Effects of hypoxic storage on the efficacy of gamma irradiation in abrogating lymphocyte proliferation and on the quality of gamma-irradiated red blood cells in additive solution 3.

BACKGROUND: Gamma irradiation of blood products is used to prevent transfusion-associated graft-versus-host disease by inhibiting the proliferation of lymphocytes that are implicated in the disease. Gamma irradiation also damages the red blood cells (RBCs). It is unknown whether hypoxia reduces the efficacy of gamma irradiation in inhibiting lymphocyte proliferation (LP). The objectives of the study were to investigate the effects of hypoxia on gamma irradiation-induced inhibition of LP and on the in vitro properties of RBCs. MATERIALS AND METHODS: Forty-four units (300-340 ml each) of less than 8-h-old ABO-matched leukocyte reduced red cell concentrates (LR-RCC) in additive solution 3 were pooled in pairs. Peripheral blood mononuclear cells were isolated from non-leukocyte reduced RCCs and added back to the pool at a final concentration of 2 × 105 /ml. The pool was divided equally into a conventional storage bag A and a hypoxic processing and storage bag B. The units were gamma-irradiated at 25Gy on day 7 for the LP experiment and on either day 7 or 14 for the RBC quality experiments. LP was measured using a limiting dilution assay, and several in vitro metrics of RBCs were measured. RESULTS: Gamma irradiation inhibited T-lymphocyte proliferation by 4.7 × 104 -fold reduction in both hypoxic and conventional storage. The in vitro metrics of RBC quality were better preserved in hypoxic storage. DISCUSSION: T lymphocytes present in hypoxic RBC are equally susceptible to gamma irradiation as conventional storage. Hypoxic storage also reduces the deleterious effects of gamma irradiation on RBCs.

Sickle cell vaso-occlusive crisis: it's a gut feeling.

Insights in the pathogenesis of vaso-occlusive crisis in patients with sickle cell disease have changed significantly in the last decade. Various laboratory and clinical evidence have provided support to the pivotal role of activated neutrophils in this process. A recent study in murine sickle cell disease indicated that the intestinal microbiota is responsible for regulating the number of aged neutrophils, a subset of neutrophils that are overly activated. Reduction of these neutrophils in vivo protected the mice from fatal TNFα-induced vaso-occlusive crisis. In this paper, we discuss the reasons why patients with sickle cell disease may have an abnormal intestinal microbiota and how this could contribute to the development of vaso-occlusive crisis. We also highlight the recent interest in studying the intestinal microbiota of patients with sickle cell disease and suggest that the next therapeutic approach for these patients may well be in the manipulation of the intestinal microbiota to restore the individual's microbial landscape.

HCV epitope, homologous to multiple human protein sequences, induces a regulatory T cell response in infected patients.

BACKGROUND & AIMS: Spontaneous resolution of hepatitis C virus (HCV) infection depends upon a broad T cell response to multiple viral epitopes. However, most patients fail to clear infections spontaneously and develop chronic disease. The elevated number and function of CD3(+)CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg)) in HCV-infected patients suggest a role of Treg cells in impaired viral clearance. The factors contributing to increased Treg cell activity in chronic hepatitis C cases remain to be delineated. METHODS: Immunoinformatics tools were used to predict promiscuous, highly-conserved HLA-DRB1-restricted immunogenic consensus sequences (ICS), each composed of multiple T cell epitopes. These sequences were synthesized and added to cultures of peripheral blood mononuclear cells (PBMCs), derived from patients who resolved HCV infection spontaneously, patients with persistent infection, and non-infected individuals. The cells were collected and following 5days incubation, quantified and characterized by flow cytometry. RESULTS: One immunogenic consensus sequence (ICS), HCV_G1_p7_794, induced a marked increase in Treg cells in PBMC cultures derived from infected patients, but not in patients who spontaneously cleared HCV or in non-infected individuals. An analogous human peptide (p7_794), on the other hand, induced a significant increase in Treg cells among PBMCs derived from both HCV-infected and non-infected individuals. JanusMatrix analyses determined that HCV_G1_p7_794 is comprised of Treg cell epitopes that exhibit extensive cross-reactivity with the human proteome. CONCLUSIONS: A virus-encoded peptide (HCV_G1_p7_794) with extensive human homology activates cross-reactive CD3(+)CD4(+)CD25(+)FoxP3(+) natural Treg cells, which potentially contributes to immunosuppression and to the development of chronic hepatitis C.

