Anti-ICOS mAb Targets Pathogenic IL-17A-expressing Cells in Canine Model of Chronic GVHD.

BACKGROUND: Chronic graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality in transplant patients. We have previously shown that 3 doses of an anti-inducible costimulator (ICOS) mAb transiently ameliorated symptoms and extended survival of dogs affected by chronic GVHD over that of control dogs. The purpose of this study was to specifically correlate changes in T-cell populations in the peripheral blood with anti-ICOS treatment and chronic GVHD progression and regression to reach a better understanding of the mechanism of the disease and prioritize future studies. METHODS: Peripheral blood cells from canines transplanted with DLA-mismatched bone marrow and peripheral blood mononuclear cells to generate chronic GVHD were analyzed by flow cytometry using a panel of antibodies specific to helper and cytolytic T cells. RESULTS: Chronic GVHD was specifically associated with an increase in CD4+ICOS+ cells, ICOS+ cells expressing IL-17A, and CD8+ cells generating granzyme B. Treatment with anti-ICOS mAb at onset of chronic GVHD symptoms specifically targeted IL-17A+-expressing cells, transiently relieved symptoms, and lengthened survival but was unable to reduce the percentage of CD8+ T-cells expressing granzyme B. CONCLUSIONS: These studies suggested a role for both CD4+ and CD8+ T cells in pathogenesis of chronic GVHD in the canine model. We propose that future studies should focus on further extending survival by developing a treatment that would control both CD4+ and CD8+ T cells.

COVID-19 and the policy sciences: initial reactions and perspectives.

The world is in the grip of a crisis that stands unprecedented in living memory. The COVID-19 pandemic is urgent, global in scale, and massive in impacts. Following Harold D. Lasswell's goal for the policy sciences to offer insights into unfolding phenomena, this commentary draws on the lessons of the policy sciences literature to understand the dynamics related to COVID-19. We explore the ways in which scientific and technical expertise, emotions, and narratives influence policy decisions and shape relationships among citizens, organizations, and governments. We discuss varied processes of adaptation and change, including learning, surges in policy responses, alterations in networks (locally and globally), implementing policies across transboundary issues, and assessing policy success and failure. We conclude by identifying understudied aspects of the policy sciences that deserve attention in the pandemic's aftermath.

Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody.

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/-, CD8+/-, CD4-, CD21-, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Anti-Inducible Costimulator Monoclonal Antibody Treatment of Canine Chronic Graft-versus-Host Disease.

In murine model systems inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS mAb could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors after total body irradiation. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared with control dogs. Within the limitations of the number of study dogs we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD.

Science diplomacy and transnational governance impact.

Science diplomacy is coming to the fore as a formidable dimension of interstate power relations. As the challenges of the world increasingly transcend borders, so too have researchers and innovators forged international coalitions to resolve global pathologies. In doing so, new channels of influence and opportunity have opened up for states alongside the 'traditional' modes of foreign diplomacy. Understanding how these channels influence global socio-economic outcomes is thereby crucial for scholars interested in the still-ambiguous structure and processes of global governance. This article advances understanding of the domains of science diplomacy by drawing attention to the 'political intercostalities' of state actors, scientific communities and other transnational actors within the new architectures of global governance. Here we trace the growing array of informal international associations alongside transgovernmental policy networks and 'global public-policy partnerships' that deal with highly specialised and technical matters of international policy and how they are drawn into science diplomacy. This article thus presents a research agenda for a particular mode of 'impact' in politics and international studies.

A Canine Model of Chronic Graft-versus-Host Disease.

In long-term survivors of allogeneic hematopoietic cell transplantation (HCT), chronic graft-versus-host disease (GVHD) is the major cause of morbidity and mortality and a major determinant of quality of life. Chronic GVHD responds poorly to current immunosuppressive drugs, and while T cell depletion may be preventive, this gain is offset by increased relapse rates. A significant impediment to progress in treating chronic GVHD has been the limitations of existing animal models. The goal of this study was to develop a reproducible comprehensive model of chronic GVHD in the dog. Ten recipient dogs received 920 cGy total body irradiation, infusion of marrow, and an infusion of buffy coat cells from a dog leukocyte antigen (DLA)-mismatched unrelated donor. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, 11) and cyclosporine. The duration of cyclosporine administration was limited to 80 days instead of the clinically used 180 days. This was done to contain costs, as chronic GVHD was expected to develop at earlier time points. All recipients were given ursodiol for liver protection. One dog had graft failure and 9 dogs showed stable engraftment. Eight of the 9 developed de novo chronic GVHD. Dogs progressed with clinical signs of chronic GVHD over a period of 43 to 164 (median, 88) days after discontinuation of cyclosporine. Target organs showed the spectrum of chronic GVHD manifestations that are typically seen clinically. These included lichenoid changes of the skin, fasciitis, ocular involvement (xerophthalmia), conjunctivitis, bronchiolitis obliterans, salivary gland involvement, gingivitis, esophageal involvement, and hepatic involvement. Peripheral blood lymphocyte surface antigen expression of CD28 and inducible costimulator was elevated in dogs with GHVD compared with those in normal dogs, but not significantly so. Serum levels of IL-8 and monocyte chemotactic protein-1 in GVHD-affected dogs at time of euthanasia were elevated, whereas levels of IL-15 were depressed compared with those in normal dogs. Results indicate that the canine model is well suited for future studies aimed at preventing or treating chronic GVHD.

