The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide.

We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.     

Assessment of clinical significance of anti-Ge in an untransfused man

A 19-year-old, untransfused Melanesian man from Papua New Guinea was admitted to the hospital for repair of an atrial septal defect. His serum contained an alloantibody that reacted strongly on the indirect antiglobulin test and was identified as anti-Ge. Gerbich-negative blood was transfused following urgent surgery. A 51Cr red cell survival study performed 2 weeks after surgery yielded zero survival of Gerbich-positive cells after 24 hours. A monocyte-driven, antibody-dependent, cell-mediated cytotoxicity assay performed on both pretransfusion and posttransfusion serum samples and on concentrated serum showed less than 1 percent specific lysis of Gerbich-positive cells. This did not correlate with the indication of clinical significance predicted by the 51Cr study. Red cell adherence and phagocytosis, not evident in a monocyte monolayer assay using native serum, were demonstrable in 16 percent of monocytes by the use of concentrated serum.     

Regulatory T Cells Exhibit Decreased Proliferation but Enhanced Suppression After Pulsing With Sirolimus

Although regulatory T cells (Tregs) suppress allo‐immunity, difficulties in their large‐scale production and in maintaining their suppressive function after expansion have thus far limited their clinical applicability. Here we have used our nonhuman primate model to demonstrate that significant ex vivo Treg expansion with potent suppressive capacity can be achieved and that Treg suppressive capacity can be further enhanced by their exposure to a short pulse of sirolimus. Both unpulsed and sirolimus‐pulsed Tregs (SPTs) are capable of inhibiting proliferation of multiple T cell subpopulations, including CD4⁺ and CD8⁺ T cells, as well as antigen‐experienced CD28⁺CD95⁺ memory and CD28^?CD95⁺ effector subpopulations. We further show that Tregs can be combined in vitro with CTLA4‐Ig (belatacept) to lead to enhanced inhibition of allo‐proliferation. SPTs undergo less proliferation in a mixed lymphocyte reaction (MLR) when compared with unpulsed Tregs, suggesting that Treg‐mediated suppression may be inversely related to their proliferative capacity. SPTs also display increased expression of CD25 and CTLA4, implicating signaling through these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue to enhance Treg‐based suppression of allo‐immunity, in a manner amenable to large‐scale ex vivo expansion and combinatorial therapy with novel, costimulation blockade‐based immunosuppression strategies.     

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation

We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.     

