Immunocompetent Lymphocytes Research Articles

Neurons Are a Primary Driver of Inflammation via Release of HMGB1.

Recent data show that activation of nociceptive (sensory) nerves turns on localized inflammation within the innervated area in a retrograde manner (antidromically), even in the absence of tissue injury or molecular markers of foreign invaders. This neuroinflammatory process is activated and sustained by the release of neuronal products, such as neuropeptides, with the subsequent amplification via recruitment of immunocompetent cells, including macrophages and lymphocytes. High mobility group box 1 protein (HMGB1) is a highly conserved, well characterized damage-associated molecular pattern molecule expressed by many cells, including nociceptors and is a marker of inflammatory diseases. In this review, we summarize recent evidence showing that neuronal HMGB1 is required for the development of neuroinflammation, as knock out limited to neurons or its neutralization via antibodies ameliorate injury in models of nerve injury and of arthritis. Further, the results of study show that HMGB1 is actively released during neuronal depolarization and thus plays a previously unrecognized key etiologic role in the initiation and amplification of neuroinflammation. Direct targeting of HMGB1 is a promising approach for novel anti-inflammatory therapy.

Expression of terminal deoxynucleotidyl transferase in the pharyngeal and palatine tonsils in local infectious and inflammatory diseases of the upper respiratory tract and pharynx in childhood

Along with central immune organs, the peripheral lymphoepithelial organs of the pharynx are actively involved in protecting the body from infections. Adaptive, or induced, immunity occurs during the postnatal ontogenesis of immunocompetent lymphocytes, which includes the secondary somatic recombination of the V genes with the participation of recombination-activating gene (RAG) and terminal deoxynucleotidyl transferase (Tdt) proteins. This publication discusses the results of detection of Tdt-positive cells in the pharyngeal and palatine tonsils of children of different ages, who had been operated on for adenoid vegetations and chronic tonsillitis. Attention is drawn to the localization of Tdt+ cells, the level of Tdt expression, an attempt to clarify the phenotype, destination, and place in the diagnostic arrays of functional markers when an adaptive immunity is generated in children.

The origin of prostate gland-secreted IgA and IgG

The prostate secretes immunoglobulin (Ig) A (IgA) and IgG; however, how immunoglobulins reach the secretion, where the plasma cells are located, whether immunoglobulins are antigen-specific and where activation of the adaptive response occurs are still unknown. Immune cells, including CD45RA+ cells, were scattered in the stroma and not organized mucosae-associated lymphoid-tissue. IgA (but not IgG) immunostaining identified stromal plasma cells and epithelial cells in non-immunized rats. Injected tetramethylrhodamine-IgA transcytosed the epithelium along with polymeric immunoglobulin receptor. Oral immunization with ovalbumin/mesopourous SBA-15 silica adjuvant resulted in more stromal CD45RA+/IgA+ cells, increased content of ovalbumin-specific IgA and IgG, and the appearance of intraepithelial CD45RA+/IgG+ cells. An increased number of dendritic cells that cooperate in other sites with transient immunocompetent lymphocytes, and the higher levels of interleukin-1β, interferon-γ and transforming growth factor-β, explain the levels of specific antibodies. Nasal immunization produced similar results except for the increase in dendritic cells. This immunomodulatory strategy seems useful to boost immunity against genitourinary infections and, perhaps, cancer.

Photochemical inactivation of lymphocytes by riboflavin with visible light for TA-GVHD prevention