Anti-CD28 Antibody-Initiated Cytokine Storm in Canines.

BACKGROUND: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT). METHODS: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs. Pharmacokinetics, pathology, cytokine release, and the crystal structure of 1C6 Fv were evaluated. RESULTS: Within an hour of an IV injection of the 1C6 mAb, the dogs became leukopenic and developed a steroid-refractory cytokine storm. Two of the dogs developed high fevers, one experienced diffuse alveolar hemorrhage, and another developed gastrointestinal hemorrhage. The cytokine storm was characterized by elevated plasma levels of MCP-1, IP-10, IL-10, IL-6, and TNF-α. In addition, one dog showed elevated levels of IL-2, IL-8, and IL-18. In contrast, infusion of 1C6 Fab was well tolerated without any side effects. Dry-coating 1C6 mAb onto tissue culture plates induced CD3-independent proliferation and TNF-alpha production. Crystal structure analysis revealed that 1C6 binds to canine CD28 in a manner different than previously reported for conventional agonistic or superagonistic antibodies. CONCLUSIONS: These results indicate that dogs and humans develop a similar cytokine storm following infusion ofanti-CD28 mAb, providing an appropriate large animal for further study. 1C6 Fab warrants evaluation as a tolerance-inducing reagent in the canine model of allogeneic HCT.

Inducible costimulator (ICOS) up-regulation on activated T cells in chronic graft-versus-host disease after dog leukocyte antigen-nonidentical hematopoietic cell transplantation: a potential therapeutic target.

BACKGROUND: Inducible costimulator (ICOS), a member of the CD28 family of costimulatory molecules, is induced on CD4 and CD8 T cells after their activation. ICOS functions as an essential immune regulator and ICOS blockade is a potential approach to immune modulation in allogeneic transplantation. Here, we describe the expression profile of ICOS in dogs and determine whether ICOS expression is up-regulated during chronic graft-versus-host disease (GVHD) and host-versus-graft reactions in the canine hematopoietic cell transplantation model. METHODS: Monoclonal antibodies (mAbs) against cell surface-expressed ICOS were produced and tested in vitro for suppression of canine mixed leukocyte reactions (MLR). Expression of ICOS on CD3 cells was evaluated by flow cytometry using peripheral blood, lymph nodes, and splenocytes obtained from dogs undergoing graft-versus-host and host-versus-graft reactions. RESULTS: Canine ICOS was expressed in an inducible pattern on T cells activated by concanavalin A, anti-CD3 mAb in combination with anti-CD28 mAb, and alloantigen stimulation. Immunosuppressive effects of ICOS blockade were observed in MLR using peripheral blood mononuclear cells from dog leukocyte antigen-nonidentical dogs. Immunosuppressive effects of ICOS blockade were observed in MLR when anti-ICOS was combined with suboptimal concentrations of cytotoxic T-lymphocyte antigen 4-Ig or cyclosporine. ICOS expression was significantly up-regulated on T cells in dogs undergoing graft rejection or chronic GVHD after allogeneic hematopoietic cell transplantation. CONCLUSIONS: These studies suggest that ICOS plays a role in graft rejection and GVHD in an outbred animal model, and ICOS blockade may be an approach to prevention and treatment of chronic GVHD.

Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation.

BACKGROUND: It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function. METHODS: The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb. RESULTS: One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells. CONCLUSION: These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

Establishment of long-term tolerance to SRBC in dogs by recombinant canine CTLA4-Ig.

