Cytokine-Induced Killer (CIK) Cells, In Vitro Expanded under Good Manufacturing Process (GMP) Conditions, Remain Stable over Time after Cryopreservation.

Cytokine-induced killer (CIK) cells are advanced therapy medicinal products, so their production and freezing process has to be validated before their clinical use, to verify their stability as a drug formulation according to the good manufacturing practice (GMP) guidelines. We designed a stability program for our GMP-manufactured CIK cells, evaluating the viability, identity and potency of cryopreserved CIK cells at varying time periods from freezing, and compared them with fresh CIK cells. We evaluated the effects of the cryopreservation method, transportation, and the length of time of different process phases (pre-freezing, freezing and post-thawing) on the stability of CIK cells. This included a worst case for each stage. The expanded CIK cells were viable for up to 30 min from the addition of the freezing solution, when transported on dry ice within 48 h once frozen, within 60 min from thawing and from 12 months of freezing while preserving their cytotoxic effects. The reference samples, cryopreserved simultaneously in tubes and following the same method, were considered representative of the batch and useful in the case of further analysis. Data obtained from this drug stability program can inform the accurate use of CIK cells in clinical settings.

A cross-sectional evaluation of HIV testing practices among women in the rural Dominican Republic.

BACKGROUND: The Dominican Republic (DR) ranks among nations with the highest burden of HIV in the Caribbean. Cultural and gender roles in rural areas of the DR may place women at increased HIV risk. However, little is known about sexual health and HIV testing behaviors among women in the rural DR. METHODS: We conducted a needs assessment among a systematic sample of adult women in a rural DR community in 2016. Demographic and behavioral attributes related to HIV testing, sexual health, and healthcare utilization were evaluated. Poisson regression analysis was used to identify demographics and behaviors associated with having had a previous HIV test. Significance was defined as a p-value < 0.05. RESULTS: Among 105 women evaluated, 77% knew someone with HIV and 73% of women reported that they would be very or extremely likely to take an HIV test if offered. Only 68% reported a previous HIV test, including 47% who were tested over 2 years prior. Barriers to HIV testing included low risk perception (23%), distance or requisite travel (13%), and discomfort being tested (11%). Women who had never been tested for HIV were more likely than those who had been tested to be older (p = 0.03), to have a lower level of education (p = 0.04), and to have never been tested for other sexually transmitted infections (STI; p <  0.01). In the Poisson multiple regression model, the only significant predictor of having had an HIV test was having had an STI test (p = 0.03). CONCLUSIONS: In the rural DR, numerous barriers contribute to low prevalence of HIV testing among women. Most women report willingness to have an HIV test and many engage in routine health care, indicating that this population may benefit from incorporating HIV testing and other sexual health promotion activities into routine medical care.

Correction to: Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

Following publication of the original article [1], the authors reported that all of the authors' names were processed incorrectly so that their given and family names were interchanged. In this Correction the correct author names are shown. The original publication of this article has been corrected.

Analysis of Mesenchymal Stromal Cell Engraftment After Allogeneic HSCT in Pediatric Patients: A Large Multicenter Study.

The mesenchymal stem cell (MSC) role after allogeneic hematopoietic stem cell transplantation (HSCT) is still a matter of debate; in particular, MSC engraftment in recipient bone marrow (BM) is unclear. A total of 46 patients were analyzed for MSC and hemopoietic stem cell engraftment after HSCT. The majority of patients had the BM as the stem cell source, and acute leukemia was the main indication for HSCT. Mesenchymal and hematopoietic stem cell chimerism analysis was carried out through specific polymorphic tandemly repeated regions. All patients reached complete donor engraftment; no evidence of donor-derived MSC engraftment was noted. Our data indicate that MSCs after HSCT remain of recipient origin despite the following: (i) myeloablative conditioning; (ii) the stem cell source; (iii) the interval from HSCT to BM analysis; (iv) the underlying disease before HSCT; and (v) the patients' or the donors' age at HSCT.

Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

BACKGROUND: Cytokine-induced killer (CIK) cells are a very promising cell population raising growing interest in the field of cellular antitumor therapy. The aim of our study was to validate the most advantageous expansion method for this advanced therapy medicinal product (ATMP) and to translate it from preclinical field to good manufacturing practices (GMP). GMP ensures that ATMP are consistently produced and controlled to the quality standards required to their intended use. For this reason, the use of the xenogenic sera tended to be minimized by GMP for their high variability and the associated risk of transmitting infectious agents. RESULTS: We decided to replace Fetal Bovine Serum (FBS), largely used as medium supplement for CIKs expansion, with other culture media. Firstly, Human Serum (HS) and Human Pool Plasma (HPP) were tested as medium supplements giving not compliant results to acceptance criteria, established for CIKs, probably for the great batch to batch variability. Consequently, we decided to test three different serum free expansion media: X-VIVO 15, (largely used by other groups) and Tex Macs and Cell Genix GMP SCGM: two GMP manufactured media. We performed a validation consisting in three run-sand even if the small number of experiments didn't permit us to obtained statistical results we demonstrated that both X-VIVO 15 and Tex Macs fulfilled the quality standards in terms of cellular growth, viability and identity while Cell Genix GMP SCGM resulted not compliant as it caused some technical problems such as high mortality. CONCLUSION: In conclusion, these preclinical validation data lay the bases for a GMP-compliant process to improve the CIKs expansion method.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). METHODS: Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. RESULTS: The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. CONCLUSIONS: We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.

Validation of analytical methods in compliance with good manufacturing practice: a practical approach.

BACKGROUND: The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range. METHODS: For the endotoxin test we used a kinetic chromogenic LAL test. As this is a limit test for the control of impurities, in compliance with International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, we evaluated the specificity and detection limit.For the immunophenotype test, an identity test, we evaluated specificity through the Fluorescence Minus One method and we repeated all experiments thrice to verify precision. The immunophenotype validation required a performance qualification of the flow cytometer using two types of standard beads which have to be used daily to check cytometer reproducibly set up. The results were compared together.Collected data were statistically analyzed calculating mean, standard deviation and coefficient of variation percentage (CV%). RESULTS: The LAL test is repeatable and specific. The spike recovery value of each sample was between 0.25 EU/ml and 1 EU/ml with a CV% < 10%. The correlation coefficient (≥ 0.980) and CV% (< 10%) of the standard curve tested in duplicate showed the test's linearity and a minimum detectable concentration value of 0.005 EU/ml.The immunophenotype method performed thrice on our cell therapy products is specific and repeatable as showed by CV% inter -experiment < 10%. CONCLUSIONS: Our data demonstrated that validated analytical procedures are suitable as quality controls for the batch release of cell therapy products.Our paper could offer an important contribution for the scientific community in the field of CTPs, above all to small Cell Factories such as ours, where it is not always possible to have CFR21 compliant software.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting.

BACKGROUND: The quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests' accuracy, precision, repeatability, linearity and range. METHODS: As the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator. RESULTS: All the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128. CONCLUSIONS: Our data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution).

Alternate-day fasting reverses the age-associated hypertrophy phenotype in rat heart by influencing the ERK and PI3K signaling pathways.

The age-related increased impedance in large arteries overloads the senescent heart, and the myocardial phenotype is hypertrophic. Together with qualitative changes observed in the senile heart, this can be responsible for impaired diastolic function. A restricted diet providing adequate nutrient intake, e.g. alternate-day fasting (ADF), has been shown to extend life-span and decrease incidence and progression of age-associated diseases in laboratory rodents, and to ameliorate some metabolic markers of aging in rhesus monkeys and humans. This study reports an age-related increase of some biological and morphological hypertrophy markers in the rat heart, together with increased plasma BNP, a well known marker of heart failure. The tissue modifications might likely be related to hyper-activation of two of the signaling pathways associated with myocardial pathological hypertrophy: ERK1/2 and PI3Kγ. Increased ERK1/2 activation might be in part related to the disturbance of STAT3, with a consequent decrease of SOCS3. In this context, the down-modulation of ERK1/2 and PI3Kγ signaling, together with the restoration of STAT3 activity and SOCS3 content, both observed with ADF, might help to reduce pathological hypertrophy stimuli and to rescue an important cardioprotective pathway, possibly opening new preventive and therapeutic perspectives in age-related heart failure.

