Hemophagocytic lymphohistiocytosis induced by Toxoplasma gondii infection diagnosed by a bone marrow biopsy and DNA next-generation sequencing in an allogeneic hematopoietic stem cell transplant recipient.

In the United States, toxoplasmosis following allogeneic hematopoietic stem transplant (allo-HCT) is very rare with a rate only between 0.5% and 2%. The reported rates of hemophagocytic lymphohistiocytosis (HLH) following allo-HCT range between 0.3% and 17%. Secondary HLH due to toxoplasmosis infection is extremely rare. Herein, we report a case of secondary HLH due to toxoplasmosis following allo-HCT. The diagnosis was reached by a bone marrow biopsy and confirmed by DNA next generation sequencing and immunohistochemical (IHC) staining. The IHC staining included CD1a, a stain previously known to react with cells infected by Leishmania, here we show CD1a staining of macrophages infected with Toxoplasma gondii. Our report highlights the utility of bone marrow biopsy in diagnosing parasitic infection underlying HLH in post-transplant settings. The pre-transplant evaluation of patients from low endemic countries, is a great opportunity to obtain a travel history to determine the risks and the preventative measures against opportunistic infections including toxoplasmosis.

Reduced-Intensity Conditioning Followed by Related and Unrelated Allografts for Hematologic Malignancies: Expanded Analysis and Long-Term Follow-Up.

Reduced-intensity conditioning (RIC) extends the curative potential of allogeneic hematopoietic cell transplantation (HCT) to patients with hematologic malignancies unable to withstand myeloablative conditioning. We prospectively analyzed the outcomes of 292 consecutive patients, median age 58 years (range, 19 to 75) with hematologic malignancies treated with a uniform RIC regimen of cyclophosphamide, fludarabine, and total body irradiation (200 cGy) with or without antithymocyte globulin and cyclosporine and mycophenolate mofetil graft-versus-host disease (GVHD) prophylaxis followed by allogeneic HCT at the University of Minnesota from 2002 to 6. Probability of 5-year overall survival was 78% for patients with indolent non-Hodgkin lymphoma, 53% for chronic myelogenous leukemia, 55% for Hodgkin lymphoma, 40% for acute myelogenous leukemia, 37% for myelodysplastic syndrome, 29% for myeloma, and 14% for myeloproliferative neoplasms. Corresponding outcomes for relapse were 0%, 13%, 53%, 37%, 39%, 75%, and 29%, respectively. Disease risk index (DRI) predicted both survival and relapse with superior survival (64%) and lowest relapse (16%) in those with low risk score compared with 24% survival and 57% relapse in those with high/very-high risk scores. Recipient cytomegalovirus (CMV)-positive serostatus was protective from relapse with the lowest rates in those also receiving a CMV-positive donor graft (29%). The cumulative incidence of 2-year nonrelapse mortality was 26% and was lowest in those receiving a matched sibling graft at 21%, with low (21%) or intermediate (18%) HCT-specific comorbidity index, and was similar across age groups. The incidence of grades II to IV acute GVHD was 43% and grades III to IV 27%; the highest rates were found in those receiving an unrelated donor (URD) peripheral blood stem cell (PBSC) graft, at 50%. Chronic GVHD at 1 year was 36%. Future approaches incorporating alternative GVHD prophylaxis, particularly for URD PBSC grafts, and targeted post-transplant antineoplastic therapies for those with high DRI are indicated to improve these outcomes.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein.

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

Alternative donor hematopoietic cell transplantation for Fanconi anemia.