BACKGROUND: Blockade of the CD28 costimulatory molecule by recombinant human cytotoxic T lymphocyte (CTL)-associated antigen (CTLA4)-Ig or CD40-CD154 interaction with the monoclonal antibody 5C8 together with donor-specific transfusion led to enhanced engraftment in the canine model of dog leukocyte antigen (DLA)-identical marrow transplantation after 1 Gy total body irradiation. To reduce or eliminate total body irradiation conditioning regimens, we have sought to develop canine specific reagents. METHODS: We have created a fusion protein of the extracellular domain of canine (c) CTLA-4 linked to the hinge-CH2-CH3 domains of canine IgG1 in a pcDNA3.1+ vector. Chinese hamster ovarian cells were cotransfected with CTLA4-Ig vector and a dihydrofolate reductase-containing vector. Stable, high producing clones were generated. RESULTS: Cell binding and mixed leukocyte reactions indicated no significant differences in activity between cCTLA4-Ig and human CTLA4-Ig. Mixed leukocyte reaction data indicated that combinations of cCTLA4-Ig and the monoclonal antibody 5C8 were superior in blocking H-thymidine uptake compared to either reagent alone. In dogs, the circulating half-life of cCTLA4-Ig was approximately 7 days with no immune response against the fusion protein. Finally, two injections of cCTLA4-Ig effectively tolerized two dogs against eight consecutive challenges with sheep red blood cells, given over 330 days as indicated by a complete block of IgG antibody production. Tolerance was broken in one of the two dogs when a ninth injection of sheep red blood cell was given subcutaneously in incomplete Freund's adjuvant. CONCLUSION: cCTLA4-Ig is an effective nonimmunogenic blocking reagent of the CD28 costimulatory pathway in dogs and is a promising reagent for studies of tolerance induction in hematopoietic cell transplantation in the canine model.

Development and in vitro characterization of canine CD40-Ig.

We recently reported that blockade of the CD40-CD154 ligand interaction with the cross-reacting mouse anti-human CD154 antibody, 5c8, together with donor-specific transfusion led to enhanced but not completely successful engraftment in a canine model of DLA-identical marrow transplantation after 100cGy total body irradiation (TBI). In order to improve the transplantation outcomes, we sought to develop a canine-specific reagent. To that end, we fused the extracellular domain of the canine CD40 with a mouse IgG2a Fc tail and tested the immunosuppressive effectiveness of the fusion protein in mixed leukocyte reactions. The extracellular domain of canine CD40 was fused with the Fc portion of mouse IgG2a in a pcDNA3.1+vector. Dhfr-deficient CHO cells were co-transfected with the CD40-Ig vector and a dhfr-containing vector. Stable, high producing clones were selected under increasing methotrexate concentrations. The fusion protein was purified, tested in mixed leukocyte reactions, and its immunosuppressive effect compared to that of the anti-CD154 antibody 5c8. The transfected cell line produced a CD40-Ig dimer whose identity was confirmed by mass spectroscopy. The purified canine CD40-Ig blocked mixed leukocyte reactions at a concentration of 1nM, which was 10 times more effective than the anti-CD154 antibody. Canine CD40-Ig is more immunosuppressive than the anti-human CD154 antibody 5c8 in canine mixed leukocyte reactions and may be more effective in vivo in a model of marrow transplantation.

Comparative biodistributions of pretargeted radioimmunoconjugates targeting CD20, CD22, and DR molecules on human B-cell lymphomas.

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SA)-conjugated antibodies (Abs), followed by clearing agent and radiolabeled biotin is a promising method that can increase the effectiveness of RIT, while decreasing the toxicities associated with directly labeled Abs. Although CD20 has been the traditional target antigen for RIT of non-Hodgkin lymphoma (NHL), studies targeting HLA DR and CD22 have yielded promising results. Targeting all 3 antigens at once may further augment the effect of PRIT. This study compares the targeting of Ramos, Raji, and FL-18 lymphoma xenografts with either anti-CD20 Ab/SA (1F5/SA), anti-HLA DR Ab/SA (Lym-1/SA), anti-CD22 Ab/SA (HD39/SA), or all 3 conjugates in combination, followed 24 hours later by a biotin-N-acetyl-galactosamine clearing agent, and 3 hours after that by (111)In-DOTA-biotin. The Ab/SA conjugate yielding the best tumor uptake and tumor-to-normal organ ratios of radioactivity varied depending on the target antigen expression on the cell line used, with 1F5/SA and Lym-1/SA yielding the most promising results overall. Also, the best tumor-to-normal organ ratios of absorbed radioactivity were obtained using single conjugates optimized for target tumor antigen expression rather than the combination therapy. This study highlights the importance of screening the antigenic expression on lymphomas to select the optimal reagent for PRIT.

