Cytohesin-associated scaffolding protein (CASP) is a substrate for granzyme B and ubiquitination.

Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.

Cytohesin-associated scaffolding protein (CASP) is involved in migration and IFN-γ secretion in natural killer cells.

Natural Killer (NK) cells are highly mobile, specialized sub-populations of lymphocytic cells that survey their host to identify and eliminate infected or tumor cells. They are one of the key players in innate immunity and do not need prior activation through antigen recognition to deliver cytotoxic packages and release messenger chemicals to recruit immune cells. Cytohesin associated scaffolding protein (CASP) is a highly expressed lymphocyte adaptor protein that forms complexes with vesicles and sorting proteins including SNX27 and Cytohesin-1. In this study we show that by using stably integrated shRNA, CASP has a direct role in the secretion of IFN-γ, and NK cell motility and ability to kill tumor cells. CASP polarizes to the leading edge of migrating NK cells, and to the immunological synapse when engaged with tumor cells. However, CASP is not associated with cytotoxic granule mediated killing. CASP is a multi-faceted protein, which has a very diverse role in NK cell specific immune functions.

A review of piscine islet xenotransplantation using wild-type tilapia donors and the production of transgenic tilapia expressing a "humanized" tilapia insulin.

Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long-term normoglycemia and provide human-like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T-cell-dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co-stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.

Cloning and molecular characterization of the glucose transporter 1 in tilapia (Oreochromis niloticus).

Facilitative glucose transporters (GLUTs) are responsible for passively transporting monosaccharides across the plasma membrane. We sequenced and characterized the Nile tilapia (Oreochromis niloticus) GLUT-1 (tGLUT-1) cDNA and genomic DNA. Using rapid amplification of the cDNA ends (RACE), two tGLUT-1 transcripts were detected differing in the length of the 3' untranslated region, 2851 and 4577 bp. Translated tGLUT-1 is a 490 amino acid product, which shares 74% homology with that of humans. Computer analysis of the amino acid sequence predicted 12 transmembrane domains, which are conserved in the GLUT-1 of various species. The tGLUT-1 gene spans more than 11 kb, and similar to the mammalian GLUT-1 genes has a 10 exon, 9 intron organization. Potential promoter regulatory elements have some similarity to those recorded for human, mouse, and rat GLUT-1 genes. Tissue expression studies revealed both GLUT-1 transcripts in liver, Brockmann bodies (BB), heart, small intestine, adipose tissue, white and red muscle, gill, spleen, pituitary gland, and brain. The highest level of expression was detected in tilapia heart, followed by BB, brain, and muscle. Protein based food and glucose had minor or no effects on the level of tGLUT-1 expression in most tissues. The tGLUT-1 mRNA level was significantly induced by glucose and food only in white muscle. Current results suggest that tGLUT-1 is similar to the GLUT-1 of other teleost species and mammals at the genomic, mRNA, and amino acid levels, supporting the concept that tGLUT-1 functions as a ubiquitous basal level glucose transporter.

Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax.

BACKGROUND: Melanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax (VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination. METHODS: C57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232-240, TRP-2: 181-188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT. RESULTS: Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-gamma producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-gamma producing splenocytes to one CTL epitopes (TRP2:180-188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice. CONCLUSION: A single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors.

Rejection of large HPV-16 expressing tumors in aged mice by a single immunization of VacciMax encapsulated CTL/T helper peptides.

The incidence of cancer increases significantly in later life, yet few pre-clinical studies of cancer immunotherapy use mice of advanced age. A novel vaccine delivery platform (VacciMax,VM) is described that encapsulates antigens and adjuvants in multilamellar liposomes in a water-in-oil emulsion. The therapeutic potential of VM-based vaccines administered as a single dose was tested in HLA-A2 transgenic mice of advanced age (48-58 weeks old) bearing large palpable TC1/A2 tumors. The VM-based vaccines contained one or more peptides having human CTL epitopes derived from HPV 16 E6 and E7. VM formulations contained a single peptide, a mixture of four peptides or the same four peptides linked together in a single long peptide. All VM formulations contained PADRE and CpG as adjuvants and ISA51 as the hydrophobic component of the water-in-oil emulsion. VM-formulated vaccines containing the four peptides as a mixture or linked together in one long peptide eradicated 19-day old established tumors within 21 days of immunization. Peptide-specific cytotoxic cellular responses were confirmed by ELISPOT and intracellular staining for IFN-gamma producing CD8+ T cells. Mice rendered tumor-free by vaccination were re-challenged in the opposite flank with 10 million HLF-16 tumor cells, another HLA-A2/E6/E7 expressing tumor cell line. None of these mice developed tumors following the re-challenge. In summary, this report describes a VM-formulated therapeutic vaccine with the following unprecedented outcome: a) eradication of large tumors (> 700 mm3) b) in mice of advanced age c) in less than three weeks post-immunization d) following a single vaccination.

