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.

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.

Activation of antigen-induced lymphocyte proliferation by interleukin-15 without the mitogenic effect of interleukin-2 that may induce human immunodeficiency virus-1 expression.

The newly identified cytokine, IL-15 enhanced antigen-induced proliferation of PBMC obtained from HIV-1-seropositive subjects. When compared to IL-2 which enhanced both spontaneous and antigen-induced lymphocyte proliferative responses, IL-15 rarely increased spontaneous lymphocyte proliferation. Additionally, in cultures of lymphocytes obtained from 15 HIV-1-infected patients with < 300 circulating CD4- lymphocytes/microliter IL-15 induced significant HIV-1 expression (46, 21, and 71 pg/ml) in only 3 of 15 experiments and IL-2 induced significant HIV-1 expression (range 16- > 5000 pg/ml) in 11 of 15 experiments (P < 0.01, Fischer's exact test). Simultaneous assays of cytokine-induced spontaneous lymphocyte proliferation and HIV-1 expression revealed similar dose-response relationships for induction of HIV-1 and lymphocyte proliferation by IL-2. Thus, IL-15 helps to correct the impaired proliferative response of CD4+ lymphocytes from HIV-1-infected persons without the mitogenic effect of IL-2 that also may induce HIV-1 expression.

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.

Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer.

An ineffective tumor-specific immune response from inadequate/incompetent antigen presentation could contribute to the failure in tumor control and its dissemination. Dendritic cells (DCs) have been shown to present antigen from apoptotic cells. We hypothesized that Flt3-ligand (Flt3L) therapy, which expands DCs in vivo, in combination with local tumor radiotherapy (RT), should improve antigen presentation from dying, irradiated tumor cells. RT + Flt3L reduced pulmonary metastases in a murine model of Lewis lung carcinoma and significantly improved survival in C57Bl/6 mice with established footpad tumors. Mice treated with Flt3L alone showed delayed tumor growth but eventually succumbed to tumor progression. The combination therapy of RT + Flt3L failed to impact survival in immunodeficient athymic mice, implicating the role of T cells in prolonging survival. These results support an attractive strategy of sequential RT and immunotherapy with Flt3L to enhance tumor antigen presentation, which may produce therapeutic responses against disseminated cancer and improvement in survival.

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.

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.

CD30 ligand in lymphoma patients with CD30+ tumors.

PURPOSE: CD30 ligand (CD30L), which is expressed on resting B and activated T lymphocytes, can induce cell death in several CD30+ cell lines. Patients with CD30+ tumors (Hodgkin's disease and Ki-1+ non-Hodgkin's lymphoma) frequently have elevated soluble CD30 (sCD30) levels in their serum, which correlates with a poor prognosis. The role of sCD30 in protecting tumor cells from CD30L-mediated cell death and the pattern of CD30L expression on human peripheral-blood lymphocytes (PBLs) of normal donors and patients with CD30+ tumors are investigated. MATERIALS AND METHODS: CD30L surface protein expression was determined by two-color flow cytometry on PBLs of patients with CD30+ tumors and normal individuals. CD30L levels were determined on subsets of PBLs before and after stimulation with phytohemagglutinin (PHA), anti-CD3 antibody, or CD40L. sCD30 was measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic activity of membrane-bound CD30L was tested in a CD30+ cell line by the annexin V-binding method. RESULTS: Unstimulated T lymphocytes of normal donors and patients with lymphoma rarely expressed CD30L surface protein, but were able to express it after stimulation with PHA or anti-CD3 antibody. Resting B cells of patients with CD30+ tumors had lower levels of detectable surface CD30L compared with normal donors (mean, 55% and 80.6%, respectively; P = .0008). Patients with high levels of serum sCD30 had lower detectable levels of CD30L on their PBLs (R2 = .72, P = .0008) and exogenous sCD30 blocked membrane-bound CD30L-mediated apoptosis in a CD30+ cell line. CONCLUSION: In patients with CD30+ tumors, sCD30 can decrease the availability of CD30L on PBLs. Blocking the apoptosis-inducing activity of CD30L by its soluble receptor may explain how CD30+ tumors escape immunosurveillance and may be related to the reported poor prognosis of patients who have elevated sCD30 levels.

Neutralizing monoclonal antibodies to the AIDS virus.

The production of neutralizing monoclonal antibodies (MAbs) will permit the exact localization of neutralizing epitopes on the AIDS virus, HIV-1. We describe the properties of seven MAbs to the envelope of the LAV-1 isolate. Five MAbs recognise the central portion of gp110, amino acids 279-472, and four of these are capable of high-titre neutralization of HIV-1, by infection inhibition, syncytial inhibition and vesicular stomatitis virus (VSV) pseudotype neutralization. One of the two MAbs to gp41 inhibits syncytium formation. Neutralization, live cell immunofluorescence and immunoprecipitation of gp110 are type-specific and restricted to HIV-1 isolates closely related to LAV-1.

Treatment of psychiatric illness by stereotactic cingulotomy.

