clinical trials. METHODS: We used a recursive partitioning technique to analyze survival in 1578 patients entered in three Radiation Therapy Oncology Group malignant glioma trials from 1974 to 1989 that used several radiation therapy (RT) regimens with and without chemotherapy or a radiation sensitizer. This approach creates a regression tree according to prognostic variables that classifies patients into homogeneous subsets by survival. Twenty-six pretreatment characteristics and six treatment-related variables were analyzed. RESULTS: The years). Patients younger than 50 years old were categorized by histology (astrocytomas with anaplastic or atypical foci [AAF] versus glioblastoma multiforme [GBM]) and subsequently by normal or abnormal mental status for AAF patients and by performance status for those with GBM. For patients aged 50 years or older, performance status was the most important variable, with normal or abnormal mental status creating the only significant split in the poorer performance status group. Treatment-related variables produced a subgroup showing significant differences only for better performance status GBM patients over age 50 (by extent of surgery and RT dose). Median survival times were 4.7-58.6 months for the 12 subgroups resulting from this analysis, which ranged in size from 32 to 256 patients. CONCLUSIONS: This approach permits examination of the interaction between prognostic variables not possible with other forms of multivariate analysis. IMPLICATIONS: The recursive partitioning technique can be employed to refine the stratification and design of malignant glioma trials.

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.

Histopathological comparison of the effects of hematoporphyrin derivative on two different murine tumors using computer-enhanced digital video fluorescence microscopy.

A comparison study of the effects of hematoporphyrin derivative (HPD) photoradiation therapy on two different mouse sarcoma tumor model systems (RIF-1 + EMT-6) was performed. Twenty-four h after i.v. administration of HPD, the responses to total laser light doses of 50-400 J/cm2 were evaluated by histological examination and the uptake and distribution of HPD using a computer enhanced digital video fluorescence microscopy technique. In response to total laser light dose (630 nm) of 50-400 J/cm2, 40 mice with RIF-1 tumor showed only minimal superficial tumor necrosis upon histological examination and a 9-12% increase in maximal tissue fluorescence. In contrast, 40 mice with EMT-6 tumor showed marked areas of patchy coagulation necrosis and vascular hemorrhage at doses as low as 50 J/cm2 and essentially total tumor destruction at total light doses of 150 J/cm2 or more. A 59-74% increase in maximal tissue fluorescence was observed using digital video fluorescence microscopy. It is concluded that the greater efficacy of treatment in the EMT-6 tumor as compared to the RIF-1 tumor was due to the greater localization of HPD as demonstrated by digital video fluorescence microscopy.

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.

Mechanisms underlying reduced growth rate in C3HBA mammary adenocarcinomas recurring after single doses of x-rays or fast neutrons.

C3HBA mammary tumors were irradiated with 3000 rads of 250-kVp X-rays or 1000 rads of 8-MeV neutrons, doses of radiation matched for producing equal growth delay. At 14 days postirradiation, tumors were regrowing at a reduced rate relative to controls. Cell kinetic parameters were examined using percentage of labeled mitoses techniques, and blood vessel spacing and tumor architecture were examined histologically to determine whether the mechanisms underlying growth rate changes were the same after neutron as after photon irradiation. The tumor volume-doubling time at 14 days posttreatment is similar in both irradiated groups (TD=117 hr for neutron-irradiated tumors, 132 hr for X-irradiated tumors) and is approximately twice as long as the doubling time of 61.4 hr in control tumors in the same size range. Control and X-irradiated tumors have median cell cycle durations of 19.3 and 18.5 hr, respectively; the more slowly growing X-irradiated tumors have a reduced growth fraction and increased cell loss factor. Regrowing neutron-irradiated tumors have a median cell cycle of 27.2 hr, with calculated growth fraction and cell loss factor values intermediate between those for control and X-irradiated tumors. Scatter in the percentage of labeled mitoses data makes it difficult to determine whether the cell cycle durations are significantly different. The average distance from tumor parenchymal interphase cells to the nearest recognizable blood vessel is nearly identical in the two irradiated groups and for both groups is significantly greater than interphase to vessel distance in controls. The average distance in irradiated tumors approaches the maximal distance for O2 diffusion in mouse adenocarcinomas of a corded structure surrounding a central blood vessel. Both neutron- and X-irradiated tumors contain more necrosis and fewer viable-appearing parenchymal cells than do control tumors of the same size. The similar growth rate and growth delay in this tumor after 3000 rads of X-rays of 1000 rads of neutrons occur in the face of possibly different cell cycle durations and seem related to similar circulatory system inadequacies which limit growth and are expressed as greater average cell-to-blood-vessel distance and increased cell loss leading to necrosis, indicating oxygen or nutrient deprivation.

Estimation of tumor growth fraction in murine tumors by the primer-available DNA-dependent DNA polymerase assay.

The tumor growth fraction measured by the percentage labeled mitoses method has been determined in transplantable solid and ascites murine tumors, the latter being measured at different times after transplantation. These values were compared to an in vitro, autoradiographic assay that determines the fraction of cells in a given population (primer-available DNA-dependent DNA polymerase index) that have both nuclear DNA-dependent DNA polymerase and DNA capable of acting as primer-template. It appears that almost all cells with a short G1 phase duration (less than 19 hr) that are within the proliferative cycle are primer-available DNA-dependent DNA polymerase positive. The results of the comparison indicate that the primer-available DNA-dependent DNA polymerase index estimation of growth fraction is very nearly identical to the growth fraction measured by the percentage labeled mitoses method.