Historically, alternative donor hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) patients resulted in excessive morbidity and mortality. To improve outcomes, we made sequential changes to the HCT conditioning regimen. A total of 130 FA patients (median age, 9.0 years; range, 1-48) underwent alternative donor HCT at the University of Minnesota between 1995 and 2012. All patients received cyclophosphamide (CY), single fraction total body irradiation (TBI), and antithymocyte globulin (ATG) with or without fludarabine (FLU), followed by T-cell-depleted bone marrow or unmanipulated umbilical cord blood transplantation. The addition of FLU enhanced engraftment 3-fold. The incidence of grades 2-4 acute and chronic graft-versus-host disease was 20% and 10%, respectively. Severe toxicity was highest in patients >10 years of age or those with a history of opportunistic infections or transfusions before HCT. Mortality was lowest in patients without a history of opportunistic infection or transfusions and who received conditioning with TBI 300 cGy, CY, FLU, and ATG. These patients had a probability of survival of 94% at 5 years. Alternative donor HCT is now associated with excellent survival for patients without prior opportunistic infections or transfusions and should be considered for all FA patients after the onset of marrow failure. These studies were registered at http://www.clinicaltrials.gov as NCT00005898, NCT00167206, and NCT00352976.

Circulating angiogenic factors associated with response and survival in patients with acute graft-versus-host disease: results from Blood and Marrow Transplant Clinical Trials Network 0302 and 0802.

Circulating angiogenic factors (AF) reflect tissue healing capacity, although some AF can also contribute to inflammation and are indicative of endothelial dysfunction. The AF milieu in acute graft-versus-host disease (aGVHD) has not been broadly characterized. We hypothesized that patients with abundant AF involved in repair/regeneration versus those mediating damage/inflammation would have improved outcomes. Circulating AF known predominantly for repair/regeneration (epidermal growth factor [EGF], fibroblast growth factor-1 and -2, heparin binding-EGF-like growth factor, and vascular endothelial growth factor-A [VEGF-A], -C, and -D) and for damage/inflammation (angiopoietin-2, endothelin-1, soluble endoglin [sEng], follistatin [FS], leptin, and placental growth factor [PlGF]) were measured in a discovery set of hematopoietic cell recipients with grade III and IV aGVHD and compared with controls, then validated in 2 aGVHD cohorts enrolled in Blood and Marrow Transplant Clinical Trials Network (BMT CTN) trials 0302 (n = 105, serum) and 0802 (n = 158, plasma) versus controls without aGVHD (n = 53, serum). Levels of EGF and VEGF-A were lower than in controls at the onset of aGVHD in both trials and higher with complete response to first-line aGVHD therapy in CTN 0802. FS and PlGF were elevated in aGVHD measured in either serum or plasma. At day 28 after initial aGVHD therapy, elevated FS was an independent negative prognostic factor for survival in both cohorts (hazard ratio, 9.3 in CTN 0302; 2.8 in CTN 0802). These data suggest that circulating AF are associated with clinical outcomes after aGVHD and, thus, may contribute to both pathogenesis and recovery.

Hemolytic anemia due to passenger lymphocyte syndrome in solid malignancy patients treated with allogeneic natural killer cell products.

BACKGROUND: Allogeneic natural killer (NK) cell products for treatment of solid organ malignancies were prepared by performing T (CD3+)-cell depletion on nonmobilized apheresis mononuclear cell collections. The products were not B-cell depleted. This report details two cases of passenger lymphocyte syndrome (PLS) after NK-cell infusion. CASE REPORTS: Patient 1 is a blood group A+ 56-year-old female with Stage IV ovarian carcinoma who received NK cells from an O+ donor. On day +7 she developed new hemolytic anemia. Direct antiglobulin test (DAT) was positive for immunoglobulin G and C3, and the eluate contained anti-A. Subsequently, anti-A was identified on reverse typing. She was transfused with group O red blood cells (RBCs). By day +12 she forward typed as O with anti-A and B on reverse typing. By day +42, DAT was negative with only weak anti-A on reverse typing. Patient 2 is a blood group B+ 51-year-old female with metastatic lobular breast carcinoma who received NK cells from an O+ donor. On day +7 she developed new hemolytic anemia. DAT was positive, and the eluate contained anti-A and -B. Anti-A reactivity was due to anti-A,B. The next day she developed new anti-B on reverse typing. She was transfused with O RBCs. Anti-B titer increased to a maximum of 512 on day +12. At discharge on Day +29 her anti-B titer was still 32. CONCLUSIONS: These patients developed PLS after infusion of NK cells. Because of these cases NK-cell products are now B (CD19+)-cell depleted at our institution, and this approach is recommended for other centers.

