Xenotransfusion of canine blood to cats: a review of 49 cases and their outcome.

OBJECTIVES: To describe the use of a xenotransfusion protocol, the outcome of xenotransfusion in recipient cats and to assess owner memory of the xenotransfusion. MATERIALS AND METHODS: Cats administered xenotransfusions in two hospitals between January 2016 and July 2018 were included. Adherence to xenotransfusion protocol, cause of anaemia, blood type, packed cell volume (PCV), transfusion volume, transfusion reactions, PCV 12 hours after transfusion and survival to discharge were recorded. Owners of surviving cats were questioned to assess if they remembered that a xenotransfusion had been performed. RESULTS: Forty-nine cats underwent the xenotransfusion protocol. The most common causes of anaemia were surgical blood loss (n = 17), immune-mediated haemolytic anaemia (n = 14) and neoplasia (n = 14). Median PCV before transfusion was 10%. Six cats (12%) had febrile non-haemolytic transfusion reactions. Median PCV 12 hours after transfusion was 25%. Ten cats (20%) died or were euthanased within 24 hours of xenotransfusion. A delayed haemolytic transfusion reaction occurred in 25 of 39 (64%) cats manifesting as icterus in 15 cats after a median of 1.9 days and haemolytic serum in 19 cats after a median of 2 days. Of the 18 cats alive at 1 week after discharge, 15 (83%) were still alive at a median of 173 days after xenotransfusion. All owners contacted remembered that their cats had received a xenotransfusion. CLINICAL SIGNIFICANCE: Xenotransfusion of canine packed red blood cells to cats is possible but haemolysis should be expected between 1 and 6 days after transfusion.

Loss of the xeroderma pigmentosum group B protein binding site impairs p210 BCR/ABL1 leukemogenic activity.

Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors.

Preputial urinary diversion to treat urine soaking during urination in a dog.

A young dog was presented with a history of adopting an unusual posture to urinate, resulting in urine soaking of the ventral abdomen and caudal forelimbs. The dog was initially treated surgically with cranial advancement of the prepuce, which did not resolve the problem. Further surgery was then successfully carried out to create a more caudal preputial orifice, which angled the penis ventrally when extruded, directing urine away from the body. At follow-up clinical examination, the dog was clinically normal.

Rac GTPases as key regulators of p210-BCR-ABL-dependent leukemogenesis.

Chronic myelogenous leukemia (CML) is a malignant disease characterized by expression of p210-BCR-ABL, the product of the Philadelphia chromosome. Survival of CML patients has been significantly improved with the introduction of tyrosine kinase inhibitors that induce long-term hematologic remissions. However, mounting evidence indicates that the use of a single tyrosine kinase inhibitor does not cure this disease due to the persistence of p210-BCR-ABL at the molecular level or the acquired resistance in the stem cell compartment to individual inhibitors. We have recently shown in a murine model that deficiency of the Rho GTPases Rac1 and Rac2 significantly reduces p210-BCR-ABL-mediated proliferation in vitro and myeloproliferative disease in vivo, suggesting Rac as a potential therapeutic target in p210-BCR-ABL-induced disease. This target has been further validated using a first-generation Rac-specific small molecule inhibitor. In this review we describe the role of Rac GTPases in p210-BCR-ABL-induced leukemogenesis and explore the possibility of combinatorial therapies that include tyrosine kinase inhibitor(s) and Rac GTPase inhibitors in the treatment of CML.

Conservation of the genomic structure and receptor-mediated signaling between human and rat IL-24.

IL-24/MDA-7 is a new member of the IL-10 family of cytokines, which signals through two heterodimeric receptor complexes (IL-20R1/IL-20R2 and IL-22R/IL-20R2). Previously, we identified a rat gene named mob-5, which encodes a secreted protein that shares a high degree of homology with human IL-24. Expression of mob-5 and its putative cell surface receptors was shown to be upregulated by oncogenic ras. Here we show that not only do rat mob-5 and human IL-24 share a strikingly similar genomic structure but also that the rat MOB-5 protein can bind to and signal through the human IL-24 receptors. Like human IL-24, binding of the rat MOB-5 protein to the human IL-24 receptors leads to activation of the JAK/STAT pathway, which in turn supports receptor-dependent survival and proliferation of Ba/F3 cells. Furthermore, using human colon cancer cell lines with somatic knockout of either the mutant or the wild-type k-ras allele, we demonstrate that the human IL-24 receptors also are upregulated by oncogenic ras. Taken together, these results provide strong experimental evidence that MOB-5 is indeed the rat homolog of human IL-24.