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice

Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF⁺ macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT.The use of immunotherapy (IT) in cancer has recently resulted in impressive responses. Yet, the usages of IT regimens, especially those involving cytokine therapies, have also resulted in the induction of severe systemic toxicities often not previously characterized in their preclinical animal studies. Because such toxicities often require intensive care management of patients, the causes for the discrepancies in observations between clinical and preclinical toxicity merit further study.Cancer has been considered a disease of aging, with persons over 65 accounting for over 60% of newly diagnosed malignancies (Balducci and Ershler, 2005). Consequences of aging include gradual decreases in immunological function, thymic involution leading to decreased naive T cell output, restriction within T cell repertoire diversity, increases in genomic instability, and increases in cellular senescence (Campisi, 2013). However, despite the majority of patients being of this demographic, the overwhelming majority of preclinical studies are performed in young inbred mice. We recently demonstrated that treatment of young mice with a combination IT regimen consisting of agonistic monoclonal antibody to CD40 (αCD40) given in conjunction with IL-2 resulted in synergistic anti-tumor effects and was well tolerated (Murphy et al., 2003). Yet, administration of the same regimen in an aged cohort resulted in 100% lethality within two days of treatment (Bouchlaka et al., 2013). Importantly, the mortality in the aged was perpetuated by an aberrant cytokine storm mirroring a systemic inflammatory response syndrome (SIRS), which resulted in multi-organ damage (Bouchlaka et al., 2013). These systemic toxicities were closely representative of those previously noted clinically with FDA-approved immunostimulatory IT therapeutics such as high-dose IL-2 and IFN-α (Lee and Margolin, 2011).Aging is associated with a gradual decline in immunological function but is also accompanied by the coincidence of a systemic, chronic, and low-grade proinflammatory response termed inflammaging that leads to increased systemic cytokine levels, namely IL-1β, IL-6, and TNF (Franceschi, 2007). Emerging data suggests that inflammaging may be influenced by age-associated changes in body mass composition such as decreased lean muscle mass, increased adiposity, and increases in overall body mass index (BMI; Franceschi et al., 2000). Whether adiposity plays a role in the generation of systemic toxicities or whether it is primarily an age-associated occurrence remains unknown.Like aging, hallmark to the obese microenvironment is the development of a low-grade chronic “meta-inflammatory” state. Excess adiposity is associated with increased infiltration of macrophages and proinflammatory mediators such as dendritic cells, NK cells, and T cells into fat depots that ultimately results in environmental remodeling. Consequences include increased circulating levels of C-reactive protein (CRP), IL-6, leptin, and TNF (Ronti et al., 2006; Vázquez-Vela et al., 2008). Importantly, the immunomodulatory effects of increased adipose tissue extends through the ability for both adipocytes and the stromal vascular fraction to express a variety of immunologically important surface receptors, such as the leptin receptor, IL-6 receptor, and both p55 and p75 TNF receptors (Ailhaud, 2000). Systemic consequences of increased adiposity are also seen at distance sites through premature induction of aging-associated changes such as thymic involution, which leads to the restriction of naive T cell output and ultimately restriction of the T cell repertoire (Yang et al., 2009).Collectively, the field of IT has made great progress in the last decade toward the development of therapeutic candidates against cancer. Yet, these candidates have been met in the clinic with the induction of severe, often limiting, systemic toxicities that hinder their usage. Additionally, given the rise of obesity within society, as well as the estimation that cancer is a disease primarily of the aged, using lean and young models may not accurately reflect patient outcomes. Therefore, this study seeks to establish the consequences of increased adipose tissue accumulation throughout aging upon IT-induced toxicities. Here, we find that adiposity results in a skewing toward increased levels of proinflammatory M1 macrophages within the peritoneal cavity and visceral adipose tissues that ultimately result in heightened production of proinflammatory cytokines, namely TNF, during strong immune stimulation with IT. Importantly, aged ad libitum (AL)–fed mice and young obese mice succumb to TNF-mediated pathological responses culminating in rapid lethality, which are prevented through caloric restriction in the aged or through either macrophage depletion or TNF blockade.     

Aspirin does not inhibit adenosine diphosphate-induced platelet alpha- granule release

The involvement of metabolites of arachidonic acid in platelet-dense granule secretion and secondary platelet-platelet interactions is well characterized. However, their role in heterotypic interactions dependent on alpha-granule secretion is less well understood. Using platelet-surface expression of P-selectin as a marker of alpha-granule secretion, we have shown that: (1) aspirin treatment of platelets at doses that block dense granule secretion does not inhibit alpha-granule secretion to adenosine diphosphate (ADP); (2) synergism between epinephrine and ADP in the induction of P-selectin expression is similarly unaffected by aspirin; and (3) the ability of P-selectin to mediate adhesion of activated platelets to monocytes and polymorphonuclear lymphocytes in whole blood is also unchanged by aspirin treatment. To further explore the mechanisms responsible for platelet alpha-granule secretion, we have shown that inhibition of Na+/H+ exchange by either acidification of the extracellular medium or amiloride treatment blocked ADP-induced P-selectin expression. In contrast, incubation with the platelet lipoxygenase inhibitor 5,8,11- eicosatrynoic acid, by itself and with aspirin, did not decrease ADP- induced P-selectin expression. We conclude that platelet alpha-granule secretion in response to ADP is dependent on intact Na+/H+ exchange but is independent of the lipoxygenase- and cyclooxygenase-dependent metabolites of arachidonic acid.