Comparison of a tetravalent single-chain antibody-streptavidin fusion protein and an antibody-streptavidin chemical conjugate for pretargeted anti-CD20 radioimmunotherapy of B-cell lymphomas.

The efficacy of radioimmunotherapy (RIT) for patients with relapsed non-Hodgkin lymphoma (NHL) is limited by nonspecific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled anti-CD20 antibodies (Abs). Pretargeted RIT using a covalent conjugate of the 1F5 anti-CD20 Ab with streptavidin (SA) has been shown to augment the efficacy of RIT and decrease toxicity compared with a directly labeled 1F5 Ab. We have engineered a tetravalent singlechain 1F5 (scFv)4SA fusion protein and compared it to the 1F5-SA conjugate. Athymic mice bearing Ramos lymphoma xenografts received either the conjugate or fusion protein, followed 20 hours later by a biotin-N-acetyl-galactosamine clearing agent, followed 4 hours later by 111In-DOTA-biotin. After 24 hours, 11.4% +/- 2.1% of the injected dose of radionuclide was present per gram of tumor (% ID/g) using 1F5 (scFv)4SA compared with 10.8% +/- 2.5% ID/g with 1F5 Ab-SA. Superior tumor-to-normal organ ratios of radioactivity were consistently seen using the fusion protein compared with the chemical conjugate (eg, tumor-to-blood ratio > 65:1 after 48 hours with the fusion protein, but < 7:1 with the conjugate). More than 90% of lymphomabearing mice could be cured with minimal toxicity using either reagent followed by 1200 muCi (44.4 MBq) 90Y-DOTA-biotin.

E-Prescribing collaboration in Massachusetts: early experiences from regional prescribing projects.

Massachusetts payers and providers have encouraged clinician usage of e-Prescribing technology to improve patient safety, enhance office practice efficiencies, and reduce medical costs. This report describes three early pilot e-Prescribing projects as case studies. These projects identified the e-Prescribing needs of clinicians, illustrated key issues that made implementation difficult, and clarified the impact of various types of functionality. The authors identified ten key barriers: (1) previous negative technology experiences, (2) initial and long-term cost, (3) lost productivity, (4) competing priorities, (5) change management issues, (6) interoperability limitations, (7) information technology (IT) requirements, (8) standards limitations, (9) waiting for an "all-in-one solution," and (10) confusion about competing product offerings including hospital/Integrated Delivery System (IDN)-sponsored projects. In Massachusetts, regional projects have helped to address these barriers, and e-Prescribing activities are accelerating rapidly within the state.

Regulatory and policy barriers to effective clinical data exchange: lessons learned from MedsInfo-ED.

MedsInfo-ED is a proof-of-concept clinical data exchange project that uses prescription claims data to deliver patient medication history to emergency department clinicians at the point of care. This patient safety initiative, while limited in scope and scale, has been crucial in identifying numerous policy and regulatory barriers to successful clinical data exchange. The lessons learned and strategies to overcome the barriers are the focus of this paper. Through commitment and effective collaboration, MA-SHARE was able to address some of these barriers that are embedded in existing government regulations and corporate business practice.

Ferrocene encapsulated within symmetric dendrimers: a deeper understanding of dendritic effects on redox potential.

Ferrocene has been encapsulated within a symmetric ether-amide dendritic shell and its redox potential monitored in a variety of solvents. The dendritic effect generated by the branched shell is different in different solvents. In less polar, non hydrogen bond donor solvents, attachment of the branched shell to ferrocene increases its E(1/2), indicating that oxidation to ferrocenium (charge buildup) becomes thermodynamically hindered by the dendrimer, a result explained by the dendrimer providing a less polar medium than that of the surrounding electrolyte solution. The effect of electrolyte concentration on redox potential was also investigated, and it was shown that the concentration of "innocent" electrolyte has a significant effect on the redox potential by increasing the overall polarity of the surrounding medium. Dendritic destabilization of charge buildup is in agreement with the majority of reported dendritic effects. A notable exception to this is provided by the asymmetric ferrocene dendrimers previously reported by Kaifer and co-workers, in which the branching facilitated oxidation, and it is proposed that in this case the dendritic effect is generated by a different mechanism. Interestingly, in methanol, the new symmetric ferrocene dendrimer exhibited almost no dendritic effect, a result explained by the ability of methanol to interact extensively with the branched shell, generating a more open superstructure. By comparison of all the new data with other reports, this study provides a key insight into the structure-activity relationships which control redox processes in dendrimers and also an insight into the electrochemical process itself.