Transfusion-associated graft-versus-host disease (TA-GVHD) is a life-threatening complication caused by the input of a number of immunocompetent allogeneic lymphocytes. This study focus on the photochemical effects of riboflavin excited by visible light (RB+L) treatment on human lymphocytes, to study the feasibility of using RB+L treatment to prevent adverse immune reactions caused by transfused lymphocytes. 100μM riboflavin was added to lymphocyte suspensions. After exposure to 400–580nm visible light with a total energy of 40J/mL, cells were cultured and the ability of proliferation and cytokine secretion were assayed upon stimuli. Meanwhile, lymphocytes were also treated by gamma-irradiation as parallel to testify the inactivation effect of RB+L. Results showed that γ-irradiation and RB+L treated cells showed a decline in cell viability. After stimulation of phytohemagglutinin (PHA) or anti-CD3 together with anti-CD28, proliferative ability of RB+L treated cells was strongly inhibited when compared to untreated cells. The inhibitive rates of proliferation in RB+L group were also higher than those of cells treated by γ-irradiation. Results of CFSE assays also illustrated hardly any cell division of RB+L and γ-irradiation treated lymphocytes. Besides low level productions of IL-4 and IL-12, cytokine production of TNF-α, IFN-γ and IL-10 by incubation with PHA or IL-1β, IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ stimulated by anti-CD3 plus anti-CD28 were suppressed after treatment of RB+L significantly. It was suggested that RB+L/γ-irradiation treatment induced cell apoptosis. These results indicated that RB+L treatment functionally inactivated lymphocytes by inhibiting cell proliferation and cytokine production. RB+L might be an alternative for TA-GVHD prevention.

IL-1 and T Helper Immune Responses

CD4 T cells play a critical role in mediating adaptive immunity to a variety of pathogens as well as in tumor immunity. If not adequately regulated, CD4 T cells can be also involved in autoimmunity, asthma, and allergic responses. During TCR activation in a particular cytokine milieu, naïve CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, and Th17, as defined by their pattern of cytokine production and function. IL-1, the prototypic proinflammatory cytokine, has been shown to influence growth and differentiation of immunocompetent lymphocytes. The differential expression of IL-1RI on human CD4 T cell subsets confers distinct capacities to acquire specific effector functions. In this review, we summarize the role of IL-1 on CD4 T cells, in terms of differentiation, activation, and maintenance or survival.

Double-Unit Umbilical Cord Blood Transplantation Induces MHC-Matched Single-Unit Dominance with Development of Immunocompetent Lymphocytes in Recipient Mice

Abstract 4097 Background:Double-unit umbilical cord blood cell (dUCBC) transplantation has emerged as an effective strategy for improving the engraftment of umbilical cord blood stem cells in the bone marrow of recipients. Due to a lack convenient animal models, analyses of the differentiation capacity of dUCBC in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In the present study, we evaluated the characteristics of immune reconstitution induced by dUCBC transplantation in mice. Materials and Methods:Natural killer cells were depleted from female C57BL/6 (B6) [H-2b] recipient mice by intraperitoneal administration of rabbit anti-asialo GM1 polyclonal antibody 1 day before transplantation. On the following day, the lethal X-ray-irradiated B6 recipients were given transplants of three different combinations of dUCBC {group (1) GFP-Tg B6 [H-2b] and BALB/c [H-2d]; group (2) GFP-Tg B6 [H-2b] and C3H [H-2k]; group (3) BALB/c [H-2d] and C3H [H-2k]}, each combination containing an equal number of cells. At 16 weeks after transplantation, reconstitution of immune cells was evaluated by flow cytometric analysis utilizing specific antibodies against lineage markers such as CD3 (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes). The donor origin of each lineage population was determined by anti-H-2Kk (for C3H) and/or H-2Kd (for BALB/c) antibody staining. GFP+ lineage cells were identified as being of B6 donor origin. Skin grafting was then performed in all recipients to assess the functional maturity of the newly developed T and B cells induced by dUCBC transplantation. Results:The survival rate at 16 weeks after transplantation was 73% (8/11) for case (1), 92% (12/13) for case (2), and 50% (3/6) for case (3). In the great majority of cases (1) and (2), in which dUCBC were administered as a stem cell source, the MHC-matched single unit from GFP-Tg B6 acts as the sole source of long-term hematopoiesis (75% (6/8) for case (1); 100% (12/12) for case (2)). CD3+ T cell peripheral blood chimerism from BALB/c was observed in two of the eight B6 survival recipient mice in case (1) at an early stage of hematopoiesis, predicting the long-term engrafting unit. On the other hand, hematopoiesis in case (3) with fully allogenenic dUCBC transplantation was reconstituted by the B6 recipients' own X-ray-resistant hematopoietic stem cells (HSC). Our results indicate that MHC-matched UCBC-HSC predominantly engraft in the recipient's bone marrow after dUCBC transplantation. However, the nature of this selective mechanism remains largely unknown. In all cases, alloreactive cytotoxic cells in recipient may participate in such selection. In dUCBC transplantation, the included allogeneic cells probably act as stimulators for promoting the differentiation and maturation of MHC-matched HSC through activation of certain types of signal transduction (for example, through cytokine secretion). Currently, we are investigating the possible presence of alloreactive cytotoxic cells in bone marrow. Functionally, these recipients were tolerant of skin grafted from B6, whereas they rejected skin from BALB/c and C3H within 20 days, indicating that both CD4+ helper and CD8+ killer T cells were functionally mature in the recipient mice. Correspondingly, only the alloantibody to BALB/c and C3H was produced in the recipients. One of two chimeric recipient mice in case (1) reacted to only C3H skin with T and B cells. Conclusions:dUCBC transplantation clearly rescued mice that had been subjected to lethal X-ray irradiation. Furthermore, our observations indicate that T and B cells derived from dUCBCs transplants are immunologically fully competent with the ability to distinguish self from non-self MHC antigens. However, a clear understanding of the mechanisms underlying the predominant engraftment of MHC-matched HSCs in the recipient's bone marrow will be necessary. Disclosures:No relevant conflicts of interest to declare.

