Pain control and functional improvement in patients treated by autologous conditioned serum after failure of platelet rich plasma treatments in knee osteoarthritis.

OBJECTIVE: To assess the efficacy of autologous conditioned serum (ACS) for the treatment of patients with knee osteoarthritis after failure of medical treatments and platelet rich plasma (PRP) injections. BACKGROUND: Knee osteoarthritis is the most common form of arthritis. Prior to prosthetic surgery these patients might benefit from medical treatments, physiotherapy, and in case of their ineffectiveness, from autologous blood component injections. METHODOLOGY: We have treated 30 patients with Kellgren-Lawrence I-III knee osteoarthritis with ACS after failure of standard medical treatments/physiotherapy and platelet rich plasma (PRP) injections for a full cycle, within the previous year from enrollment. RESULTS: ACS administration was performed in all patients with mild side effects and produced prompt (1 month) improvements of VAS and Lequesne scales in 67% of patients and this result persisted at 6 and 12 months. No relationship between the rate of response and Kellgren-Lawrence scale at enrollment was observed whilst responders had a significantly higher amount of interleukin-1 receptor antagonist (IL1-RA) in ACS as compared to nonresponders. CONCLUSION: The present study confirms the efficacy of ACS in pain control and functional recovery of patients with knee osteoarthritis resistant to medical and PRP treatment. These results were obtained in a well-defined cohort of resistant patients and seem to be related with IL1-RA content in injected ACS.

Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway.

CIK cells are a subset of effector lymphocytes endowed with a non-MHC restricted anti-tumor activity making them an appealing and promising cell population for adoptive immunotherapy. CIK are usually generated ex-vivo by initial priming with Interferon-γ (IFN-γ) and monoclonal antibody against CD3 (anti-CD3), followed by culture in medium containing Interleukin-2 (IL-2). Interleukin-15 (IL-15) shares with IL-2 similar biological functions and recently it has been reported to induce CIK with increased anti-leukemic potential. The aim of the study was to compare the killing efficacy of CIK generated by IL-2 alone or IL-2 and IL-15 toward tumor targets of different origins, leukemic cells and malignant cells from epithelial solid tumors. CIK bulk cultures were examined for cell proliferation, surface phenotype and cytotoxic potential against tumor cell lines K562, HL60, HeLa and MCF-7. The results showed that IL-15 is able to induce a selective expansion of CIK cells, but it is less effective in sustaining CIK cell proliferation compared to IL-2. Conversely, our data confirm and reinforce the feature of IL-15 to induce CIK cells with a potent cytotoxic activity mostly against tumor cells from epithelial solid malignancies via NKG2D-mediated mechanism.

A new standardized clinical-grade protocol for banking human umbilical cord tissue cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses. STUDY DESIGN AND METHODS: We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. RESULTS: We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques. CONCLUSION: In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

BACKGROUND: Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. METHODS: PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. RESULTS: PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL. CONCLUSION: The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol.

BACKGROUND AIMS: We have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safe. METHODS: Five patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2-3 weeks in culture, a median of 4.65 × 10(6) immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 days. RESULTS: ITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3(+) CD16(+) CD56(+) CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10(6) CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1-13) from the first infusion of CIK cells. CONCLUSIONS: This study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.

Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with ß-thalassaemia.

BACKGROUND: The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content. MATERIALS AND METHODS: Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 µm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan). RESULTS: Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50-70%), percentage haemolysis (<0.8/unit), white cell count (<1×10(6)) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%. CONCLUSIONS: The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some "low dose" normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units.

Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures.

BACKGROUND: Cytokine-induced killer (CIK) cells are typically differentiated in vitro with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol. METHODS: Peripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of bona fide regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the in vitro cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells. RESULTS: CIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells. CONCLUSIONS: TG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.

Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells.

Defects in chromosome segregation play a critical role in producing genomic instability and aneuploidy, which are associated with congenital diseases and carcinogenesis. We recently provided evidence from immunofluorescence and electron microscopy studies that merotelic kinetochore orientation is a major mechanism for lagging chromosomes during mitosis in PtK1 cells. Here we investigate whether human primary fibroblasts exhibit similar errors in chromosome segregation and if at least part of lagging chromosomes may arise in cells entering anaphase in the presence of mono-oriented chromosomes. By using in situ hybridization with alphoid probes to chromosome 7 and 11 we showed that loss of a single sister is much more frequent than loss of both sisters from the same chromosome in anatelophases from human primary fibroblasts released from a nocodazole-induced mitotic arrest, as predicted from merotelic orientation of single kinetochores. Furthermore, the lagging of pairs of separated sisters was higher than expected from random chance indicating that merotelic orientation of one sister may promote merotelic orientation of the other. Kinetochores of lagging chromosomes in anaphase human cells were found to be devoid of the mitotic checkpoint phosphoepitopes recognized by the 3F3/2 antibody, suggesting that they attached kinetochore microtubules prior to anaphase onset. Live cell imaging of H2B histone-GFP-transfected cells showed that cells with mono-oriented chromosomes never enter anaphase and that lagging chromosomes appear during anaphase after chromosome alignment occurs during metaphase. Thus, our results demonstrate that the mitotic checkpoint efficiently prevents the possible aneuploid burden due to mono-oriented chromosomes and that merotelic kinetochore orientation is a major limitation for accurate chromosome segregation and a potentially important mechanism of aneuploidy in human cells.