Insulin expression in the brain and pituitary cells of tilapia (Oreochromis niloticus).

While the presence of immunoreactive insulin in the central nervous system of many vertebrate species is well known, the origin of brain insulin is still debated. In this study, we applied RT-PCR, quantitative RT-PCR (qRT-PCR), and Northern hybridization to examine expression of the insulin gene in different tissues of an adult teleost fish, the Nile Tilapia (Oreochromis niloticus). We found that the insulin gene is transcribed at a high level in Brockmann bodies (pancreatic islet organs) and at a low level in the brain and pituitary gland. In the brain, insulin transcripts were detected in all areas by qRT-PCR and in situ hybridization. The highest level of insulin mRNA was found in the hypothalamus. The level of insulin transcription in the pituitary gland was 6-fold higher than that in the brain and 4.6-fold higher than that in the hypothalamus. Furthermore, insulin mRNA and immunoreactive insulin-like protein was detected in the pituitary gland using in situ hybridization, immunohistochemistry, and Western blot analysis. Our results indicate that in adult tilapia insulin expression is not restricted to the endocrine pancreatic cells, but also occurs in endocrine cells of the pituitary gland and in the neuronal cells of the brain, suggesting that the brain/pituitary gland might represent extrapancreatic origin of insulin production.

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene.

Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish.

Survey of zona pellucida antigens for immunocontraception of cats.

The purpose of this study was to screen a panel of native zona pellucida (ZP) antigens isolated from five mammalian species for immunocontraceptive activity in the cat (Felis catus). Native soluble-isolated ZP (SIZP) was prepared from the ovaries of cows (bZP), cats (fZP), ferrets (feZP), dogs (cZP), and mink (mZP). Vaccines were constructed using SIZP from each of the above species encapsulated in liposomes suspended in saline and emulsified with Freund's complete adjuvant (SpayVac). Female cats were immunized once (n = 3 cats per group). Serum was collected for determination of antibody titers against SIZP and for binding of antibodies to feline ovaries. All cats responded to immunization by producing anti-SIZP antibodies. The most immunogenic SIZP in cats was from mink, followed by feZP, cZP, and fZP in descending order. Antibodies had low reactivity for fZP, and no reactivity against feline ovaries was detected by immunohistochemistry. A breeding trial was commenced 20 weeks after immunization. All cats became pregnant, averaging 4.1 +/- 0.7 viable kittens per litter. We have previously shown that porcine SIZP is not an effective antigen for immunocontraception of cats. In this study, SIZP from five other mammalian species were immunogenic in the cat, but ZP antibodies failed to bind to fZP in situ, and fertility was not impeded.

Development of the islets, exocrine pancreas, and related ducts in the Nile tilapia, Oreochromis niloticus (Pisces: Cichlidae).

Pancreatic development and the relationship of the islets with the pancreatic, hepatic, and bile ducts were studied in the Nile tilapia, Oreochromis niloticus, from hatching to the onset of maturity at 7 months. The number of islets formed during development was counted, using either serial sections or dithizone staining of isolated islets. There was a general increase in islet number with both age and size. Tilapia housed in individual tanks grew more quickly and had more islets than siblings of the same age left in crowded conditions. The pancreas is a compact organ in early development, and at 1 day posthatch (dph) a single principal islet, positive for all hormones tested (insulin, SST-14, SST-28, glucagon, and PYY), is partially surrounded by exocrine pancreas. However, the exocrine pancreas becomes more disseminated in older fish, following blood vessels along the mesenteries and entering the liver to form a hepatopancreas. The epithelium of the pancreatic duct system from the intercalated ducts to the main duct entering the duodenum was positive for glucagon and SST-14 in 8 and 16 dph tilapia. Individual insulin-immunopositive cells were found in one specimen. At this early stage in development, therefore, the pancreatic duct epithelial cells appear to be pluripotent and may give rise to the small islets found near the pancreatic ducts in 16-37 dph tilapia. Glucagon, SST-14, and some PPY-positive enteroendocrine cells were present in the intestine of the 8 dph larva and in the first part of the intestine of the 16 dph juvenile. Glucagon and SST-14-positive inclusions were found in the apical cytoplasm of the mid-gut epithelium of the 16 dph tilapia. These hormones may have been absorbed from the gut lumen, since they are produced in both the pancreatic ducts and the enteroendocrine cells. At least three hepatic ducts join the cystic duct to form the bile duct, which runs alongside the pancreatic duct to the duodenum.