Concurrent Allorecognition Has a Limited Impact on Posttransplant Vaccination

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.

Allogeneic Stem Cell Transplant Versus Tandem High-Dose Melphalan for Front-Line Treatment of Deletion 13q14 Myeloma – An Interim Analysis of the German DSMM V Trial.

Abstract 51 BackgroundAllogeneic stem cell transplantation (allo SCT), a treatment modality based on transfer of immunocompetent donor lymphocytes offers curative potential to subjects with a variety of hematological cancers. In multiple myeloma (MM), high-dose melphalan followed by autologous stem cell transplantation (auto SCT) is adopted as a standard of care. However, it remains palliative since virtually all patients (pts) relapse and renders allo SCT an option of interest. Deletion of chromosome 13q14 (13q-) in MM has been shown to negatively impact prognosis. Therefore, improvement of therapy for 13q- pts is highly desirable. Patients and methods A prospective two-arm multi-center trial (DSMM V) was set up by our group to compare tandem high-dose melphalan 200 mg/m2 (HD Mel) with a reduced intensity conditioning allo-SCT after one cycle of HD Mel for 13q- MM. Eligibility criteria were 13q- on bone marrow FISH analysis; age up to 60 years; newly diagnosed MM in Salmon and Durie stages II and III; and measurable disease. Allocation to either treatment arm was by availability of an HLA-matched (one mismatch allowed) volunteer related (VRD) or unrelated donor (VUD). Initially, all pts received four cycles of anthracycline/dexamethasone-based induction followed by chemomobilization of peripheral blood stem cells (PBSCT) and one cycle of HD Mel. Allogeneic SCT was performed after preparation with fludarabine (30 mg/m2 for 3 consecutive days) and melphalan 140 mg/m2. ATG was administered for VUD transplants. Results 199 pts with a median age of 53 (range, 30 – 60) years were enrolled between October 2002 and March 2007 and included in this interim analysis. Sixty-seven percent had stage III disease. Allo SCT was performed in 126 of 199 pts (63%), 76 of whom (60%) received VUD allografts. The remaining 73 subjects uniformely received tandem HD Mel. Pts following allo SCT were more likely to achieve CR (59%) when compared to tandem HD Mel (32%; p=.003) within one year after end of therapy. Similarly, overall response rate was significantly higher with allo SCT (91% versus 86%; p=.003). Of note, depth of response to allo SCT was not associated with presence of acute graft-versus-host disease (GVHD): 62% CR with grades II to IV GVHD vs 58% CR with grades 0 and I (p=.75). Treatment-related mortality (TRM) at 2 years from allo SCT was 16/126 (12.7%). At a median follow up of 25 months for tandem HD Mel and 34 months for allo SCT, projected 3-year overall survival is 72% (auto) and 60% (auto/allo SCT; p=0.22), respectively. Conclusions This is the largest trial on first-line allogeneic stem cell transplant in MM so far. Our interim results show a higher CR rate in FISH 13q- subjects undergoing allo SCT when compared to tandem HD Mel. Despite a majority of allografts in our study being delivered from unrelated donors, TRM was comparable to trials confined to sibling transplants. At a relatively short follow-up, there is not yet a difference between both arms regarding OS, albeit longer follow-up may be important as previously described. This as well as analysis of the impact of donor type and chronic GVHD on outcome will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Neuropeptides and thymic hormones in the Xenopus thymus