The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice.

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Reduced-intensity conditioning followed by related allografts in hematologic malignancies: long-term outcomes most successful in indolent and aggressive non-Hodgkin lymphomas.

Reduced-intensity conditioning (RIC) extends the curative potential of allogeneic hematopoietic cell transplantation (HCT) to patients with hematologic malignancies unable to withstand myeloablative conditioning. We prospectively analyzed the outcomes of 123 patients (median age, 57 years; range, 23-70 years) with hematologic malignancies treated with a uniform RIC regimen of cyclophosphamide, fludarabine, and total-body irradiation (200 cGy) with or without antithymocyte globulin followed by related donor allogeneic HCT at the University of Minnesota between 2002 and 2008. The cohort included 45 patients with acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS), 27 with aggressive non-Hodgkin lymphoma (NHL), 8 with indolent NHL, 10 with Hodgkin lymphoma (HL), 10 with myeloma, and 23 with acute lymphocytic leukemia, chronic myelogenous leukemia, other leukemias, or myeloproliferative disorders. The probability of 4-year overall survival was 73% for patients with indolent NHL, 58% for those with aggressive NHL, 67% for those with HL, 30% for those with AML/MDS, and only 10% for those with myeloma. Corresponding outcomes for relapse in these patients were 0%, 32%, 50%, 33%, and 38%, and those for progression-free survival were 73%, 45%, 27%, 27%, and 10%. The incidence of treatment-related mortality was 14% at day +100 and 22% at 1 year. The incidence of grade II-IV acute graft-versus-host disease was 38% at day +100, and that of chronic graft-versus-host disease was 50% at 2 years. Multivariate analysis revealed superior overall survival and progression-free survival in patients with both indolent and aggressive NHL compared with those with AML/MDS, HL, or myeloma. Worse 1-year treatment-related mortality was observed in patients with a Hematopoietic Cell Transplantation Comorbidity Index score ≥ 3 and in cytomegalovirus-seropositive recipients. These results suggest that (1) RIC conditioning was well tolerated by an older, heavily pretreated population; (2) patients with indolent and aggressive NHL respond well to RIC conditioning, highlighting the importance of the graft-versus-lymphoma effect; and (3) additional peri-transplantation manipulations are needed to improve outcomes for patients with AML/MDS or myeloma receiving RIC conditioning before HCT.

Acadesine inhibits tissue factor induction and thrombus formation by activating the phosphoinositide 3-kinase/Akt signaling pathway.

OBJECTIVE: Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. CONCLUSION: Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Facilitating resolution of life-threatening acute GVHD with human chorionic gonadotropin and epidermal growth factor.

Acute graft-versus-host disease (aGVHD) is a potentially fatal complication of allogeneic hematopoietic cell transplantation that fails to improve with intense immunosuppression in some patients. We hypothesized that urinary-derived human chorionic gonadotropin (uhCG) could help facilitate resolution of life-threatening aGVHD when added as supportive care via 2 potential mechanisms: immunomodulation (akin to its role in pregnancy) and supplementation of epidermal growth factor (EGF; to aid in epithelial repair). In a phase 1 study, 26 participants received subcutaneous injections of uhCG in addition to standard immunosuppression (13 receiving initial therapy for high-risk aGVHD [according to the Minnesota criteria] and 13 receiving second-line therapy). Participants underwent serial blood testing for biomarkers of hormone response, immune modulation, and aGVHD activity on study. uhCG was well tolerated, with no dose-limiting toxicities. Sixty-two percent of patients in the high-risk cohort and 54% of patients in the second-line cohort had a complete response at study day 28. Plasma EGF was elevated sixfold (from 4 to 24 pg/mL; P = .02) at 6 hours postdose in the high-risk cohort, in contrast to no peak in plasma EGF in the more severe second-line cohort. After 1 week of uhCG, patients reported a twofold increase in the regulatory T cell to conventional T-cell ratio, suggesting immune modulation despite high-dose steroids. Responding patients reported significantly lower plasma amphiregulin and higher plasma butyrate levels at study completion, suggesting improvement in mucosal damage over time. uhCG is a novel, safe, supportive therapy, proceeding to phase 2 testing at 2000 units/m2 in high-risk aGVHD. This study was registered at www.clinicaltrials.gov as #NCT02525029.