Effect of CD40 ligand on the course of murine histoplasmosis.

CD40 ligand-CD40 ligation is important in the development of T-cell-mediated immune responses. The purpose of this study was to examine the role of CD40L in recovery from histoplasmosis using a murine model of intratracheally induced infection. B6C3F1 mice were infected intratracheally with Histoplasma capsulatum yeast and monitored for clearance of the organism from the lungs and spleen. CD40L treatment was begun on either day -2 or +2 post inoculation and continued until day 14 in CD4-depleted animals and from day -2 to day +4 in non-immunosuppressed animals. Amphotericin B treatment was begun four days following inoculation and given every other day for 10 days. CD40L reduced fungal burden by less than one log when started two days before infection but did not act synergistically with low-dosage amphotericin B (0.2 mg kg(-1) qod) in CD4 depleted mice. Low-dose amphotericin B, CD40L, and the combination of the two failed to lower the fungal burden in a second experiment using a more virulent isolate of the same strain of H. capsulatum in CD4-depleted mice. Furthermore, CD40L did not increase the concentrations of IFN-gamma, IL-12 or IL-10 in the lungs or spleens of infected animals. In summary, CD40L had minimal or no effect on the course of infection in this murine model of histoplasmosis.

High-affinity T helper epitope induces complementary helper and APC polarization, increased CTL, and protection against viral infection.

Natural viral proteins do not always make optimal vaccines. We have found that sequence modification to increase epitope affinity for class II MHC molecules (epitope enhancement) can improve immunogenicity. Here we show first that a higher-affinity helper epitope-enhanced HIV vaccine not only induces more cytotoxic T lymphocytes (CTLs), but also skews helper cells toward Th1 cytokine production and protects against HIV-1 recombinant vaccinia viral challenge. Furthermore, we elucidate a novel mechanism in which the higher-affinity vaccine induces dramatically more effective helper cells with a higher level of CD40L per helper cell and more positive cells, which in turn more effectively conditions dendritic cells (DCs) for CTL activation in a second culture. The improved helper cells also induce much greater IL-12 production by DCs, accounting for the reciprocal T helper polarization to Th1, and increase costimulatory molecule expression. Thus, increasing affinity for class II MHC results in a complementary interaction in which T helper and antigen-presenting cells polarize each other, as well as increase CTL, and provide greater vaccine efficacy against viral infection.

Efficiency of cross presentation of vaccinia virus-derived antigens by human dendritic cells.

Dendritic cells (DC) utilize at least two pathways to process viral antigens onto MHC class I molecules. The conventional endogenous route is used to acquire antigens from both infectious and non-replicating virions. Exogenous pathways are used by DC to acquire and "cross-present" antigens derived from virus-infected donor cells that by themselves lack the ability to activate T cells directly. We analyzed the role of this pathway for antigens derived from vaccinia, a virus which inhibits DC maturation and causes extensive apoptosis of infected cells, yet is highly immunogenic. Using recombinant vaccinia virus encoding the influenza matrix protein as model vector, DC were shown to cross-present vaccinia-derived antigens from both apoptotic and necrotic infected cells to antigen-specific CD8(+) T cells. Efficient cross presentation required uptake of dead cells by immature DC and exposure to maturation stimuli, especially CD40 ligand. The responding CD8(+) T cells secreted IL-2 and IFN-gamma, proliferated and developed into cytotoxic effectors. Quantification of the cross presentation of vaccinia-derived antigens showed this pathway to be highly efficient, corresponding to a peptide pulse of 10-100 nM. While monocytes also phagocytosed apoptotic and necrotic cells, they were far less efficient at cross-presenting vaccinia-derived antigens to CD8(+) T cells. The ability of DC to cross-present vaccinia-derived antigens from infected apoptotic cells or necrotic cell lysates, bypasses the deleterious effects of direct infection of DC and provides one explanation for this pathogen's immunogenicity.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein.