T-cell development is characterised by a complex series of events in the thymus, which results in the development of self-restricted immunocompetent lymphocytes. We have previously reported the expression of neuropeptides in the thymus of various species, highlighting the evolutionary importance of neuroendocrine interactions in thymocyte development. Despite the many physiological and functional similarities in their immune systems, no study has addressed the importance of neuropeptides and thymic hormones in T-cell development in Xenopus. Immunohistochemical analysis revealed that the neuropeptides substance P, neuropeptide Y, somatostatin, calcitonin gene related peptide, and vasoactive intestinal polypeptide and the thymic hormones thymosin alpha1, thymosin beta4, and thymopoietin are found in the Xenopus thymus. This was further corroborated by RT-PCR. Furthermore, double staining revealed that neuropeptides and thymic hormones are coexpressed within the epithelial cell component of the thymus. These results show that neuropeptides and thymic hormones are expressed in the thymus of Xenopus, and suggest that they are likely to play a role in T-cell development.

Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS 2) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

Inactivation of Lymphocytes in Blood Products Using Riboflavin Photochemical Treatment with Visible Light

Immunocompetent lymphocytes present in blood products are an important cause of immune reactions following blood transfusion such as transfusion-associated graft-versus-host disease (TA-GVHD). In this study the effects of riboflavin photochemical treatment (RPT) on lymphocyte proliferation and cytokine production in vitro were measured to establish whether RPT of blood products can be used to prevent immune reactions resulting from transfused lymphocytes. Lymphocytes and riboflavin were added together in medical PVC transparent bags and then exposed to visible light. Control lymphocytes were exposed to light in the absence of riboflavin. Lymphocytes exposed to riboflavin photochemical treatment (RPT-lymphocytes) and control lymphocytes were tested for the proliferative ability and the production of several cytokines upon stimulation with antigens. Upon stimulation by phytohemagglutinin (PHA) the proliferation of RPT-lymphocytes was inhibited. Using flow cytometry it was shown that RPT-lymphocytes were unable to enter the S-phase of the cell cycle following PHA stimulation. The level of cytokines present in the supernatant of RPT-lymphocytes after stimulation by antigens was significantly lower than those present in the corresponding supernatant of control lymphocytes. RPT with visible light inactivates lymphocytes, inhibits lymphocyte proliferation and the production of cytokines. It appears to be a promising method to prevent immune reactions such as TA-GVHD.

Development of Immunocompetent Lymphocytes In Vivo From Murine Umbilical Cord Blood Cells

Reports concerning the immunological functions of lymphocytes derived from umbilical cord blood cell (UCBC) have been limited. In murine syngeneic transplantation system using green fluorescent protein transgenic donors, UCBC-derived lymphocytes were studied for their immunological competence. Hematopoietic stem cells (HSC) among UCBC differentiated in the recipients into phenotypically mature T and B lymphocytes. The T lymphocytes were capable of specific recognition of major histocompatibility complex/peptide complex and of the subsequent activation. The antigen-induced CD4(+) T cells produced lymphokine upon in vitro antigen stimulation. CD8(+) T cells simulated in the mixed lymphocyte culture could lyze specific target cells. Furthermore, RAG2(-/-) mice reconstituted with UCBC mounted specific antibody responses to T-dependent antigen comparable to those by bone marrow chimeras and also rejected allogeneic skin grafts. Collectively the data indicated that T and B lymphocytes derived from UCBC-HSC are fully competent in immunological terms.