The treatment of 198 psychiatrically disabled patients with stereotactic cingulotomy was evaluated prospectively for a mean follow-up of 8.6 years. Patients with major affective disorders and anxiety disorders fared the best, with a return to normal functioning in the majority. Patients with obsessive-compulsive disorders, schizophrenia, and personality disorders improved less predictably, with an uneven improvement in functioning that required active ongoing psychiatric treatment. Low mortality and morbidity, a reduction of violent behavior, a possible reduction of suicidal risk, and a lessening of the intractable suffering of chronic psychiatric illness all indicate that cingulotomy can be an effective, safe treatment for patients with affective disorders that are unresponsive to all other forms of therapy.

Effects of interleukin-15 on in vitro human T cell proliferation and activation.

Interleukin-15 (IL-15) has been reported to have many activities on T cell populations, including a potential role in improving antigen-specific proliferation in HIV-1 disease. We tested this response in healthy adults by studying the response of T cell populations after stimulation with medium, tetanus, cytomegalovirus (CMV) antigens in cultures from 21 volunteers. IL-15 caused a dose-dependent increase in medium and antigen-induced proliferation. The expansion was due to CD8>natural killer (NK)>CD4 lymphocytes and memory > naive cells. The IL-15-stimulated CD8 cells had increased levels of the activation markers CD69 and DR. The published CMV-induced expression of CD57 on CD8+ cells was increased in CMV seronegative and seropositive subjects by IL-15. IL-15 appears to be a stimulator of T cell populations in healthy adults and may be useful in settings to enhance nonspecific NK activity or antigen-specific CD8 activity.

Anti-idiotypic antibody to the V3 domain of gp120 binds to vimentin: a possible role of intermediate filaments in the early steps of HIV-1 infection cycle.

Although the CD4 molecule is the major cellular receptor for human immunodeficiency virus (HIV), several lines of evidence suggest participation of additional molecules that are engaged after the binding of HIV to the CD4 receptor and that may facilitate viral entry into the target cell. Some of the post-CD4 binding, perfusion events involve the third hypervariable region (V3 loop) of the viral envelope protein gp120. To identify cellular proteins that interact with the V3 loop, we chose as a probe an antiidiotypic monoclonal antibody (MAb), anti-id2, which was prepared against the neutralizing MAb 110.4 that binds the V3 domain in the envelope glycoprotein gp120 of the LAI isolate of HIV-1. Anti-id2 reacted specifically with a 55- to 60-kDa protein in human T cell and monocytoid cell lines, and in a mouse melanoma cell line. This protein was identified immunologically and by protein sequence analysis as vimentin, an intermediate filament protein of lymphoid and other cells of mesodermal origin. Antiserum raised against vimentin inhibited nuclear translocation of HIV-1 DNA following infection of monocytes and CD4+ T cells with live virus, and reduced the amount of HIV-1 gag-specific RNA in the nuclei of monocytes following inoculation with HIV-1 pseudovirions. These data suggest that vimentin may participate in the early steps of HIV-1 replication, perhaps during the uptake of HIV-1 preintegration complexes into the nuclear compartment.

Variable region gene utilization and mutation in a group of neutralizing murine anti-human immunodeficiency virus type 1 principal neutralizing determinant antibodies.

The heavy (VH) and light (VL) chain variable region nucleotide sequences of four neutralizing anti-human immunodeficiency virus type 1 (HIV-1) murine monoclonal antibodies (mAbs) were determined. These mAbs bind to native gp120, recombinant gp120, and a linear HIV-1 principal neutralizing determinant (PND) peptide that spans amino acid 308-328. Three mAbs that bind to the same linear determinant, 110.3, 110.4, and 110.5, all use the same VL gene elements, a VK21 gene and JK2. These three mAbs also share the same VKJK junctional diversity and specific somatic mutations. They have identical VL immunoglobulin gene rearrangement patterns on Southern blot. Two of the antibodies, 110.4 and 110.5, also use the same VH gene elements, SB32-D-JH4, and have identical VD and DJ junctions and N sequences. Two different anti-HIV-1 PND murine mAbs reported by others, BAT123 and 0.5 beta, also use VK21-JK2, and BAT123 also uses the SB32 VH gene element. Although 110.3 uses the same VL region gene as 110.3 and 110.4, it uses a different VH gene that appears to be a member of the 7183 VH family. 110.6, an mAb that recognizes a discrete, overlapping PND compared to 110.3, 110.4, and 110.5, uses entirely different VH and VL gene elements and has unique immunoglobulin VH and VL rearrangement patterns. Our data, taken together with reports of the BAT123 and 0.5 beta mAb sequences, suggest that the murine antibody response to HIV-1 PND may be restricted to a small subset of VH and VL gene elements.

Current concepts in thoracic drainage systems.

Thoracic drainage systems are currently marketed in many varieties, resulting in significant cost and complicating patient management. Realistic needs have been identified from a survey of thoracic surgeons. These are: (1) clear plastic chest catheters with multiple drainage holes in sizes 28, 32, and 36F for adults and 16, 20, and 24F for infants and children; (2) serrated plastic connectors that can be sized at operation; (3) connecting tubes of clear plastic 6 feet long with a diameter of 1/2 inch; (4) a single graduated volume-collecting bottle of 1- to 2-liter capacity that can be emptied and marked, with a separate waterseal component and an associated manometer; and (5) a highflow vacuum source. A drainage system with these characteristics should be safe, effective, simple, and less costly.

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.

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.