M Arras

Experimental analysis and modelling of in vitro proliferation of mesenchymal stem cells

Objectives:  Stem cell therapies based on differentiation of adult or embryonic stem cells into specialized ones appear to be effective for treating several human diseases. This work addresses the mathematical simulation of proliferation kinetics of stem cells.

Exploring the Role of Killer Cell Immunoglobulin-Like Receptors and Their HLA Class I Ligands in Autoimmune Hepatitis

Background Natural killer cells are involved in the complex mechanisms underlying autoimmune diseases but few studies have investigated their role in autoimmune hepatitis. Killer immunoglobulin-like receptors are key regulators of natural killer cell-mediated immune responses. Methods and Findings KIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 114 patients diagnosed with type 1 autoimmune hepatitis and compared with a group of 221 healthy controls. HLA Class I and Class II antigen frequencies were compared to those of 551 healthy unrelated families representative of the Sardinian population. In our cohort, type 1 autoimmune hepatitis was strongly associated with the HLA-B18, Cw5, DR3 haplotype. The KIR2DS1 activating KIR gene and the high affinity HLA-C2 ligands were significantly higher in patients compared to controls. Patients also had a reduced frequency of HLA-Bw4 ligands for KIR3DL1 and HLA-C1 ligands for KIR2DL3. Age at onset was significantly associated with the KIR2DS1 activating gene but not with HLA-C1 or HLA-C2 ligand groups. Conclusions The activating KIR gene KIR2DS1 resulted to have an important predictive potential for early onset of type 1 autoimmune hepatitis. Additionally, the low frequency of the KIR-ligand combinations KIR3DL1/HLA-Bw4 and KIR2DL3/HLA-C1 coupled to the high frequency of the HLA-C2 high affinity ligands for KIR2DS1 could contribute to unwanted NK cell autoreactivity in AIH-1.

In vitro ovine articular chondrocyte proliferation: experiments and modelling

This study focuses on analysis of in vitro cultures of chondrocytes from ovine articular cartilage. Isolated cells were seeded in Petri dishes, then expanded to confluence and phenotypically characterized by flow cytometry. The sigmoidal temporal profile of total counts was obtained by classic haemocytometry and corresponding cell size distributions were measured electronically using a Coulter Counter. A mathematical model recently proposed ( 1 ) was adopted for quantitative interpretation of these experimental data. The model is based on a 1‐D (that is, mass‐structured), single‐staged population balance approach capable of taking into account contact inhibition at confluence. The model’s parameters were determined by fitting measured total cell counts and size distributions. Model reliability was verified by predicting cell proliferation counts and corresponding size distributions at culture times longer than those used when tuning the model’s parameters. It was found that adoption of cell mass as the intrinsic characteristic of a growing chondrocyte population enables sigmoidal temporal profiles of total counts in the Petri dish, as well as cell size distributions at ‘balanced growth’, to be adequately predicted.

T cell tyrosine phosphorylation response to transient redox stress

Reactive Oxygen Species (ROS) are crucial to multiple biological processes involved in the pathophysiology of inflammation, and are also involved in redox signaling responses. Although previous reports have described an association between oxidative events and the modulation of innate immunity, a role for redox signaling in T cell mediated adaptive immunity has not been described yet. This work aims at assessing if T cells can sense redox stress through protein sulfhydryl oxidation and respond with tyrosine phosphorylation changes. Our data show that Jurkat T cells respond to -SH group oxidation with specific tyrosine phosphorylation events. The release of T cell cytokines TNF, IFNγ and IL2 as well as the expression of a number of receptors are affected by those changes. Additionally, experiments with spleen tyrosine kinase (Syk) inhibitors showed a major involvement of Syk in these responses. The experiments described herein show a link between cysteine oxidation and tyrosine phosphorylation changes in T cells, as well as a novel mechanism by which Syk inhibitors exert their anti-inflammatory activity through the inhibition of a response initiated by ROS.

Absence of activating killer immunoglobulin-like receptor genes combined with hepatitis C viral genotype is predictive of hepatocellular carcinoma

Killer immunoglobulin-like receptors and their human leukocyte antigen class I ligands have a critical role in natural killer cell response to viral pathogens and tumors. To investigate whether killer immunoglobulin-like receptor genes could influence the chronic course of hepatitis C virus infection and/or progression to hepatocellular carcinoma we retrospectively analyzed a cohort of 228 patients transplanted for hepatitis C virus-induced cirrhotic end stage liver disease, combined or not with hepatocellular carcinoma. We found that patients completely lacking activating killer immunoglobulin-like receptor genes had a high risk of developing hepatocellular carcinoma. Hepatitis C viral genotype and viral load are other risk factors that can influence the course of chronic hepatitis C virus infection. In our study, the risk conferred by hepatitis C viral genotypes was enhanced in patients lacking activating killer immunoglobulin-like receptors. These results point to an important role for activating killer immunoglobulin-like receptors in the control of hepatitis C virus infection and progression to hepatocellular carcinoma. In clinical practice, assessment of killer immunoglobulin-like receptor and hepatitis C viral genotype combinations should allow for more accurate monitoring of patients with chronic hepatitis C virus infection.