Cyclosporine A: A Review of Current Oral and Intravenous Delivery Systems

ABSTRACTAs early as 1978, the immunosuppressive effect of cyclosporine A (CsA), a metabolite of the fungus Tolypocladium inflatum (), was reported to be effective in inhibiting organ rejection in patients receiving kidney transplants from mismatched cadaver donors () and in the treatment of graft-versus-host disease in patients with acute leukemia following bone marrow transplants (). Today, CsA is still indicated to prevent rejection following solid organ transplantations, prevent and treat graft-vs-host disease following bone marrow transplants, and has also been used in the treatment of autoimmune disease such as psoriasis, rheumatoid arthritis, and nephrotic syndrome (). The effectiveness of CsA is derived from its ability to specifically and reversibly inhibit immunocompetent lymphocytes in the G0 and G1 phase of the cell cycle. The T-helper cells are the main target, but suppression of the T‐suppressor cells also occurs. The production and release of lymphokines, including interleukin-2 are also inhibited (). CsA can be administered intravenously as well as orally in the form of a solution or a soft gelatin capsule. The following review will focus on the evolution of the emulsion-based oral formulations from the first generation as Sandimmune® to the second generation Neoral®, both products of Novartis Pharmaceutical, as well as on the Sandimmune® commercial intravenous formulation. The potential of alternative delivery systems, including micelles, micro- and nanoparticles, and liposomes, will also be discussed.

Transfusion Associated Graft Versus Host Disease (TA-GVHD) Following Liver Transpalantation.

We report clinical experience of diagnosis and treatment in four cases underwent TA-GVHD after liver transplant in 550 cases carried out in our hospital. The four patients, mean 38 years and male, with liver cancer were performed liver transplant. The immunosuppressive drugs of FK506 and MMF were given. Recovery were uneventful and liver function transferred into normal after operation. One to four weeks after transplant they complained of increasing fatigue and developed high fever, then presented skin rash from head and trunk to limbs, diarrhea, abnormal liver function (ALT mean 260 IU/L, AST mean 180 IU/L), and pancytopenia. The bone marrow biopsy showed a poorly erythroid, abnormal myeloid and megakaryocyte cell lines. Serum IgG and IgM antibodies of parvovirus B19, CMV (pp65), and EBV (IgM) were negative. Vitamin B12 and folate levels in serum were normal. Skin biopsy showed that epidermis loose and a lot of lymphocytes and monocytes infiltrate into tissue between dermis and epidermis. At clinical diagnosis of TA-GVHD the patients were protected in isolation room and FK506 and MMF were transferred or adjusted. The corticosteroid medication, dexamethasone and methyprednisolone, were given for controlling TA-GVHD progress. The other regimen consist of preventing infection with antibiotics and immunoglobulin, promoting hematopoietic recovery with G-CSF, preventing bleed and improving anemia with gamma irradiation of blood components when necessary. After two to three weeks the temperature recovered normally, skin rash disappeared, diarrhea stopped eventually, and liver function again recovered in three patients following normally up one year. The other one died from respiratory failure due to complicated pneumonia in the three weeks late. TA-GVHD is a rare complication caused by immunocompetent lymphocytes in blood used in transplant period. Although the incidence rate (<0.01%) was very low in our practice, severe TA-GVHD threatening to life can result in recipients. The diagnosis of TA-GVHD depended on clinical manifestation, skin biopsy, HLA typing and DNA analysis, if possible, between donor blood and host. The diagnosis should be distinguished from liver transplant associated GVHD and virus infection. The diagnosis of TA-GVHD can be established based mainly on their clinical manifestation, skin biopsy, and effective treatment. Because transfusion of blood were different donor resources, HLA typing and DNA analysis were difficult to be tested. Since liver transplant associated GVHD does not cause abnormal liver function, whether or not abnormal liver function is important key at difference diagnosis with TA-GVHD. Also, the virus infection should be ruled out because virus test are negative. Although the prognosis of TA-GVHD is pessimism according to literature, the three out of them in our report were effectively cured. Especially, treatment of corticosteroid medication are very important regimen. Adjusting immunosuppressive drugs, anti-infection, preventing bleed and promoting hematopoietic recovery are also essential. The gamma irradiation of blood components can be used for preventing TA-GVHD.

