A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product.

BACKGROUND: Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. MATERIALS AND METHODS: The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. RESULTS: MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. CONCLUSIONS: MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Extracorporeal photopheresis as an immunomodulatory agent: Haematocrit-dependent effects on natural killer cells.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Leukapheresis for autologous stem cell transplantation: Comparative study of two different thawing methods WSCFD(®) Stem Cell Fast Thawer KW versus 37 °C thermostatic bath.

BACKGROUND: Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. AIM OF THE STUDY: We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD(®) Stem Cell Fast Thawer KW. MATERIALS AND METHODS: The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. RESULTS: We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD(®) versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD(®) and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10(4) ± 98.3 total CFU using WSCFD(®) versus 592 × 10(4) ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. CONCLUSIONS: WSCFD(®) can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

Reversal of human allergen-specific CRTH2+ T(H)2 cells by IL-12 or the PS-DSP30 oligodeoxynucleotide.

BACKGROUND: The chemoattractant receptor homologous molecule expressed on T(H)2 cells (CRTH2) is a receptor for prostaglandin D(2), which among human T cells is selectively expressed by T(H)2 and type 2 cytotoxic effectors. OBJECTIVE: Our purpose was to assess whether the cytokine production profile of T(H)2 effectors could be reversed by exploiting their selective expression of CRTH2. METHODS: CRTH2(+) T cells were purified from the blood of allergic subjects, stimulated with the specific allergen in the absence or presence of IL-12, and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma after antigen or polyclonal stimulation. RESULTS: Both IL-12 and the PS-DSP30 oligodeoxynucleotide enabled CRTH2(+) allergen-stimulated T(H)2 cells to produce IFN-gamma. This change in the profile of cytokine production by T(H)2 cells from allergic subjects was related to the upregulation of IL-12 receptor beta2 chain and was associated with the loss of CRTH2. CONCLUSIONS: These data demonstrate that the cytokine production pattern of fully differentiated T(H)2 effectors can be changed to a less polarized profile, thus providing the physiologic basis for new immunotherapeutic strategies in allergic disorders.

Genetic and environmental factors contributing to the onset of allergic disorders.

Evidence has been accumulated to suggest that allergen-reactive Th2 cells play a triggering role in the activation and/or recruitment of IgE antibody-producing B cells, mast cells and eosinophils, the cellular triad involved in allergic inflammation. Recently, chemokines and chemokine receptors involved in such Th2-type response have been also defined. Th2 cells represent the polarized arm of the effector-specific responses that contribute to the protection against gastrointestinal nematodes and act as regulatory cells for chronic and/or excessive Th1-mediated responses. Th2 cells are generated from precursor naive Th cells when they encounter the specific antigen in an IL-4-containing microenvironment. The question of how these Th2 cells are selected in atopic patients is also unclear. Both the nature of the T cell receptor signalling provided by the allergen peptide ligand and a disregulation of IL-4 production likely concur to determine the Th2 profile of allergen-specific Th cells, but the genetic unbalanced IL-4 production is certainly overwhelming. Some gene products selectively expressed in Th2 cells or selectively controlling the expression of IL-4 have recently been described. These findings allow to suggest that the upregulation of genes controlling IL-4 expression and/or abnormalities of regulatory mechanisms of Th2 development and/or function may be responsible for Th2 responses against allergens in atopic people. The increasing prevalence of allergy in developed countries suggests that environmental factors acting either before or after birth also contribute to regulate the development of Th2 cells and/or their function. The reduction of infectious diseases in early life due to increasing vaccinations, antimicrobial treatments as well as changed lifestyle are certainly important in influencing the individual outcome in the Th response to ubiquitous allergens. Moreover, the recent evidence that bacterial DNA or oligodeoxynucleotides containing unmethylated 'CpG motifs' promote the development of Th1 cells via the production of immunomodulatory cytokines (namely IL-12, IL-18 and IFNs) by professional antigen-presenting cells confirms previous epidemiological data. The new insight into the pathophysiology of T cell responses in atopic diseases provides exciting opportunities for the development of novel immunotherapeutic strategies.

Phosphorothioate oligodeoxynucleotides promote the in vitro development of human allergen-specific CD4+ T cells into Th1 effectors.