KIR and their HLA Class I ligands: Two more pieces towards completing the puzzle of chronic rejection and graft loss in kidney transplantation

Background Kidney transplantation is a life-saving treatment for patients with end-stage renal disease. However, despite progress in surgical techniques and patient management, immunological rejection continues to have a negative impact on graft function and overall survival. Incompatibility between donors and recipients for human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) generates a series of complex cellular and humoral immune response mechanisms that are largely responsible for rejection and loss of graft function. Within this context, a growing amount of evidence shows that alloreactive natural killer (NK) cells play a critical role in the immune response mechanisms elicited by the allograft. Killer immunoglobulin-like receptors (KIRs) are prominent mediators of NK cell alloreactivity. Methods and findings A cohort of 174 first cadaveric kidney allograft recipients and their donors were selected from a total cohort of 657 transplanted patients for retrospective immunogenetic analyses. Patients with HLA Class II mismatches were excluded. HLA Class I allele frequencies were compared among patients with chronic rejection, patients with stable graft function and a group of 2388 healthy controls. Activating and inhibitory KIR gene frequencies, KIR haplotypes, KIR-HLA ligand matches/mismatches and combinations of recipient KIRs and donor HLA Class I ligands were compared among patients with and without chronic rejection and a group of 221 healthy controls. Patients transplanted from donors homozygous for HLA-C1 antigens had a significantly higher risk for chronic rejection than patients transplanted from donors homozygous or heterozygous for HLA-C2 antigens or with epitopes belonging to the HLA-Bw4 ligand group. The Kaplan-Meier curves obtained by dividing the patients into 3 groups according to the presence or absence of one or both of the combinations of recipient KIRs and donor HLA ligands (rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4) showed a significantly higher cumulative incidence of chronic rejection in the group of patients completely lacking these functional units. These patients showed a progressively stronger decline in modification of diet in renal disease-estimated glomerular filtration rate. Conclusions KIR genotyping should be performed at the time of enrolment of patients on the waiting list for organ transplantation. In our study, a significantly higher risk of chronic rejection after kidney transplantation was observed when recipient (r) and donor (d) pairs completely lacked the two functional rKIR-dHLA ligand combinations rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4. This immunogenetic profile corresponds to low levels of NK cell inhibition. Therefore, patients with this high risk profile could benefit from immunosuppressive therapy aimed at reducing NK-cell cytotoxicity.

HLA-G molecules and clinical outcome in Chronic Myeloid Leukemia

The human leukocyte antigen-G (HLA-G) gene encodes a tolerogenic protein known to promote tumor immune-escape. We investigated HLA-G polymorphisms and soluble molecules (sHLA-G) in 68 chronic myeloid leukemia (CML) patients. Patients with G*01:01:01 or G*01:01:02 allele had higher value of sHLA-G compared to G*01:01:03 (109.2±39.5 vs 39.9±8.8 units/ml; p=0.03), and showed lower event free survival (EFS) (62.3% vs 90.0%; p=0.02). The G*01:01:03 allele was associated with higher rates and earlier achievement of deep molecular response (MR)4.5 (100% vs 65%, median of 8 vs 58 months, p=0.001). HLA-G alleles with higher secretion of sHLA-G seem associated with lower EFS, possibly because of an inhibitory effect on the immune system. Conversely, lower levels of sHLA-G promoted achievement of MR4.5, suggesting increased cooperation with immune system.

Successful and safe stem cell mobilization using plerixafor in a patient with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Treatment of patients aff ected by Philadelphia chromosome- positive acute lymphoblastic leukemia (PhALL) continues to be problematic in spite of the better complete response rates (75 - 95%) achieved with induction regimens based on tyrosine kinase inhibitors (TKIs), used either alone or in combination with conventional cytotoxic chemotherapy agents (1). Th e presence of the Philadelphia chromosome (Ph) remains a primary indication for hematopoietic stem cell transplant (HSCT), but for patients lacking a suitable donor other post-induction strategies need to be investigated. Reports published during the imatinib era, albeit limited in size and number, suggest that autologous HSCT needs to be reconsidered for patients with Phacute lymphoblas- tic leukemia (ALL) with low or negative molecular residual disease (MRD) at the end of remission-induction therapy (2,3). Unfortunately, patients older than 60 years and

Long-term efficacy and tolerance of rituximab for post-transfusional alloimmune haemolytic anaemia in a thalassaemia patient.