Differential detergent fractionation for non-electrophoretic bovine peripheral blood monocyte proteomics reveals proteins involved in professional antigen presentation

Professional antigen presenting cells (APC), dendritic cells (DC) and their myeloid progenitors, monocytes/macrophages are critical controllers of innate and adaptive immunity. Here we show that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. We identify 53 bovine proteins involved in immune function of professional APC. In particular, 13 adhesion molecules, three toll-like receptors (TLR1, 6 and 8), three antigen uptake-related proteins (including mannose receptor [MR] precursor), and eight actin-like proteins involved in active endocytosis were identified. In addition, MHC class I and II-related proteins, cytokines, active substances and growth factors have been identified. We conclude that the DDF approach can provide interpretable and meaningful functional information concerning protein expression profiles associated with monocyte activation, transformation into macrophages and/or immature DC, and maturation of monocyte-derived DC in the presence of multiple bovine pathogens.

Diagnosis of transfusion‐associated graft‐vs.‐host disease: the importance of short tandem repeat analysis

Transfusion-associated graft-vs.-host disease (TA-GvHD) can occur following transfusion of blood products containing immunocompetent lymphocytes, usually from HLA homozygous donors, into immunocompromised patients sharing one HLA haplotype with the donor. The diagnosis of TA-GvHD may be delayed due to the initial nonspecific clinical features involved. Investigations to detect the presence of donor-derived cells in the blood and/or affected tissues of the recipient are essential to confirm the diagnosis. We report the investigation of suspected TA-GvHD using short tandem repeat (STR) analysis, to detect the presence of donor cells (chimerism), in an immunocompetent patient admitted for coronary artery bypass surgery. Peripheral blood and skin biopsies (from affected and nonaffected sites) from the patient and peripheral blood samples from the implicated donors were taken for HLA typing and STR analysis. STR analysis revealed the presence of donor material in the patient's peripheral blood sample and in DNA extracted from the affected skin biopsy but not the unaffected biopsy, suggesting lymphocytes from this donor were responsible for the development of TA-GvHD. Furthermore, HLA typing results supported the diagnosis of TA-GvHD. These data demonstrate the use of STR and HLA analysis as effective tools in the diagnosis of TA-GvHD.

Ionising Irradiation Influences Platelet Adhesion and Aggregation under High Flow Conditions.

Transfusion-associated graft-versus-host disease (GVHD) is a dramatic immunologic reaction mediated by transfused immunocompetent lymphocytes directed against an immunocompromised host. GVHD shows a rapid onset and is characterized by severe symptoms and a poor response to treatment (mortality rate > 90%). Prophylactic gamma irradiation of cellular blood components is the most efficient and reliable way to prevent GVHD. The currently recommended standard dose of gamma irradiation of blood products is 25 Gy. While irradiated blood components including platelet concentrates have been extensively transfused over the past 25 years, only few studies (with controversy results) evaluated the effect of ionising irradiation on platelet morphology and function. In this study, we examined the effect of a standard irradiation dose of 25Gy on platelet function using a platelet function analyzer (PFA-100). Citrate-anticoagulated blood was obtained in duplicate from twelve healthy blood donors. One sample was immediately irradiated according to a standard protocol (25 Gy), the other specimen was used as control. Platelet function of irradiated and control samples was evaluated using PFA-100. This device applies a high shear flow system to simulate the conditions under which platelets are subjected at the site of a damaged blood vessel wall. Anticoagulated whole blood was passed through the membranes coated with either collagen and epinephrine (Col/Epi) or collagen and ADP (Col/ADP) under hemodynamic conditions similar to those of small capillaries. Platelets adhere and aggregate across the aperture until the flow ceases, evaluated by the closure time (CT, sec). Statistical analysis was performed using a standard software (Sigma Plot v. 8; Student's paired t test). Analysis of platelet function with Col/Epi cartridges showed that irradiated samples had a prolonged closure time compared to corresponding non irradiated controls (144 ± 21 sec vs. 120 ± 18 sec, p<0.05). The averaged CT prolongation was 19±5%. Analysis of CT using Col/ADP cartridges showed also prolongated CT (averaged prolongation 7%) in seven of nine irradiated samples compared to non irradiated controls (97 ± 14 sec vs. 103 ± 13 sec, p<0.05). Our results suggest that a standard dose of ionising irradiation for prevention of GVHD has a significant influence on platelet function. The biochemical nature by which this platelet dysfunction following irradiation is caused, remains to be assessed in further detail.