We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.

Low levels of productive HIV infection in Langerhans cell-like dendritic cells differentiated in the presence of TGF-beta1 and increased viral replication with CD40 ligand-induced maturation.

Langerhans cells (LC) represent dendritic cells (DC) within mucosal epithelium that are purported initial targets for HIV following sexual exposure to virus. Here, morphologic, phenotypic, functional and HIV infection experiments were performed using monocyte-derived DC cultured in the presence of GM-CSF, IL-4 and TGF-beta1 (G4T-DC), GM-CSF and IL-4 (G4-DC), and G4T-DC incubated for an additional 3 days with CD40 ligand (CD40L-DC). G4T-DC, which demonstrated characteristics of immature LC, could be productively infected by either R5- or X4-HIV strains. Infection levels, however, were markedly lower than those observed in immature G4-DC. Surprisingly, CD40L-DC, which demonstrated features of mature LC, could be productively infected with HIV at higher levels than immature G4T-DC. Productive HIV infection in these three DC populations correlated positively with cell surface expression of CD4, CCR5 and CXCR4. We suggest that low levels of HIV infection in LC-like G4T-DC indicate an inefficient mechanism by which HIV can initially infect individuals, perhaps explaining the relative difficulty in becoming infected during sexual exposure to virus. In addition, enhanced HIV infection in LC-like G4T-DC following CD40L treatment suggests a mechanism by which inflammatory CD40L(+) T cells, if present in mucosal tissue, could lead to increased HIV transmission rates.

Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents.

Dendritic cells (DCs) can efficiently acquire foreign antigen(s) from apoptotic cells and induce MHC class I-restricted, antigen-specific CTLs. An accumulation of DCs within solid tumor masses in situ has been associated indirectly with a more favorable prognosis. Therefore, DCs may offer an efficient means for triggering immune responses within tumors, particularly in those masses containing significant apoptosis. We examined whether delivery of DCs could, alone, impact on the progressive growth of a tumor with a relatively high apoptotic index. We detected significant early apoptosis within the mass of a s.c. growing murine MT-901 breast carcinoma. DCs could efficiently engulf MT-901 tumor apoptotic cells in vitro. Intratumoral injections of syngeneic but not allogeneic DCs resulted in significant inhibition of MT-901 tumor growth. Histological examination of the tumor revealed intense mononuclear cell infiltration during and after DC injections. Tumor growth inhibition was relatively radiosensitive and dependent on host-derived CD8+ T cells. The baseline level of tumor apoptosis could be increased substantially by tumor necrosis factor alpha administration, leading to a greater DC-mediated antitumor effect. The antitumor effect could also be enhanced by first pulsing DCs with the foreign helper protein, keyhole limpet hemocyanin, prior to intratumoral delivery and combining it with the systemic administration of interleukin 2. Splenocytes from treated animals showed heightened levels of specific CTL activity and production of cytokines. The level of in situ tumor apoptosis appears to play a critical role in DC-mediated antitumor effects. The potential implication of these findings in DC-based tumor therapy strategies is discussed.

In vivo CD40-CD154 (CD40 ligand) interaction induces integrated HIV expression by APC in an HIV-1-transgenic mouse model.

Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with PLASMODIUM: chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.

Functional monocyte-derived dendritic cells can be generated in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) remains an incurable disease. Although modern available treatments are able to induce disease regression, relapse almost inexorably occurs. Therefore, novel therapeutic strategies aimed at reducing the disease relapse rate are very much needed. Among these, the induction of tumour-associated antigen-specific cytotoxic T lymphocytes (CTL), through either DNA vaccines or injection of idiotype pulsed dendritic cells (DCs), has been actively investigated with encouraging preliminary results in B-cell malignancies. As the CLL B lymphocyte characteristically expresses low amounts of surface immunoglobulin (Ig) and T cells from these patients have been reported to display impaired functional activity, there are concerns related to the possibility of generating specific cytotoxic antitumoral T cells in this disease. In addition, no information is presently available regarding the functional ability of CLL-derived DCs. In the present work, freshly purified monocytes from CLL patients and normal donors were induced to differentiate in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 serum-free medium and compared for their morphological, phenotypic and functional characteristics. Our results demonstrate that: (1) functional DCs can be generated from CLL patients with similar phenotype and function to those observed from normal donors; (2) in contrast to normal control subjects, monocyte-derived DCs from CLL patients spontaneously secrete endogenous IL-10; and (3) interferon (IFN)-gamma in combination with CD40L plays a major role in priming DCs from CLL patients for IL-12 and IL-15 production. Overall, these results indicate that it is possible to derive functionally competent DCs from circulating monocytes in CLL patients.

