Non-invasive monitoring of ultrasound-stimulated microbubble radiation enhancement using photoacoustic imaging.

Modulation of the tumour microvasculature has been demonstrated to affect the effectiveness of radiation, stimulating the search for anti-angiogenic and vascular-disrupting treatment modalities. Microbubbles stimulated by ultrasound have recently been demonstrated as a radiation enhancer when used with different cancer models including PC3. Here, photoacoustics imaging technique was used to assess this treatment's effects on haemoglobin levels and oxygen saturation. Correlations between this modality and power doppler assessments of blood flow, and histology measurements of vascular integrity and cell death were also investigated. Xenograft prostate tumours in SCID mice were treated with 0, 2, or 8 Gy radiation combined with microbubbles exposed to 500 kHz ultrasound at a peak negative pressure of 0, 570, and 750 kPa. Tumours were assessed and levels of total haemoglobin, oxygen saturation were measured using photoacoustics before and 24 hours after treatment along with power doppler measured blood flow. Mice were then sacrificed and tumours were assessed for cell death and vascular composition using immunohistochemistry. Treatments using 8 Gy and microbubbles resulted in oxygen saturation decreasing by 28 ± 10% at 570 kPa and 25 ± 29% at 750 kPa, which corresponded to 44 ± 9% and 40 ± 14% respective decreases in blood flow as measured with power doppler. Corresponding histology indicated 31 ± 5% at 570 kPa and 37 ± 5% at 750 kPa in terms of cell death. There were drops in intact vasculature of 15 ± 2% and 20 ± 2%, for treatments at 570 kPa and 750 kPa. In summary, photoacoustic measures of total haemoglobin and oxygen saturation paralleled changes in power doppler indicators of blood flow. Destruction of tumour microvasculature with microbubble-enhanced radiation also led to decreases in blood flow and was associated with increases in cell death and decreases in intact vasculature as detected with CD31 labeling.

Ultrasound-activated microbubble cancer therapy: ceramide production leading to enhanced radiation effect in vitro.

Blood vessels within tumours represent a key component for cancer cell survival. Disruption of these vessels can be achieved by inducing vascular endothelial-cell apoptosis. Moreover, endothelial cell apoptosis has been proven to be enhanced by ceramide-increasing drugs. Herein, we introduce a novel therapeutic approach which uses ultrasound-stimulated microbubbles used in combination with radiation to cause a rapid accumulation of ceramide in endothelial cells in-vitro. We also test this modality directly with other cell types as a general method of killing cancer cells. Human umbilical vein endothelial cells (HUVEC), acute myeloid leukemia cells (AML), murine fibrosarcoma cells (KHT-C), prostate cancer cells (PC3), breast cancer cells (MDA-MB-231) and astrocytes were used to evaluate this mechanism of inducing cell death. Survival was measured by clonogenic assays, and ceramide content was detected using immunohistochemistry. Exposure of cell types to ultrasound-stimulated bubbles alone resulted in increases in ceramide for all cell types and survivals of 12 ±â€…2%, 65 ±â€…5%, 83 ±â€…2%, 58 ± 4%, 58 ±â€…3%, 18 ±â€…7% for HUVEC, AML, PC3, MDA, KHT-C and astrocyte cells, respectively. Results from selected cell types involving radiation treatments indicated additive treatment enhancements and increases in intracellular ceramide content one hour after exposure to ultrasound-activated microbubbles and radiation. Endothelial cell survival decreased from 8 ±â€…1% after 2 Gy of radiation treatment alone and from 12 ±â€…2% after ultrasound and microbubbles alone, to 1 ±â€…1% with combined treatment. In Asmase +/+ astrocytes, survival decreased from 56 ±â€…2% after 2 Gy radiation alone and from 17 ±â€…7% after ultrasound and microbubbles alone, to 5 ±â€…2% when combined. Using ASMase deficient astrocytes (Asmase -/- ) and Sphingosine-1-phosphate (S1P), we also demonstrate that ultrasound-activated microbubbles stimulate ASMase activity and ceramide production. These findings suggest that ultrasound-stimulated microbubbles could be used as a new biomechanical method to enhance the effects of radiation.

The fish endocrine pancreas: review, new data, and future research directions in ontogeny and phylogeny.

The literature on the ontogeny and phylogeny of the endocrine pancreas of ray-finned fishes is summarized since the latest review in fish [Youson, J.H., Al-Mahrouki, A.A., 1999. Review. Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Gen. Comp. Endocrinol. 116, 303-335]. A basic description and a demonstration of the diversity of the fish islet organ is provided through new immunohistochemical data on islet tissue from a basal teleost, an osteoglossomorph, and a more derived teleost, a perciforme. Unlike the previous review, the present report provides a review and discussion of the utility of sequence data of insulin, somatostatin, and NPY- and glucagon-family peptides in phylogenetic analyses of jawed and jawless fishes. The present study also provides the first comparative analysis of sequences of preprohormones of endocrine peptides from closely related basal teleost species. Some nucleotide and deduced amino acid sequence data for preprosomatostatins (PPSS-I and/or -II) are compared for four species of bonytongues, Osteoglossomorpha, and with PPSSs of the white sucker, Catostomus commersoni, representing Cypriniformes, a more generalized teleost order. Phylogenetic analysis of deduced amino acid sequences of the PPSSs of these species and others from databases indicates good support for the monophyly of Osteoglossomorpha and some support for the present taxonomic grouping of the osteoglossomorphs examined, and also the white sucker. However, PPSS may have limited phylogenetic utility due to the relative short sequence, particularly in resolving relationships among lineages that diverged over a short period of time. Since in the few fish species examined we have just touched the surface in describing the diversity of structure of the islet organ, and likely the nature of the products of its cells, this report promotes the continued study of this organ.