Hematopoietic lineage-restricted minor histocompatibility antigen HA-1 in graft-versus-leukemia activity after donor lymphocyte infusion.

Immunocompetent alloreactive donor lymphocytes directed against minor histocompatibility antigens are supposed to be responsible for graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity after allogeneic stem cell transplantation. The authors describe the detection of HA-1-specific T cells by peptide-loaded dimers and flow cytometry in the peripheral blood of a patient in complete remission but without GvHD after donor lymphocyte infusion for chemotherapy-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia. The HA-1-specific T cells were sorted and an alloreactive, polyclonal T-cell line with specific lytic activity against HA-1-positive target cells, including leukemic cells, was established. Although P190 bcr/abl peptide-specific CD8positive T cells were detected in the peripheral blood at the same time, these T cells could not be expanded. Furthermore, no P190 bcr/abl peptide-specific T-cell response could be induced in vitro, even when peptide-loaded dendritic cells were used as stimulator cells. The authors conclude that in the absence of GvHD, HA-1-specific rather than P190 bcr/abl-specific T cells are responsible for ongoing GvL activity.

Extracorporeal photopheresis in the treatment of graft-vs-host disease.

Graft-vs-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. GVHD was first described by Barnes and Mole as a fatal disease manifest by skin changes, diarrhea, and runting in irradiated mice given allogeneic spleen cells. Early human allogeneic bone marrow transplant patients were noted to manifest a similar syndrome of dermatitis, hepatitis, and enteritis occurring within the first 100 days after infusion of allogeneic stem cells. The etiology of GVHD is related to alloreactivity between immunocompetent donor lymphocytes and transplantation alloantigens on host tissues. The incidence of acute GVHD (aGVHD) ranges from 27% to 50% after sibling-matched related donors and from 42% to 72% after unrelated donor bone marrow or peripheral blood stem cell transplants. Despite aggressive immunosuppressive therapies, including high-dose corticosteroids, cyclosporine A, antithymocyte globulin, tacrolimus, and mycophenolate mofetil, the mortality from acute GVHD remains high. Chronic GVHD (cGVHD) is a syndrome of immune dysregulation whose onset has been arbitrarily defined as occurring at least 100 days after allogeneic stem cell infusion. Clinical features of cGVHD are distinguished from aGVHD in that they more closely resemble autoimmune disorders. Histopathologic changes include sclerodermatous skin changes from collagen deposition, pulmonary fibrosis, esophageal dysfunction, dry mouth or mucocutaneous ulcerations, cholestasis, and myositis or fasciitis and are thought to be initiated, in part, by autoantibodies to cell surface and intracellular proteins. The incidence of cGVHD ranges from 30% in siblingmatched donors to over 70% in matched unrelated donor transplants. Factors associated with cGVHD include increased donor and recipient age, prior aGVHD, and the use of alloimmune female donors. Conventional therapeutic approaches for cGVHD, including corticosteroids and immunosuppressive agents, have demonstrated limited efficacy in patients with extensive disease. Oral methoxsalen followed by ultraviolet A therapy (PUVA), while effective in alleviating the symptoms of chronic skin GVHD, has had no impact on visceral involvement. Novel agents demonstrating activity include T-cell directed therapies, such as humanized anti-CD25 antibody (dacluzimab) and pentostatin, as well as anti-TNF-a antibody (infliximab) and thalidomide. While aGVHD is understood as a disease of acute alloreactivity, the etiology of cGVHD is controversial and is believed to be either an extension of aGVHD and/ or a result of dysfunctional immune reconstitution with generation of autoantibodies and tissue autoreactive Tcell clones. Acute GVHD is believed to be a Th1-driven disease wherein inflammatory cytokines, such as IL-2 and IFN-c as well as the monokines IL-1 and TNF-a, contribute to tissue damage. In murine models, donor CD4-enriched cells of Th2 phenotype have been shown to prevent aGVHD without affecting engraftment, suggesting that they play a role in downregulating the Th1 response, and, in other studies, administration of a Th1 inhibitor (TAK-603) markedly reduced the mortality associated with aGVHD. Chronic GVHD has been proposed by others to be a Th2-mediated disease, but recent studies demonstrating upregulation of Th1 cytokines IFN-c and IL-12 in peripheral blood mononuclear cells implicate, in part, a Th1-derived mechanism similar to that seen in acute alloreactivity.