Cellular immunotherapy for refractory hematological malignancies.

BACKGROUND: Acute myeloid leukemia (AML) and other aggressive refractory hematological malignancies unresponsive to upfront therapy remain difficult conditions to treat. Often, the focus of therapy is centered on achieving complete remission of disease in order to proceed with a consolidative stem cell transplant. At issue with this paradigm is the multitude of patients who are unable to achieve complete remission with standard chemotherapeutic options. A major benefit of transplantation is the graft versus tumor effect that follows successful engraftment. However, with this graft versus tumor effect comes the risk of graft versus host disease. Therefore, alternative treatment options that utilize immunotherapy while minimizing toxicity are warranted. Herein, we propose a novel treatment protocol in which haploidentical peripheral blood stem cells are infused into patients with refractory hematological malignancies. The end goal of cellular therapy is not engraftment but instead is the purposeful rejection of donor cells so as to elicit a potent immune reaction that appears to break host tumor tolerance. METHODS/DESIGN: The trial is a FDA and institutional Rhode Island Hospital/The Miriam Hospital IRB approved Phase I/II study to determine the efficacy and safety of haploidentical peripheral blood cell infusions into patients with refractory hematological malignancies. The primary objective is the overall response rate while secondary objectives will assess the degree and duration of response as well as safety considerations. Patients with refractory acute leukemias and aggressive lymphomas over the age of 18 are eligible. Donors will be selected amongst family members. Full HLA typing of patients and donors will occur as will chimerism assessments. 1-2x108 CD3+ cells/kilogram will be infused on Day 0 without preconditioning. Patients will be monitored for their response to therapy, in particular for the development of a cytokine release syndrome (CRS) that has been previously described. Blood samples will be taken at the onset, during, and following the cessation of CRS so as to study effector cells, cytokine/chemokine release patterns, and extracellular vesicle populations. Initially, six patients will be enrolled on study to determine safety. Provided the treatment is deemed safe, a total of 25 patients will be enrolled to determine efficacy. DISCUSSION: Cellular Immunotherapy for Refractory Hematological Malignancies provides a novel treatment for patients with relapsed/refractory acute leukemia or aggressive lymphoma. We believe this therapy offers the immunological benefit of bone marrow transplantation without the deleterious effects of myeloablative conditioning regimens and minus the risk of GVHD. Laboratory correlative studies will be performed in conjunction with the clinical trial to determine the underlying mechanism of action. This provides a true bench to bedside approach that should serve to further enrich knowledge of host tumor tolerance and mechanisms by which this may be overcome. TRIAL REGISTRATION: NCT01685606.

Treatment of whole blood with riboflavin plus ultraviolet light, an alternative to gamma irradiation in the prevention of transfusion-associated graft-versus-host disease?

BACKGROUND: Exposure of blood products to gamma irradiation is currently the standard of care in the prevention of transfusion-associated graft-versus-host disease (TA-GVHD). Regulatory, technical, and clinical challenges associated with the use of gamma irradiators are driving efforts to develop alternatives. Pathogen reduction methods were initially developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and white blood cells (WBCs). To date, systems for pathogen and WBC inactivation of products containing red blood cells are less well established than those for platelets and plasma. STUDY DESIGN AND METHODS: In this study, the in vitro and in vivo function of WBCs present in whole blood after exposure to riboflavin plus ultraviolet light (Rb-UV) was examined and compared to responses of WBCs obtained from untreated or gamma-irradiated blood by measuring proliferation, cytokine production, activation, and antigen presentation and xenogeneic (X-)GVHD responses in an in vivo mouse model. RESULTS: In vitro studies demonstrated that treatment of whole blood with Rb-UV was as effective as gamma irradiation in preventing WBC proliferation, but was more effective in preventing antigen presentation, cytokine production, and T-cell activation. Consistent with in vitro findings, treatment with Rb-UV was as effective as gamma irradiation in preventing X-GVHD, a mouse model for TA-GVHD. CONCLUSION: The ability to effectively inactivate WBCs in fresh whole blood using Rb-UV, prior to separation into components, provides the transfusion medicine community with a potential alternative to gamma irradiation.

