Viskam Wijewardana

Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells

Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

Effect of IL-12 on canine dendritic cell maturation following differentiation induced by granulocyte-macrophage CSF and IL-4.

IL-12 is a cytokine produced by dendritic cells (DC) and activates cytotoxic T cells and NK cells against tumors. We investigated the effect of IL-12 on DC. When peripheral blood monocytes were incubated with canine granulocyte-macrophage (GM)-CSF and IL-4, expression of MHC class II, CD1a, CD80 and CD86 was significantly elevated, but the cell morphology was that of immature DC. By adding canine IL-12 to the DC-inducing culture, expression of CD80 and CD1a was significantly enhanced, and the cell morphology shifted to that of mature DC. T cell stimulation by cultured cells was also significantly enhanced by the presence of IL-12. These results show that IL-12 significantly promotes the maturation and activation of canine DC following the induction of differentiation by GM-CSF and IL-4.

Induction of fertile oestrus in bitches by an intranasal spray of gonadotrophin- releasing hormone agonist

BITCHES are monoestrous, and ovulate only once or twice a year at fiveto 12-month intervals. A reliable method to induce ovulation and control the timing of pregnancy would aid the clinical management of prolonged anoestrus or infertility. Several protocols using various gonadotrophins have been used in attempts to induce oestrus and ovulation in anoestrous bitches (Renton and others 1984, Nakao and others 1985, England and Allen 1991). These protocols were not very effective, and would require further improvement to enable practical application. Fertile oestrus in anoestrous dogs has been induced successfully by the pulsatile administration of gonadotrophin-releasing hormone (GnRH) via an indwelling infusion catheter (Vanderlip and others 1987). However, many pet owners are reluctant to subject their animals to this procedure. In 1998, the authors presented an alternative approach to the pulsatile infusion of GnRH, using a depot-injectable form of the potent GnRH agonist leuprolide acetate (Inaba and others 1998). In human beings, the intranasal administration of a GnRH agonist is used as a painless, simple and practical method for ovarian stimulation in in vitro fertilisation (Elgendy and others 1998), central precocious puberty (Sizonenko and others 1990) and endometriosis (Franssen and others 1989). This short communication describes a study to determine whether an intranasal spray of leuprolide acetate can induce fertile oestrus in mature, anoestrous bitches, and to investigate the effect of a combination of leuprolide acetate administered intranasally and oestrogen administered orally. Twenty laboratory-bred, anoestrous beagle bitches, two to six years of age, were housed and studied in accordance with National Institutes of Health guidelines, the regulations of the local Institutional Animal Care and Use Committee, and with accepted veterinary medical practice. They were fed a commercial dog food once daily and were given water ad libitum. The time of the most recent oestrus of each bitch was known. The bitches were divided into three treatment groups: the first group, of seven bitches, was treated daily with 3·6 μg of an intranasal spray of a sustained-release formulation of leuprolide acetate (Leupron Depot; Takeda Chemical Industries). The second group, also of seven bitches, received 0·3 mg/kg estradiol-17β orally, once daily for three days, and then a daily intranasal spray of leuprolide acetate as described above. Six untreated bitches were studied as controls. The treatment with intranasal leuprolide acetate was continued either until the onset of oestrus or for up to a maximum of 14 days. The bitches were observed daily for vulval swelling and oestrous behaviour, and a vaginal smear was taken every day. The interoestrous interval was calculated as the interval between the first day of each standing oestrus. The bitches were allowed to breed on the first day of oestrus and undergo pregnancy and parturition; the length of gestation was calculated from the day of mating. Blood samples were collected daily by jugular venepuncture from the start of the treatment until the end of oestrus, in order to detect ovulation: ovulation was considered to have occurred if the level of progesterone in the plasma was more than 2 ng/ml (Wanke and others 1997). The concentration of progesterone in the plasma was measured by radioimmunoassay as described by Inaba and others (1998). The sensitivity of the assay was 10 pg per tube, and the intra-assay variation was less than 11·5 per cent. All the samples were assayed in duplicate in the same assay. The data were evaluated by analysis of variance, followed by Fisher’s protected least significant difference post hoc analysis, using the Stat View computer program (Abacus Concepts). The significance level was set at P<0·05. Behavioural signs of oestrus were induced in three of the bitches treated with leuprolide acetate alone and five of the bitches treated with leuprolide acetate and oestrogen (Table 1). These three leuprolide acetate-treated bitches showed a strong pro-oestrous and oestrous response, including normal oestrous behaviour, and accepted natural mating. Their plasma progesterone concentrations rose above 2 ng/ml during an apparently normal oestrus, and all three became pregnant and delivered normal litters. In one bitch, the vulval swelling and discharge were slight and lasted only for two to three days. The five bitches treated successfully with the combination of estradiol-17β and leuprolide acetate showed evidence of oestrus, became pregnant and delivered normal litters. All the bitches in which oestrus was not induced returned to normal oestrus within three months. The interoestrous interval of the bitches in which oestrus was induced was significantly shorter than that of the control dogs (Table 1). The litter sizes did not differ significantly between bitches that underwent induced or spontaneous oestrus (Table 1). No sign of fetal resorption or abortion was observed. The length of pro-oestrus, oestrus and gestation did not differ significantly between the groups. To the authors’ knowledge, this is the first report of the successful induction of oestrus in bitches by treatment with an intranasal spray of a GnRH agonist. One bitch appeared to respond weakly to the spray treatment, and did not allow the male to mount or show an increase in plasma progesterone above 2 ng/ml. It was assumed that the GnRH agonist stimulation was not sufficient to induce follicular growth and ovulation in this bitch. Veterinary Record (2006) 158, 378-379