Regular red blood cell transfusion is the mainstay of therapy in thalassaemia patients. Haemolytic reactions caused by autoantibodies can seriously complicate chronic transfusion therapy and is often secondary to alloimmunization, especially in patients that are late in starting transfusion therapy (Hoffman, 2006). Studies performed in Italy and Greece demonstrated that 5–10% of multiply-transfused thalassaemia patients possess antibodies against erythrocyte antigens (Rebulla & Modell, 1991). In a minority of patients, the presence of these antibodies determines clinically relevant haemolysis (Singer et al, 2000). To date, immunosuppressive drugs, such as steroids, azathioprine, cyclophosphamide or cyclosporine A, or immunomodulating agents, such as intravenous immunoglobulin, have been used, alone or in combination, with transitory and poor results. However, these therapies correlate with a high risk of infections (Ware & Rose, 1998) and the complications of protracted steroid therapy can be particularly severe in patients who already present organ impairment (heart, liver, bones, endocrine pancreas, etc.). This report describes the efficacy of rituximab in a 36-yearold man affected by b-thalassaemia intermedia who developed post-transfusional alloimmune haemolytic anaemia that was non-responsive to treatment with steroids and intravenous immunoglobulin. The patient was diagnosed at the age of 4 years and underwent splenectomy at the age of 18 years. Until the age of 31 years, his haemoglobin (Hb) levels remained within a range of 85–95 g/l. In January 2000, the patient’s Hb levels dropped to <75 g/l and regular transfusion and iron chelation therapy with desferreoxamine was started. For 3 years, direct (DAT) and indirect (IAT) Coombs assays remained negative but, starting in January 2003, they gradually became positive for warm-reactive IgG antibodies (score 4, range 1–4) and C3b/d complement fragments. A search for alloantibodies revealed the presence of antibodies directed against the C and c antigens. Although the transfused red blood cells had previously been leucodepleted, washed and tested against prohibited erythrocyte antigens, the patient experienced a sporadic haemolytic transfusion reaction at this time. Regular transfusions (2 U every 14 d; leucodepleted RBC = 175 ± 8 ml/week) were continued but within a few months the Hb levels fell from 90 to 73 g/l. This made it necessary to intensify transfusional support by reducing the intervals between transfusions (2 U every 9–11 d; 239 ± 12 ml/week). Haematochemical tests performed during this period confirmed haemolysis: lactic dehydrogenase (LDH) >3000 U/l (normal range <600 U/l), haptoglobin <70 mg/l (normal range, 30–200), total bilirubin >119 lmol/l (normal range, 3.4–22.2), direct bilirubin 17.1 lmol/l (normal range, <8.5), DAT positive, IAT panagglutinated. Flow cytometry performed on peripheral blood showed an elevated B-lymphocyte count and monocyte–macrophage activated cells. In particular, the mean fluorescence intensities for CD1a and the co-stimulatory molecule CD40 were significantly increased. Based on persistent clinical symptomatology and the lack of response to steroids and intravenous immunoglobulin, the patient was administered rituximab, 375 mg/m, once a week for 2 weeks. The drug was well tolerated and no adverse events were observed. One month after treatment with rituximab, pretransfusional Hb levels were significantly increased (92.5 ± 3.0 g/l vs. 83 ± 6.0 g/l P < 0.03) and the patient’s transfusion requirement returned to normal (leucodepleted RBC = 178 ± 12 ml/week). A significant decrease was also observed for the absolute count of erythroblasts (5.8 ± 1.3 · 10/l vs. 4.004 ± 0.9 · 10/l; P < 0.05) and the proportion of erythroblasts in the total of nucleated cells (pretransfusional erythroblasts 45.6 ± 10.5/100 nucleated cells vs. 36.1 ± 7.8/100 nucleated cells, P < 0.05). LDH and haptoglobin levels returned to normal, even if DAT and IAT remained positive. Cross-match positivity decreased from 2+ to 3 to 0.5 to 1+. Flow cytometry on peripheral blood performed 2 weeks after the second infusion of rituximab, demonstrated a marked decrease of circulating B lymphocytes (0.0015 · 10/l) and a reduced expression of the surface antigens that characterize monocyte–macrophage activation. It is likely that, by reducing the synthesis of antibodies, rituximab decreased the number of red blood cells with alloantibodies attached to their cell membrane and, consequently, the phagocytic action of macrophages and dendritic cells. Normal levels of circulating B-lymphocytes reappeared after 4 months of treatment. For 30 months, the patient had no manifestations of haemolysis and pretransfusional Hb levels, measured every 14 d, remained constantly above 90 g/l. The patient had no complications caused by immunosuppression. After 3 years, the progressive appearance of haemolysis made it necessary to repeat the treatment with two doses of rituximab, 375 mg/m. As before, the patient’s response to the drug was excellent and at the time of writing 8 months later, the patient still has no signs of haemolysis. Correspondence