Phospho-SXXE/D motif mediated TNF receptor 1-TRADD death domain complex formation for T cell activation and migration.

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1-TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase ß plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

Tumor Derived Extracellular Vesicles Modulate Gene Expression in T cells.

Extracellular vesicles (EVs) are excreted from all cells in the human body and are heterogeneous lipid bilayer particles containing proteins, RNA, DNA, and other cargo. T cells are primary players of the adaptive immune system and have a significant role in anti-cancer immunotherapies. Tumor derived EVs have diverse effects on host cells including modulating the immune response. In this study, we investigate the role acute myeloid leukemia (AML) derived EVs have on modulation of gene expression levels in three different T cell populations (CD8+, CD4+, and CD4 + CD39 + T cells). AML derived EVs modulate gene expression levels in all three cell populations with transcripts associated with major immunoregulatory pathways being significantly altered. Genes whose expression were significantly upregulated or downregulated were analyzed for gene ontogeny category expression and for the impact of the changes on specific immunoregulatory pathways. CD8 + T cells were modulated to a larger extent than other T cell populations and gene expression levels associated with proliferation and differentiation were influenced by AML-derived EVs.

Developments in the prevention of transfusion-associated graft-versus-host disease.

The transfusion of blood products can result in a variety of consequences, including transfer of pathogens and the induction of immune responses, such as the almost invariably fatal transfusion-associated graft-versus-host disease (TA-GVHD). Exposure of blood products to γ-irradiation is currently the standard of care for the prevention of TA-GVHD. Regulatory, technical and clinical challenges associated with the use of γ-irradiators are driving efforts to develop alternatives. Initially, pathogen reduction methods were developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and leucocytes. These methods have been found to be as effective as γ-irradiation in preventing T lymphocyte proliferation and TA-GVHD responses. Additional benefits of pathogen reduction protocols potentially include inhibition of antigen presentation, cytokine production and activation of lymphocytes.

Inhibition of histone deacetylases preserves myocardial performance and prevents cardiac remodeling through stimulation of endogenous angiomyogenesis.

We have previously shown that the inhibition of histone deacetylases (HDACs) protects the heart against acute myocardial ischemia and reperfusion injury. We also demonstrated that HDAC inhibition stimulates myogenesis and angiogenesis in a cultured embryonic stem cell model. We investigate whether in vivo inhibition of HDAC preserves cardiac performance and prevents cardiac remodeling in mouse myocardial infarction (MI) through the stimulation of endogenous regeneration. MI was created by ligation of the left descending artery. Animals were divided into three groups: 1) sham group, animals that underwent thoracotomy without MI; 2) MI, animals that underwent MI; and 3) MI + trichostatin A (TSA), MI animals that received a daily intraperitoneal injection of TSA. In addition, infarcted mice received a daily intraperitoneal injection of TSA (0.1 mg/kg), a selective HDAC inhibitor. 5-Bromo-2-deoxyuridine (50 mg/kg) was delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, the MI hearts showed a reduction in ventricular contractility. HDAC inhibition increased the improvement of myocardial functional recovery after MI, which was associated with the prevention of myocardial remodeling and reduction of myocardial and serum tumor necrosis factor α. HDAC inhibition enhanced the formation of new myocytes and microvessels, which was consistent with the robust increase in proliferation and cytokinesis in the MI hearts. An increase in angiogenic response was demonstrated in MI hearts receiving TSA treatment. It is noteworthy that TSA treatment significantly inhibited HDAC activity and increased phosphorylation of Akt-1, but decreased active caspase 3. Taken together, our results indicate that HDAC inhibition preserves cardiac performance and mitigates myocardial remodeling through stimulating cardiac endogenous regeneration.

Inactivation of human white blood cells in platelet products after pathogen reduction technology treatment in comparison to gamma irradiation.