Piscine islet xenotransplantation.

Tilapia, a teleost fish species with large anatomically discrete islet organs (Brockmann bodies; BBs) that can be easily harvested without expensive and fickle islet isolation procedures, make an excellent donor species for experimental islet xenotransplantation research. When transplanted into streptozotocin-diabetic nude or severe combined immunodeficient mice, BBs provide long-term normoglycemia and mammalian-like glucose tolerance profiles. However, when transplanted into euthymic recipients, the mechanism of islet xenograft rejection appears very similar to that of islets from "large animal" donor species such as the very popular fetal/neonatal porcine islet cell clusters (ICCs). Tilapia islets are more versatile than ICCs and can be transplanted (1) into the renal subcapsular space, the cryptorchid or noncryptorchid testis, or intraportally as neovascularized cell transplants; (2) as directly vascularized organ transplants; or (3) intraperitoneally after microencapsulation. Unlike the popular porcine ICCs, BBs function immediately after transplantation; thus, their rejection can be assessed on the basis of loss of function as well as other parameters. We have also shown that transplantation of tilapia BBs into nude mice can be used to study the possible implications of cross-species physiological incompatibilities in xenotransplantation. Unfortunately, tilapia BBs might be unsuitable for clinical islet xenotransplantation because tilapia insulin differs from human insulin by 17 amino acids and, thus, would be immunogenic and less biologically active in humans. Therefore, we have produced transgenic tilapia that express a "humanized" tilapia insulin gene. Future improvements on these transgenic fish may allow tilapia to play an important role in clinical islet xenotransplantation.

Getting a GRASP on CASP: properties and role of the cytohesin-associated scaffolding protein in immunity.

Cytohesin-associated scaffolding protein (CASP) is a novel human adaptor protein that participates in the assembly and recruitment of protein complexes associated with intracellular trafficking and signaling. Owing to its exclusive expression in cells of hematopoietic origin, CASP has attracted attention from many groups of researchers as a potential key contributor to molecular mechanisms governing cells of the immune system. The functional characterization of CASP has involved a wide range of experimental approaches and provided broad and interesting insights that, collectively, distinguish CASP as an important contributor for a fully functioning and rapidly responsive immune system. Protein interaction studies have demonstrated that CASP interacts with members of the ADP ribosylation factor (ARF)-activating cytohesin family and with a unique PDZ domain-containing member of the sorting nexin family of endocytic trafficking proteins. Physiological knockout studies, however, have revealed that CASP may not be an essential protein in immunity under normal conditions, but rather a streamlining protein that greatly ameliorates the efficiency of the immune system under circumstances of significant stress. Interestingly, an evolutionarily related neuronal protein, called GRP1-associated scaffolding protein, may further participate in CASP-related functions in immune cells, conferring a level of redundancy in associated molecular pathways. In this review, we summarize and critically review the current literature, bringing together common themes from a variety of studies that, when considered together, provide new insights into the nature and significance of CASP function in the broad context of immunity.

Regulation of insulin gene expression and insulin production in Nile tilapia (Oreochromis niloticus).

Compared to mammals, little is known about insulin gene expression in fish. Using transient transfection experiments and mammalian insulinoma cell lines we demonstrate that transcription of the Nile tilapia (Oreochromis niloticus) insulin gene is (a) regulated in a beta-cell-specific manner; and (b) not sensitive to the glucose stimulations. Deletion analysis of the 1575 bp 5' insulin gene flanking sequence revealed that cooperative interactions between regulatory elements within the proximal (-1 to -396 bp) and the distal (-396 bp to -1575 bp) promoter regions were necessary for induction of the beta-cell-specific transcription. Effects of glucose and arginine on endogenous insulin secretion, translation, and transcription in isolated tilapia Brockmann bodies were determined using Northern hybridization, Western analysis, and quantitative RT-PCR. Similar to the regulation of mammalian insulin, we found that increases of glucose (1-70 mM) and arginine (0.4-25 mM) induced insulin secretion. However, transcription of the insulin gene was activated only by extremely high concentrations of glucose and arginine added simultaneously. When stimulated for 24 h with low concentrations of both inducers or with either of them added separately, tilapia beta-cells were able to replenish secreted insulin and to maintain insulin stores at a constant level without elevations of the insulin mRNA levels. Since the basal level of insulin mRNA was approximately 3.7-fold higher in tilapia beta-cells than it is in mammalian beta-cells, insulin production in tilapia cells probably relies on an enlarged intracellular insulin mRNA pool and does not require the transcriptional activation of the insulin gene.