Reduced-intensity allogeneic transplant in patients older than 55 years: unrelated umbilical cord blood is safe and effective for patients without a matched related donor.

The lower morbidity and mortality of reduced-intensity conditioning (RIC) regimens have allowed allogeneic hematopoietic cell transplantation (HCT) in older patients. Unrelated umbilical cord blood (UCB) has been investigated as an alternative stem cell source to suitably HLA matched related (MRD) and adult volunteer unrelated donors. We hypothesized that RIC HCT using UCB would be safe and efficacious in older patients, and compared the treatment-related mortality (TRM) and overall survival (OS) of RIC HCT in patients older than 55 years using either MRD (n = 47) or, in patients with no 5 of 6 or 6 of 6 HLA compatible related donors, UCB (n = 43). RIC regimen consisted of total-body irradiation (TBI; 200 cGy) and either cyclophosphamide and fludarabine (n = 69), or busulfan and fludarabine (n = 16) or busulfan and cladribine (n = 5). The median age of MRD and UCB cohorts was 58 (range, 55-70) and 59 (range, 55-69) years, respectively. acute myelogenous leukemia/myelodysplastic syndrome (AML/MDS) (50%) was the most common diagnosis. All MRD grafts were 6 of 6 HLA matched to the recipient. Among patients undergoing UCB HCT, 88% received 2 UCB units to optimize cell dose and 93% received 1-2 HLA mismatched grafts. The median follow-up for survivors was 27 (range: 12-61) months. The 3-year probabilities of progression-free survival (PFS; 30% versus 34%, P = .98) and OS (43% versus 34%, P = .57) were similar for recipients of MRD and UCB. The cumulative incidence of grade II-IV acute graft-versus-host (aGVHD) disease (42% versus 49%, P = .20) and TRM at 180-days (23% versus 28%, P = .36) were comparable. However, UCB recipients had a lower incidence of chronic graft-versus-host disease (cGVHD) at 1 year (40% versus 17%, P = .02). On multivariate analysis, graft type had no impact on TRM or survival, and the HCT comorbidity index score was the only factor independently predictive for these endpoints. Our study supports the use of HLA mismatched UCB as an alternative graft source for older patients who need a transplant but do not have an MRD. The use of RIC and UCB extends the availability of transplant therapy to older patients previously excluded on the basis of age and lack of a suitable MRD. A careful review of existing comorbidities is necessary when considering older patients for HCT.

Posterior reversible encephalopathy syndrome in a postpartum woman with acute lymphoblastic leukaemia after intrathecal methotrexate.

Posterior reversible encephalopathy syndrome (PRES) is the most common neurological complication occurring in children undergoing induction chemotherapy for acute lymphoblastic leukaemia (ALL) but is increasingly recognised to occur in adults as well. Here, we report a woman who presented with B-cell ALL (B-ALL) at the time of delivery and developed PRES 1 day after receiving intrathecal (IT) methotrexate (MTX) that rapidly resolved. She subsequently received IT MTX without recurrence of neurological symptoms. This case represents the first case of PRES in a postpartum B-ALL patient receiving IT MTX, demonstrates that re-treatment with MTX in this case could be done safely and highlights the risk of PRES in adults treated for B-ALL.

Platelet factor 4: a chemokine enigma.

Platelet factor 4 (PF4) is a platelet alpha-granule protein sequenced over 25 years ago that is a founding member of the C-X-C chemokine family, yet its physiologic function has yet to be definitively established. Initial investigations focused on possible procoagulant roles for PF4 in platelet function and plasmatic coagulation. Subsequent in vitro studies have, however, described a puzzling array of other apparently unrelated biologic functions, including inhibition of angiogenesis and hematopoiesis, promotion of neutrophil adhesion, and activation, enhancement of oxy-LDL binding to the LDL receptor and stimulation of anti-coagulant activated protein C generation by the thrombomodulin/protein C system. Preliminary studies with a just-described PF4 knockout mouse line support a role for PF4 in platelet-dependent thrombosis in vivo.

NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis.

NF-kappaB transcription factors regulate the expression of tissue factor (TF), a principal initiator of the coagulation cascade. Dominant among them is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a p50 inhibitor) and genetic deletion of p50 attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. Results of the electrophoretic mobility "supershift" assay and chromatin immunoprecipitation demonstrated the direct interaction of the p50/p65 heterodimer with the NF-kappaB site of the human TF promoter. Andro-treated and p50 null mice both exhibited blunted TF expression and reduced venous thrombosis, which were recapitulated by an anti-murine TF antibody in vivo. Our findings thus indicate that regulation of TF by NF-kappaB transcription factor p50 is essential for the pathogenesis of deep vein thrombosis and suggest that specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and perhaps treating venous thrombosis.

Myeloablative hematopoietic cell transplantation for acute lymphoblastic leukemia: analysis of graft sources and long-term outcome.

PURPOSE: Analysis of hematopoietic cell transplantation (HCT) for high-risk or recurrent acute lymphoblastic leukemia (ALL) using different donor sources is confounded by variable conditioning and supportive care. PATIENTS AND METHODS: We studied 623 consecutive ALL myeloablative HCT (1980 to 2005). Donors were autologous (n = 209), related (RD; n = 245), unrelated (URD; n = 100), and umbilical cord blood (UCB; n = 69). RESULTS: After median of 8.3 years of follow-up, 5-year overall survival (OS), leukemia-free survival (LFS), and relapse were 29% (95% CI, 26% to 32%), 26% (95% CI, 23% to 29%), and 43% (95% CI, 39% to 47%), respectively. Treatment-related mortality (TRM) at 2 years was 28% (95% CI, 25% to 31%). Mismatched URD sources yielded higher TRM (relative risk [RR], 2.2; P < .01) and lower OS (RR, 1.5; P = .05) than RD or UCB HCT. Autografting yielded significantly more relapse (68%; 95% CI, 59% to 77%; P < .01) and poorer LFS (14%; 95% CI, 10% to 18%; P = .01). HCT in first complete remission (CR1) yielded significantly better outcomes than later HCT. In a 1990 to 2005 allogeneic CR1/second complete response cohort, 5-year OS, LFS, and relapse rates were 41% (95% CI, 35% to 47%), 38% (95% CI, 32% to 44%), and 25% (95% CI, 19% to 31%), respectively; 2-year TRM was 34% (95% CI, 28% to 40%). With RD, well-matched URD and UCB sources, 5-year LFS was 40% (95% CI, 31% to 49%), 42% (95% CI, 14% to 70%), and 49% (95% CI, 34% to 64%), respectively, while relapse was 31% (95% CI, 22% to 40%), 17% (95% CI, 0% to 37%), and 27% (95% CI, 13% to 41%). Acute graft-versus-host disease was associated with fewer relapses. Since 1995, we noted progressive improvements in OS, LFS, and TRM. CONCLUSION: Allogeneic, but not autologous, HCT for ALL results in durable LFS. Importantly, HCT using UCB led to similar outcomes as either RD or well-matched URD. HCT in early remission can best exploit the potent antileukemic efficacy of allografting from UCB, RD, or URD sources.

Histone deacetylase inhibitors suppress TF-kappaB-dependent agonist-driven tissue factor expression in endothelial cells and monocytes.

Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA), can regulate gene expression by promoting acetylation of histones and transcription factors. Human tissue factor (TF) expression is partly governed by a unique, NF-kappaB-related "TF-kappaB" promoter binding site. We find that TSA and four other HDACi (apicidin, MS-275, sodium butyrate, and valproic acid) all inhibit by approximately 90% TF activity and protein level induction in human umbilical vein endothelial cells stimulated by the physiologic agonists tumor necrosis factor (TNF)-alpha, interleukin-1beta, lipopolysaccharide, and HOSCN without affecting expression of the NF-kappaB-regulated adhesion molecules ICAM-1 and E-selectin. TSA and butyrate also blunt TF induction approximately 50% in vitro in peripheral blood mononuclear cells and in vivo in thioglycolate-elicited murine peritoneal macrophages. In human umbilical vein endothelial cells, TSA attenuates by approximately 70% TNF-alpha stimulation of TF mRNA transcription without affecting that of ICAM-1. By electrophoretic mobility shift assay analyses, TNF-alpha and lipopolysaccharide induce strong p65/p50 and p65/c-Rel heterodimer binding to both NF-kappaB and TF-kappaB probes. TSA nearly abolishes TF-kappaB binding without affecting NF-kappaB binding. A chromatin immunoprecipitation assay and a promoter-luciferase reporter system confirm that TSA inhibits TF-kappaB but not NF-kappaB activation. Chromatin immunoprecipitation and small interfering RNA inhibitor studies demonstrate that HDAC3 plays a significant role in TNF-alpha-mediated TF induction. Thus, HDACi transcriptionally inhibit agonist-induced TF expression in endothelial cells and monocytes by a TF-kappaB- and HDAC3-dependent mechanism. We conclude that histone deacetylases, particularly HDAC3, play a hitherto unsuspected role in regulating TF expression and raise the possibility that HDACi might be a novel therapy for thrombotic disorders.

Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge.

Pharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4(-/-)) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C-deficient (PC(+/-)) mice. Transgenic mice overexpressing human PF4 (hPF4(+)) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC(+/-) mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4(+) and PC(+/-)/hPF4(+) mice had improved survival. Further, infusion of hPF4(+) platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting.

Lymphodepletion followed by donor lymphocyte infusion (DLI) causes significantly more acute graft-versus-host disease than DLI alone.

Donor lymphocyte infusions (DLIs) can produce lasting remissions in patients with relapsed chronic myeloid leukemia (CML), but are less effective in non-CML diseases. We hypothesized that lymphodepletion, achieved with cyclophosphamide (Cy) and fludarabine (Flu), would promote in vivo expansion of the infused lymphocytes enhancing their immunologic effects. Fifteen patients with relapsed non-CML disease who received Cy/Flu/DLI were compared with 63 controls who received DLI without chemotherapy. Only the patients receiving Cy/Flu/DLI became lymphopenic at the time of DLI. Compared with controls, patients who received Cy/Flu/DLI developed significantly more grades II to IV (60% vs 24%, P = .01) and grades III to IV acute graft-versus-host disease (GVHD) (47% vs 14%, P = .01) with greater GVHD lethality. In Cy/Flu/DLI patients, T-cell proliferation was elevated at 14 days after DLI. Although these data suggest that chemotherapy-induced lymphodepletion enhances activation of donor lymphocytes, the toxicity needs to be managed before testing whether better disease control can be achieved. This trial was registered at www.clinicaltrials.gov as no. NCT00303693 and www.cancer.gov/clinicaltrials as no. NCT00167180.

Role of eosinophil peroxidase in host defense and disease pathology.

Three unusual substrates-bromide (Br(-)), nitrite (NO(2)(-)), and thiocyanate (SCN(-))-compete for oxidation by eosinophil peroxidase (EPO) in physiologic fluids in the presence of H(2)O(2) to yield, respectively, hypobromous acid (HOBr), nitrogen dioxide (NO(2)()), or hypothiocyanous acid (HOSCN). These oxidant products have strikingly different reactivities: HOBr and NO(2)() are potent, widely reactive, membrane-lytic oxidants whereas HOSCN is a weak, SH-specific oxidant that penetrates into cells and imposes an intracellular oxidant stress that can activate kinase pathways and transcription factors that profoundly influence gene expression in host cells. All three oxidants are lethal for pathogens. SCN(-) is the strongly preferred substrate for the EPO/H(2)O(2). Specific biomarkers document that EPO-dependent oxidants are generated at sites of inflammation, but direct evidence that these oxidants cause disease is confined to the observation that an EPO knockout mouse line has dramatically less pathologic damage than do wild type animals in a murine model of ulcerative colitis.