BACKGROUND: During the transfusion of blood products, the transfer of allogeneic donor white blood cells (WBCs) can mediate adverse reactions in recipients. To minimize reactions, blood components are leukoreduced and/or exposed to gamma irradiation. Nucleic acid-targeted pathogen reduction technologies (PRTs) processes are well suited for WBC inactivation. The Mirasol PRT system (CaridianBCT Biotechnologies) uses riboflavin and ultraviolet light to reduce the active pathogen load and inactivate residual WBCs in blood products used for transfusion. The aim of this study was to compare the effect of PRT treatment to gamma irradiation. MATERIALS AND METHODS: WBCs were resuspended in 100% plasma or in plasma containing 65% platelet additive solution (SSP+). A single product was split into three aliquots: control, PRT treatment, or gamma irradiation. After treatment, peripheral blood mononuclear cells were isolated from products, and cell viability and functionality were assessed in limiting dilution assays (LDAs), by CD69 expression, by proliferation, and by cytokine accumulation after lipopolysaccharide addition. RESULTS: PRT treatment and gamma irradiation reduced the ability of the T cells to proliferate by similar degrees as evidenced by a 6 log or greater reduction in the LDA and a lack of response to several stimuli. However, gamma-irradiated T cells were still capable of up regulating surface CD69, inducing a proliferative response in allogeneic responder cells, and producing levels of proinflammatory cytokines similar to those of untreated control cells, responses that PRT-treated T cells were incapable of mediating. CONCLUSIONS: These in vitro results demonstrate that PRT treatment is more effective than gamma irradiation at abrogating selected WBC immune functions.

Murine Leukemia-Derived Extracellular Vesicles Elicit Antitumor Immune Response.

BACKGROUND: Extracellular vesicles (EVs) are heterogeneous lipid bilayer particles secreted by cells. EVs contain proteins, RNA, DNA and other cargo that can have immunomodulatory effects. Cancer-derived EVs have been described as having immunomodulating effects in vivo with immunosuppressive and pro-tumor growth capabilities. However, cancer-derived EVs have also been harnessed and utilized for anti-cancer potential. METHODS: To assess the immunomodulatory effect of EVs produced by acute myeloid leukemia (AML) cells, we isolated vesicles secreted by the murine AML cell line, C1498, and investigated their effect on in vitro and in vivo immune responses. RESULTS: These leukemia-derived EVs were found to induce increased proliferation of CD3+ cells and enhanced cytolytic activity of CD3+ cells directed toward leukemic cells in vitro. Injection of leukemia-derived EVs into syngeneic naïve mice induced T cell responses in vivo and resulted in enhanced immune responses upon T cell re-stimulation in vitro. CONCLUSION: These findings indicate that C1498-derived EVs have immunomodulatory effects on cell-mediated immune responses that could potentially be utilized to facilitate anti-leukemia immune responses.

White blood cell inactivation after treatment with riboflavin and ultraviolet light.

Functional white blood cells (WBCs) in blood components may be responsible for a number of adverse transfusion effects, including transfusion-associated graft-versus-host disease (TA-GVHD), alloimmunization, and alloimmune platelet (PLT) refractoriness. TA-GVHD occurs when functional lymphocytes are transfused into a patient who is unable to mount an immune response to the human leukocyte antigen (HLA) due to HLA compatibility or immunosuppression. Alloantibodies against HLA antigens on donor WBCs and PLTs are the major cause of refractoriness to PLT transfusions in patients receiving repeated blood transfusions. Attempts to reduce these undesirable effects have included leukoreduction filters and gamma irradiation. Studies have shown that exposure of PLT concentrates to riboflavin and light (Mirasol pathogen reduction technology [PRT], CaridianBCT Biotechnologies) causes irreparable modifications of nucleic acids that result in inactivation of a wide range of pathogens as well as inhibition of the immunologic responses mediated by WBCs present in PLT concentrates. This article summarizes these studies and also reports on additional findings from the Trial to Reduce Alloimmunization to Platelets (TRAP) and Mirasol Clinical Evaluation (MIRACLE) trials. Data from in vitro studies and this clinical trial suggest that PRT treatment may be as effective as gamma irradiation in preventing TA-GVHD and more effective than leukoreduction in preventing alloimmunization.