Gene duplication in early vertebrates results in tissue-specific subfunctionalized adaptor proteins: CASP and GRASP.

CASP and GRASP are small cytoplasmic adaptor proteins that share highly similar protein structures as well as an association with the cytohesin/ARNO family of guanine nucleotide exchange factors within the immune and nervous systems respectively. Each contains an N-terminal PDZ domain, a central coiled-coil motif, and a carboxy-terminal PDZ-binding motif (PDZbm). We set out to further characterize the relationship between CASP and GRASP by comparing both their gene structures and their functional motifs across several vertebrate organisms. CASP and GRASP not only share significant protein structure but also share remarkably similar gene structure, with six of eight exons of equal length and relative position. We report on the addition of a unique amino acid within the coiled-coil motif of CASP proteins in several species. We also examine the Class I PDZbm, which is highly conserved across all classes of vertebrates but shows a functionally relevant mutation in the CASP proteins of several species of fish. Further, we determine the evolutionary relationship of these proteins both by use of phylogenetics and by comparative analysis of the conservation of genes near each locus in various chordates including amphioxus. We conclude that CASP and GRASP are the products of a relatively recent gene duplication event in early vertebrate organisms and that the evolution of the adaptive immune system and complex brain most likely contributed to the apparent subfunctionalization of these proteins into tissue-specific roles.

Polarization of endosomal SNX27 in migrating and tumor-engaged natural killer cells.

Polarization is a critical mechanism for the proper functioning of many cell types. For lymphocytes, it is essential in a variety of processes, including migration from the blood to other tissue sites and vice versa. In NK cells and CTLs, the cytotoxic granule delivery mechanism requires polarization for granule movement to the immunological synapse (IS), in killing tumor and virus-infected cells. Recently, it has become apparent that endosomes are also involved in the cytotoxic mechanism. Using an in vitro conjugation approach, we show that in NK-92 cells, endosomal Sorting Nexin 27 (SNX27) polarizes to the IS during tumor cell engagement in a distinct compartment adjacent to the cytotoxic granules. We also show that SNX27 polarizes to the apical membrane, opposite the uropod, during NK cell migration. These previously unreported results indicate that SNX27 is a participant in NK cell polarization, as a mediator or target of the mechanism.

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes.

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.

Things we have learned from tilapia islet xenotransplantation.

An islet xenotransplantation model has been developed using tilapia (Oreochromis niloticus) as the donors. Studies using this model for the treatment of experimental type 1 diabetes in mice have produced promising results including the maintenance of long-term normoglycemia and mammalian-like glucose tolerance profiles in islet graft recipients. Islet encapsulation has also provided a promising method for the prevention of graft rejection, and strains of transgenic tilapia expressing a [desThrB30] human insulin molecule have been produced. In addition to studying islet transplantation for the treatment of type 1 diabetes, these studies have also produced insights into piscine glucose homeostasis. Studies demonstrating the glucose responsiveness of tilapia islets are described. In addition, work performed by our group and by others pertaining to presence and nature of piscine glucose transporters is reviewed. Finally, studies addressing some of the broader challenges of islet xenotransplantation are discussed with particular attention paid to the post-transplantation fate of the various islet cell populations and the proteins they produce.

Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion.

Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide with half a million new cases per year. Despite the encouraging development of a preventive vaccine for HPV, a therapeutic vaccine for cervical cancer or pre-cancerous lesions remains a high priority. The preclinical study reported here used VacciMax((R)) (VM) to deliver a peptide-based vaccine composed of an HPV 16 E7-derived cytotoxic T lymphocyte (CTL) epitope fused to the T helper epitope PADRE (FP) and combined with CpG or lipopeptide adjuvant. In the study, C57BL/6 mice received 0.5million HPV 16-expressing C3 tumor cells. Mice were inoculated post-tumor challenge with a single s.c. injection of FP-CpG-VM on either day 4, 5, 6, 9, or 14. All mice that received the FP-CpG-VM vaccine were tumor-free to day 130 when the experiment was terminated. In contrast, only a minority of mice that received a control vaccine were tumor-free on day 60. Cytotoxicity assays, ELISPOT and intracellular staining for interferon (IFN)-gamma showed the immune response was specific for the selected CTL epitope. All mice that received the FP-CpG-VM vaccine remained tumor-free when re-challenged with 6million C3 cells. Cytotoxicity assays 4 months post-challenge showed that only splenocytes from mice inoculated with the FP-CpG-VM vaccine had high lysis activity. These results indicate that VacciMax((R)) causes a rapid, robust, durable and therapeutic CTL response to HPV 16 E7 protein expressing tumors.