Immunohistochemical and Molecular Biological Investigations Regarding the Pathogenesis of Extrahepatic Biliary Atresia

The pathogenetic model for biliary atresia presently most favored is that EHBA is the result of a peri- or postnatal bile duct lesion. Several authors demonstrated inflammatory infiltrations in the mesenchymal areas of the liver and thus concluded an infectious genesis. An association of rota-, reo- (and CMV) virus infection with EHBA was suspected, but the presence of these viruses in EHBA could not be reproduced. In view of this controversial debate we found it to be indicated to investigate tissue blocks from the porta hepatis and liver biopsies in children with EHBA by histo- and immunohistochemistry for the quality and quantity of leukocyte infiltrations. 31 tissue excidates of the porta hepatis were gained on the occasion of hepatoportoenterostomy, fixed in 4 % buffered formalin and embedded in paraffin. The presence of leukocyte infiltrations and their subpopulations was demonstrated by histochemical reactions and immunohistochemical staining methods using specific antibodies against surface markers. The number of leukocytes and their subpopulations was counted in three different regions of the porta hepatis, the obliterated extrahepatic bile duct, the fibrous mass of the porta hepatis and the transition zone between the fibrous mass and liver parenchyma. A statistical analysis was done. In EHBA, leukocyte infiltrations consist mainly of macrophages. Antigen-presenting cells and lymphatic cells play a minor role. Lymphatic cells could only be detected in 6 out of 31 tissue preparations. Antigen-presenting cells could only be detected via anti-F13a antibody which shows cross-reactivities, i.e. against macrophages and embryonal tissue. Evaluating the density of leukocyte infiltrations with regard to the different anatomical regions of the porta hepatis we could demonstrate that leukocyte infiltrations are scarce around the rudiment of the bile duct whereas the highest leukocyte density could be found in the fibrous mass of the porta hepatis and the intrahepatic fibrous septs interconnecting the fibrous mass of the porta hepatis with liver parenchyma. Liver parenchyma was mainly free of leukocyte infiltrations with the exception of neutrophilic granulocytes. Regardless of the subpopulations, leukocytes were mainly arranged around the bile ducts of the fibrotic septa. Most tissue preparations from children operated on during the 4th-8th week of life show only small leukocyte infiltrations and in the majority of cases no immunocompetent lymphocytes. This leads to the conclusion that a virus infection as an underlying cause for EHBA is very unlikely. Most probably, the observed leukocyte infiltrations are due to an unspecific phagocytotic activity. Comparing our results to reports from Hadchouel et al (9) and Landing et al (12) led us to believe that a pathologic immunoreaction with a possible defective antigen elimination could also be considered as a reason for EHBA.