A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment.

Dendritic cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal antigen presentation and T-cell activation. High cell surface expression of CD83 is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83 expression on DCs. We hypothesized that interleukin 12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating antigen-specific T cells. We used combinations of signaling through CD40, using CD40 ligand trimer (CD40L), and interferon gamma to demonstrate that CD83 expression is necessary but not sufficient for optimal production of IL-12 by DCs. Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals. By intracellular cytokine detection, we determined that only a subset of cells that express high levels of CD80 and CD83 generate large amounts of IL-12. DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T cell in vitro. These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.

The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses.

CD4(+) T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8(+) CTL response in murine models. Recent studies have demonstrated that CD4(+) T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4(+) T cell help in the expansion of virus-specific CD8(+) memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4(+) T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4(+) T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4(+) T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4(+) T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4(+) T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8(+) memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4(+) T cell-depleted conditions, where IL-2 is lacking.

Soluble CD40 ligand induces beta-chemokine production by macrophages and resistance to HIV-1 entry.

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication.

Effect of CD40 ligand and other immunomodulators on Pneumocystis carinii infection in rat model.

The corticosteroid-treated animal is well established as an experimental model for the study of Pneumocystis carinii pneumonitis (PCP). Latent or acquired infection with P. carinii in the murine lung progresses to fatal pneumonitis when the host is profoundly immunocompromized. In this study the effects of five immunomodulators; recombinant CD40 ligand (CD40L), bryostatin 1, recombinant FLT3 ligand (FLT3L), recombinant granulocyte colony-stimulating factor (G-CSF) and recombinant interleukin-15 (IL-15) were investigated against PCP in a dexamethasone immunosuppressed Sprague-Dawley rat model. The majority of rats (70%) treated with CD40L at the onset of dexamethasone immunosuppression were protected against PCP. When CD40L was given after 10 days of immunosuppression, only 40% of the rats resolved the infection. However, 95% of the control animals developed PCP. Immunosuppressed rats treated with bryostatin 1, an immune activator had a partial (50%) protection against P. carinii infection. In contrast, daily administration of FLT3L, IL-15 or G-CSF provided no protection against P. carinii infection.

IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process.

Leishmaniasis, a vector-borne parasitic disease, is transmitted during a sandfly blood meal as the parasite is delivered into the dermis. The parasite displays a unique immune evasion mechanism: prevention of IL-12 production within its host cell, the macrophage (i.e., where it differentiates and multiplies). Given the close proximity of skin dendritic cells (DC) to the site of parasite delivery, their critical role in initiating immune responses and the self-healing nature of Leishmania major (Lm) infection, we examined the interaction between myeloid-derived human DC and Lm metacyclic promastigotes (infectious-stage parasites) to model the early "natural" events of infection. We found that DC can take up Lm and, after this internalization, undergo changes in surface phenotype suggesting "maturation". Despite the intracellular location of the parasite and resultant up-regulation of costimulatory and class II molecules, there was no detectable cytokine release by these Lm-harboring DC. However, using intracellular staining and flow cytometry to analyze cytokine production at the single-cell level, we found that Lm-harboring DC, but not monocytes, produce large amounts of IL-12p70 in a CD40 ligand (CD40L)-dependent manner. Finally, DC generated from mononuclear cells from patients with cutaneous leishmaniasis (Lm), once loaded with live metacyclic promastigotes, were found to reactivate autologous primed T lymphocytes and induce a CD40L-dependent IFN-gamma response. Our results link the required CD40/CD40L interactions for healing with DC-derived IL-12p70 production and provide a mechanism to explain the genesis of a protective T cell-mediated response in the face of local immune evasion within the macrophage at the site of Leishmania delivery.