The endocrine cells in the gastroenteropancreatic system of the bowfin, Amia calva L.: an immunohistochemical, ultrastructural, and immunocytochemical analysis.

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species.

Molecular cloning of preproinsulin cDNAs from several osteoglossomorphs and a cyprinid.

Several preproinsulin cDNAs were isolated and characterized from four members of the Osteoglossomorpha (an ancient teleost group); Osteoglossum bicirrhosum (arawana), Pantodon buchholzi (butterfly fish), Notopterus chitala (feather fin knife fish), Hiodon alosoides (goldeye) and Gnathonemus petersii (elephantnose). In addition, we isolated and characterized the preproinsulin cDNA from Catostomus commersoni (white sucker, as a representative of a generalized teleost). The comparative analysis of the sequences revealed conservation of the cystine residues known to be involved in the formation of the disulfide bridges, as well as residues involved in the hexamer formation, except for B-17 in the butterfly fish, the arawana and the goldeye. However, the N-terminus of the B-chain was very weakly conserved among the species studied. Residues known to be significant for maintaining receptor-binding conformation and those known to comprise the receptor-binding domain were all conserved, except for a conservative substitution at B13, aspartate substituted glutamate in the arawana, goldeye, butterfly fish and white sucker, and at B16, phenylalanine substituted tyrosine in the elephantnose. Phylogenetic analysis of the sequences revealed a monophyletic grouping of the osteoglossomorphs, and showed that they were not the most basal living teleost. Comparative sequence analysis of preproinsulins among the osteoglossomorphs was useful in assessment of intergroup relationship, relating elephantnose with the feather fin knife fish and the arawana, butterfly fish, and goldeye. This arrangement of species is consistent with relationships based on other more classical parameters, except for the goldeye which was assessed as being sister to all the osteoglossomorphs. The white sucker was grouped with the common carp and both are cyprinids.

Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fish.

The morphology of the gastroenteropancreatic (GEP) system of fish was reviewed with the objective of providing the phylogenetic and ontogenetic development of the system in this vertebrate group, which includes agnathans and gnathostome cartilaginous, actinoptyerygian, and sarcopterygian fish. Particular emphasis is placed on the fish homolog of the endocrine pancreas of other vertebrates, which is referred to as the islet organ. The one-hormone islet organ (B cells) of larval lampreys is the most basic pattern seen among a free-living vertebrate, with the two-hormone islet organ (B and D cells) of hagfish and the three-hormone islet organ (B, D, and F cells) of adult lampreys implying a phylogenetic trend toward the classic four-hormone islet tissue (B, D, F, and A cells) in most other fish. An earlier stage in the development of this phylogenetic sequence in vertebrates may have been the restriction of islet-type hormones to the alimentary canal, like that seen in protochordates. The relationship of the islet organ to exocrine pancreatic tissue, or its equivalent, is variable among bony, cartilaginous, and agnathan fishes and is likely a manifestation of the early divergence of these piscine groups. Variations in pancreatic morphology between individuals of subgroups within both the lamprey and chondrichthyan taxa are consistent with their evolutionary distance. A comparison of the distribution and degree of concentration of the components of the islet organ among teleosts indicates a diffuse distribution of relatively small islets in the generalized euteleosts and the tendency for the concentration into Brockmann bodies of large (principal) islets (with or without secondary islets) in the more derived forms. The holostean actinopterygians (Amiiformes and Semiontiformes) share with the basal teleosts (osteoglossomorphs, elopomorphs) the diffuse arrangement of the components of the islet organ that is seen in generalized euteleosts. Since principal islets are also present in adult lampreys the question arises whether principal islets are a derived or a generalized feature among teleosts. There is a paucity of studies on the ontogeny of the GEP system in fish but it has been noted that the timing of the appearance of the islet cell types parallels the time that they appear during phylogeny; the theory of recapitulation has been revisited. It is stressed that the lamprey life cycle provides a good opportunity for studying the development of the GEP system. There are now several markers of cell differentiation in the mammalian endocrine pancreas which would be useful for investigating the development of the islet organ and cells of the remaining GEP system in fish.

Ultrastructure and immunocytochemistry of the islet organ of osteoglossomorpha (Teleostei).