Enhancement of anti-tumor immune responses by transfection of IFNγ gene into tumor using a novel type synthetic vector

The existence of Th1 responses in a tumor microenvironment elicits a better prognosis for the patients. Transfection of Th1 polarizing cytokines, such as IFNγ, into tumor cells is an effective way to set up an appropriate microenvironment. Using a novel type synthetic vector composed of polyamidoamine dendrons, we transfected canine IFNγ gene into canine tumor cell lines, and examined direct and indirect effects of dendritic cells (DCs) against tumor growth in vitro. A cloned canine IFNγ gene expressed functional protein that induces maturation of DCs. When the canine IFNγ gene was transfected into canine tumor cell lines using the synthetic vector, most cells secreted canine IFNγ. Secretion of IFNγ reduced with time, but was maintained for 48h. DCs incubated with the IFNγ-transfected tumor cells exhibited greater suppressive activity and induced significantly higher cytotoxic activity against the tumor cells, relative to those incubated with untransfected tumor cells and comparable dose of IFNγ. Successful transfection of IFNγ by the synthetic vector efficiently enhanced the anti-tumor immune function of DCs, and sets up a suitable microenvironment for improvement in tumor therapy.

Production of canine soluble CD40 ligand to induce maturation of monocyte derived dendritic cells for cancer immunotherapy

CD40 ligand (CD40L) expressed by activated T cells is shown to induce maturation of immature dendritic cells (DCs) and this maturation is a vital part in DC based tumor immunotherapy. We constructed an expression vector by cloning the extracellular domain of canine CD40L fused to the signal sequence of canine IL-12p40. When PBMCs were incubated with canine granulocyte-macrophage (GM) -CSF and IL-4, expression of CD86 was significantly elevated, but the majority of cells displayed the morphology of immature DCs. Following addition of the expressed canine soluble CD40L (csCD40L) to the DC-inducing culture, the cell morphology shifted to that of mature DCs, and expression of CD80, CD86, MHC class II and CD1a was significantly enhanced. This morphological change and enhancement of expression was observed even when the csCD40L was present only in the second half period of the culture. Furthermore, the csCD40L caused a significant increase in IL-12 production from DCs. These results show that the csCD40L significantly promotes the maturation and activation of canine monocyte derived DCs.

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs.

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.

Development of a vaccine based on bacteria-mimicking tumor cells coated with novel engineered toll-like receptor 2 ligands

For a successful tumor vaccine, it is necessary to develop effective immuno‐adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria‐mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll‐like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non‐specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C‐GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross‐presentation in vitro. They also induced effective tumor‐specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation‐inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen‐specific immuno‐adjuvant, with few adverse effects.

Development of effective tumor immunotherapy using a novel dendritic cell–targeting Toll-like receptor ligand

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.