Effects Induced In Vivo by Exposure to Magnetic Signals Derived From a Healing Technique.

Energy healing is a therapy said to manipulate and balance the flow of "energies" in the body. One such technique, the Bengston Healing Method (BHM), has shown some success in healing malignant tumors in animals and humans, but the mechanism of action and factors influencing therapeutic success of this method are poorly understood. In this study, we tested in vivo the antitumor potential of magnetic signals recorded during BHM healing. Balb/c mice engrafted with 4T1 breast cancer cells were exposed to this recording for 4 h/d on a weekly or daily basis for 28 days; control mice were not exposed at all. Tumors showed a trend to grow slower in the treatment versus control group during the fourth week of treatment. Elevated leukocyte counts, associated with an increase in blood levels of granulocyte-macrophage colony stimulating factor and interleukin-6, were observed in tumor-bearing mice exposed to the BHM recording but not in healthy animals exposed to the recording. This suggests that exposure to a recording of BHM may induce a biological response in tumor-bearing mice, but limited effects on tumor growth when observed within the predefined end point of 28 days. Studies involving longer end points are recommended to observe the progression of tumor growth.

Nonengraftment haploidentical cellular immunotherapy for refractory malignancies: tumor responses without chimerism.

Allogeneic bone marrow transplantation relies on immunosuppression, which controls graft-versus-host disease (GVHD) and allows engraftment at the expense of diminished graft versus-tumor (GVT) activity. Advances in hematologic transplantation have prompted the development of effective, less-toxic regimens that attempt to balance GVH and GVT immunoreactions. We analyzed the safety and efficacy of haploidentical transplantation in a Phase I/II nonimmunosuppressive, nonmyeloablative setting. A total of 41 patients with relapsed refractory cancer received 100 cGy of total body irradiation (TBI), along with an infusion of 1 x 10(6) to 2 x 10(8) CD3+ cells/kg; 29 patients received the highest dose. A postinfusional cellular graft rejection syndrome resembling engraftment syndrome was noted at the 2 highest CD3+ infusion cohorts. There were 26 patients with hematologic malignancies with 14 responses, 9 of which were major. Two of 6 patients with lymphoma remained free of disease at 76 months and 82 months, respectively; there were 5 durable complete responses and 4 partial responses in 13 patients with acute myelogenous leukemia (AML). All responses occurred outside of donor chimerism. TBI at 100 cGy followed by HLA-haploidentical immunotherapy is a biologically active therapy for patients with refractory AML and lymphoma. Possible mechanisms contributing to its effectiveness include initial GVT kill, breaking of host tolerance to tumor through cross-reactive alloreactive responses, persistent nondetectable microchimerism, or some combination of these.

Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery.

OBJECTIVE: The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery. STUDY DESIGN: Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells. RESULTS: Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis. CONCLUSION: The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.

Growth, differentiation, transplantation and survival of human skeletal myofibers on biodegradable scaffolds.

Skeletal muscle transplantation strategies for muscle repair or gene therapy involve either the injection of proliferating myoblasts followed by fusion with host myofibers or implantation of ex vivo differentiated myofibers; however, both implant procedures are associated with significant cell loss. Biodegradable porous, gas-foamed poly-lactide-co-glycolide (PLG) scaffolds have desirable characteristics for cell transfer and were used to study attachment, growth, differentiation and survival of human myogenic cells. Primary human myoblasts suspended in clinical grade extracellular matrixes (ECMs) and adhered to PLG scaffolds differentiated in vitro into high-density tropomyosin positive myofibers. An immunodeficient non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse implant model was used to study the transfer and in vivo survival of differentiated human myofibers on these scaffolds. Scaffold rigidity allowed the myofibers to be maintained under tension in vitro and following subcutaneous transplantation in vivo. Following implantation, myofiber density on the PLG scaffolds decreased linearly by 78% over a 4-week period. ECM composed of either Tisseel fibrin or Zyderm collagen type I did not significantly affect in vivo cell viability over the 4-week period. Varying PLG scaffold microsphere content (10-100%) also had little effect on cell survival in vivo. In contrast, when the residual NK cell population in the immunodeficient NOD/SCID mouse model was depleted with anti-asialo GM1 (ASGM1) antiserum, in vivo cell survival significantly increased from 22% to 34% after 4 weeks. With further improvements in cell survival, PLG scaffolds may prove useful for the implantation of primary human myofibers in future clinical applications.