Both routine electron microscopy and immunocytochemistry with protein A-gold were used to identify the cell types within the islet organs of four species of teleosts (Osteoglossum bicirrhosum, Pantodon buchholzi, Notopterus chitala, and Gnathonemus petersii) within Osteoglossomorpha, a subdivision with an ancient lineage. Four primary endocrine cell types, A, B, D, and F, were identified within the islets of the four species examined. The B- and D-cells were located mainly in the central core of the islet in the four species. In general, the A-cells were located at the islet periphery in all of the four species but in P. buchholzi and N. chitala they were also differently distributed toward the islet core. F-cells were present only at the islet periphery. Granules of B-cells in three species had a relatively homogeneous shape of the matrix core, but in O. bicirrhosum, the shape varied greatly. Variation in matrix shape of B-cell granules may indicate a different conformation of insulin molecules among at least some species of osteoglossomorphs, and this observation may have some taxonomic significance. Two somatostatin-containing (SST) D-cell types (D1 and DX) with granules of different shape were observed in all four species of osteoglossomorphs. The granules of the two D-cells immunostained either with anti-SST-25 and anti-SST-14 (D1-cells) or with anti-SST-34 (DX-cells). Immunocytochemistry confirmed that A-cells, containing glucagon-family peptides, and F-cells, containing peptides of the pancreatic polypeptide family, had granules of different shape. The cells of the islet organs of these osteoglossomorphs are more similar to those in more derived teleosts than they are to those of nonteleost actinopterygians.

Effects of insulin on lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus.

This study was designed to examine the role of insulin (INS) in regulating changes in lipid metabolism of larval and metamorphosing landlocked lamprey, Petromyzon marinus. Larvae and stage 6 metamorphosing individuals were injected intraperitoneally once per day for 2 days with either saline (0.6%), bovine INS (100 ng/g body weight), or alloxan (0.2 mg/g body weight). Insulin administration resulted in depressed plasma fatty acid (FA) levels, whereas alloxan injection elevated plasma FA levels at both life cycle intervals. In larvae, INS-induced hypolipidemia was attended by increased lipid concentration in kidney and muscle, reduced rates of lipolysis in kidney, liver, and muscle (as indicated by decreased triacylglycerol lipase activity), and, to a lesser extent, by higher rates of lipogenesis in kidney and muscle (as evidenced by higher acetyl-CoA carboxylase and/or diacylglycerol acyltransferase activities). In general, the effects of alloxan were opposite of those of INS. The alloxan-induced increase in plasma FA was supported by an enhanced rate of lipolysis in the kidney, a relatively lower rate of fatty acid synthesis in kidney, liver, and muscle, and a relatively lower renal rate of TG synthesis. In stage 6 metamorphosing lamprey, the INS-induced decline in plasma FA was attended by reduced renal and hepatic rates of lipolysis and by enhanced lipogenesis, as indicated by increased renal and hepatic rates of de novo fatty acid synthesis and hepatic and muscular rates of TG synthesis. In contrast, the increase in plasma FA induced by alloxan in stage 6 animals was supported by reduced TG synthesis in liver. Immunocytochemistry revealed that alloxan was not cytotoxic to pancreatic beta cells, suggesting that the effects of alloxan were extrapancreatic in the time frame of our study. Because insulin-induced lipogenesis and antilipolysis is similar to the pattern of lipid metabolism (phase I) displayed by lamprey during their spontaneous metamorphosis, INS may play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism.

Immunohistochemical studies of the endocrine cells within the gastro-entero-pancreatic system of Osteoglossomorpha, an ancient teleostean group.

The identification and distribution of endocrine cells within the gastro-entero-pancreatic (GEP) system of five species of the Osteoglossomorpha (Osteoglossum bicirrhosum, Scleropages jardini, Pantodon buchholzi, Notopterus chitala and Gnathonemus petersii) were analyzed by immunohistochemistry. Four immunoreactive cell types were identified within the pancreatic islets (A, B, D, and F cells), using antisera directed against mammalian insulin (m-INS), somatostatins (SST-14, SST-25), and members of the pancreatic polypeptide (aPY, NPY, PYY) and glucagon (GLU, GLP) families. The B cells were located throughout the center of the islets in the five species and, in general, D cells had a similar distribution. However, immunoreactivity to anti-somatostatins varied between four of the species and G. petersii, which showed less intensely stained D cells in the islets, but greater SST immunoreactivity in both the intestinal and the stomach epithelia than in comparable epithelia of other species. For peptides of both the pancreatic polypeptide and the glucagon families, the immunoreactivity was detected at the periphery of the islets, and there was a suggestion of an interfamily colocalization of peptides in some cells. In addition, glucagon family peptides showed a scattered immunoreactivity throughout the central portion of the islets. A moderately abundant number of cells in the intestine were immunoreactive to the PP family antisera in all five species. However, immunoreactivities to GLU, GLP, SST, and m-INS antisera were variable in intestinal cells of the species. Immunoreactivity with sera raised against m-INS and PYY was also observed in the stomach of P. buchholzi. The significance of these findings is discussed in both ontogenetic and phylogenetic contexts with respect to the GEP system in actinopterygian fishes and with respect to the possibility of variable processing of prohormones in the different organs of these osteoglossomorphs.