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.

Highly Th2-skewed cytokine profile of beta-lactam-specific T cells from nonatopic subjects with adverse drug reactions.

A positive lymphocyte transformation test to beta-lactams (beta-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to beta-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-gamma with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-gamma but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-gamma, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-gamma. These data suggest that cytokines produced by Th2 cells play an important role in all beta-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable.

Influence of both TCR repertoire and severity of the atopic status on the cytokine secretion profile of Parietaria officinalis-specific T cells.

Peripheral blood mononuclear cells (PBMC) from both nonatopic and Parietaria officinalis-sensitive donors proliferated in response to the allergen Par o 1 and developed into Par o 1-specific T cell lines and clones, which also showed reactivity for Par o 1-derived peptides. Virtually all Par o 1-specific T cell lines and large numbers of Par o 1-specific T cell clones proliferated in response to two Par o 1 nonapeptides (p92 and p96), which probably contain immunodominant epitopes of the Par o 1 allergen. Both p92- and p96-specific T cell clones showed the ability to produce IFN-gamma, but p92-specific T cell clones produced significantly lower amounts of IL-4 and IL-5 than p96-specific T cell clones, indicating that distinct epitopes, able to elicit functionally different T helper cell responses, may coexist in Par o 1. However, p92-specific T cell clones derived from atopic subjects with high IgE serum levels (high IgE producers) secreted significantly higher amounts of IL-4 and IL-5 than corresponding T cell clones generated from nonatopic subjects or patients with low IgE serum levels (low IgE producers), whereas p96-specific T cell clones secreted high IL-4 and IL-5 concentrations irrespective of whether they derived from high or low IgE producers. The addition of IL-4 and anti-IL-12 mAb to bulk culture significantly up-regulated the development of p92-specific T cells into IL-4-producing cells, whereas the addition of IL-12 and anti-IL-4 mAb shifted the differentiation of p96-specific T cells towards IFN-gamma-producing cells. Taken together, these results suggest that the cytokine profile of allergen-specific T cells is influenced by both the T cell receptor repertoire and the severity of atopic status and can be modulated, at least in vitro, by stimulation with the specific peptide in the presence, or after removal, of appropriate cytokines.

Pathogenesis and therapy of Behçet's disease.

Behçet's disease is a relapsing-remitting systemic vasculitis characterized by oral and genital ulcers, uveitis and thrombophlebitis which can involve many organs. Although its pathogenesis is not fully understood, a possible pathogenetic model can be proposed on the basis of recent studies. Genetic factors, in particular, have been investigated and the role of the genes encoding tumor necrosis factor, transporter in antigen processing proteins and MIC (MHC class I chain related) has been emphasized. In addition, a possible polarization of T lymphocytes towards the Th1 phenotype in Behçet's disease has been suggested by recent observations in experimental uveoretinitis and by preliminary data in humans. Neutrophils may also play a role in the pathogenesis of this disease, as they are attracted by macrophage-released cytokines at the site of the lesions, and thus contribute to tissue damage and self-maintenance of inflammation. New strategies for the treatment of Behçet's disease are being devised. In particular, immunosuppressive drugs used in association or in sequence may be administered to patients with particular clinical features or very severe disease.

Long term efficacy of high-dose intravenous methylprednisolone pulses in active lupus nephritis. A 21-month prospective study.

The efficacy of a single course of three high dose intravenous (i.v.) methylprednisolone (MP) pulses followed by low dose oral prednisone (PRED) was assessed in a group of patients with active lupus nephritis (LN). At 21 months after such therapeutic regimen in 10 out of 12 patients a complete clinical remission was found, in one patient a partial response with persistent moderate renal failure occurred, while one patient was refractory even to the additional administration of cyclophosphamide. The statistical analysis of repeated measures of a series of biological markers of LN, monitored over the course of the study, evidenced a significant improvement of serum creatinine (p < 0.05), C3 and C4 complement components (p < 0.05), 24-hour proteinuria (p < 0.02) and ESR values (p < 0.05). Moreover, a progressive and significant reduction of mean daily PRED dosage was reported (p < 0.05). We conclude that i.v. MP pulse therapy may exert a substantial long-term control of active LN and may induce steroid-sparing effects.