Extracellular vesicles in leukemia.

Extracellular vesicles (EV) are nano-sized membrane enclosed vehicles that are involved in cell-to-cell communication and carry cargo that is representative of the parent cell. Recent studies have highlighted the significant roles leukemia EVs play in tumor progression, and ways in which they can lead to treatment evasion, thus meriting further investigation. Leukemia EVs are involved in crosstalk between the leukemia cell and its surroundings, transforming it into a cancer favorable microenvironment. Due to the diverse biological content found in leukemia EVs, they have an assortment of effects on the cells they interact with and can be harnessed as candidates for diagnostic and therapeutic treatments. This review focuses on EVs in the context of leukemia and the means by which they modulate their microenvironment, hematopoiesis, and the immune system to facilitate malignancy. We will also address current and prospective EV-based therapeutics.

Induction of anti-leukemic responses by stimulation of leukemic CD3+ cells with allogeneic stimulator cells.

BACKGROUND: Immunotherapeutic protocols have focused on identification of stimuli that induce effective anti-leukemic immune responses. One potent immune stimulus is the encounter with allogeneic cells. Our group previously showed that the infusion of haploidentical donor white blood cells (1-2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies induced responses of varying magnitude in over half of the patients. Because donor cells were eliminated within 2 weeks in these patients, it is presumed that the responses of recipient lymphocytes were critically important in achieving prolonged anti-leukemic responses. METHODS: The role of patient CD3+ cells in anti-leukemic responses was examined by isolating peripheral blood mononuclear cells from newly diagnosed leukemic patients. Immunophenotyping was performed on these peripheral blood mononuclear cells. CD3+ cells were isolated from the peripheral blood mononuclear cells and tested for their ability to proliferate and lyse autologous leukemic cells when stimulated with unrelated allogeneic cells. RESULTS: Allostimulated CD3+ cells effectively generated cytolytic responses to autologous CD3-cells in 11/21 patients. Increased numbers of CD4+ cells expressing high levels of granzyme A, B and perforin and CD8+CD39+ cells were found in nonresponsive CD3+ cells. CONCLUSIONS: These results indicate that CD3+ cells from leukemic patients are capable of generating anti-leukemic responses when stimulated with unrelated allogeneic cells. This model can be used to identify approaches using alloreactive responses by patient lymphocytes to enhance in vivo anti-leukemic responses.

Altered levels and molecular forms of granzyme k in plasma from septic patients.

Granzyme K (GrK) is a member of a highly conserved group of potent serine proteases specifically found in the secretory granules of cytotoxic T lymphocytes and natural killer cells. Based on the report indicating that inter-alpha inhibitor proteins are the physiological inhibitors of GrK and on previous findings that showed a significant decrease in plasma inter-alpha inhibitor proteins in patients with sepsis, it was our aim to determine whether increased levels of uninhibited GrK would contribute to the development of sepsis. To test this hypothesis, a competitive enzyme-linked immunosorbent assay system was developed; and the levels of GrK were measured in plasma samples obtained from healthy controls and 2 sets of patients with sepsis: patients admitted to the emergency department with a putative diagnosis of sepsis and patients with severe sepsis enrolled in a clinical trial. In addition, the molecular form(s) of GrK present in these samples was analyzed by Western blot. The levels of GrK were significantly increased in emergency department patients compared with healthy controls and significantly decreased in patients with severe sepsis enrolled in a clinical trial compared with healthy controls. GrK was detected as high-molecular-weight protein complexes in healthy controls but as complexes of lower molecular weight in the septic patients. The decrease in complex size correlated with the appearance of a band at 26 kDa similar to the size of free GrK. Our results indicate that plasma levels of GrK could serve as a useful diagnostic marker to stage sepsis, permitting better classification of septic patients and enabling targeting of specific treatments, and may play a functional role in the development of sepsis.