Alternate-day fasting protects the rat heart against age-induced inflammation and fibrosis by inhibiting oxidative damage and NF-kB activation.

The free radical theory of aging is currently one of the most popular. In parallel, many studies have demonstrated the association of fibrosis and increased oxidative stress in the pathogenesis of some chronic human diseases, and fibrosis is often characteristic of aging tissues. One of the few interventions that effectively slow aging is calorie restriction and the protection against the age-associated increase of oxidative stress remains one of the foremost hypotheses to explain this action. As an alternative to traditional calorie restriction, another dietary regimen, termed alternate-day fasting, has also been tested, whose antiaging mechanisms have not been explored so much extensively. We thus studied the effects of alternate-day fasting, started at 2 months of age, on oxidative stress and fibrosis in the heart during aging. In the left ventricle of the heart of elderly (aged 24 months) versus young (aged 6 months) male rats we found a significant increase in oxidative stress paralleled by increased fibrosis. In parallel there was a significant increase in inflammatory cytokine levels and in NF-kB DNA binding activity with advancing age. Alternate-day fasting protected against all these age-related phenomena. These data support the hypothesis that this kind of dietary restriction protects against age-related fibrosis, at least in part by reducing inflammation and oxidative damage, and this protection can thus be considered a factor in the prevention of age-related diseases with sclerotic evolution.

Calorie restriction protects against age-related rat aorta sclerosis.

Many theories have been advanced to account for the ageing process, among which the free radical theory deserves much attention. Numerous studies have also shown an association between tissue fibrosis and oxidative stress. Of note, fibrosis may be considered a significant index of tissue ageing. Calorie restriction (CR) has been demonstrated to maintain many physiological processes in a youthful state until a very advanced age. However the anti-ageing mechanisms of CR are still not fully understood. We thus evaluated the effect of CR on oxidative damage and its relationship with fibrosis during ageing. We found a significant increase of both oxidative stress and fibrosis parameters in the aortae from aged vs. young rats. CR reversed both phenomena. CR also protected against the age-associated increase of Jun-N-terminal kinase and p-38 activities, involved in TGFbeta1 expression and signaling. On the contrary, extracellular regulated kinases did not show any age-related change. CR similarly reversed the age-related increase of AP-1 DNA binding activity and the AP-1-dependent increase of vimentin gene expression. In parallel, CR reversed the age-related morphological alterations of the aorta wall cell composition. These data further support the relationship between oxidative stress and fibrosis in different diseases and during ageing. The protection exerted by CR against fibrosclerosis might be due to a decrease of oxidative stress, with consequent decreased MAPK activity and down-regulation of AP-1 activation and of TGFbeta1 expression and signaling.

Role of 4-hydroxy-2,3-nonenal in the pathogenesis of fibrosis.

Transient activation of fibroblasts or fibroblast-like cells to proliferate and to produce elevated quantities of extracellular matrix is essential to fibrosis. This activation is regulated by several cytokines produced by various inflammation-associated cells. Among these, transforming growth factor beta1 (TGFbeta1) is considered of major importance. Many studies have shown that lipid peroxidation play a key role in the initiation and progression of fibrosis in different organs. In fact, 4-hydroxy-2,3-nonenal (HNE), the major aldehydic product of lipid peroxidation, is able to induce TGFbeta1 expression and synthesis, and activation of activator protein-1 (AP-1) transcription factor. In this study, using the murine macrophage line J774-A1, we show that these effects are strictly related to the chemical structure of HNE, since neither 2-nonenal nor nonanal are biologically active to the same extent. Moreover, we demonstrate that HNE can indeed contribute to the onset of fibrosis by stimulating AP-1 binding to DNA and consequently inducing TGFbeta1 expression, since thiol-group reagents, such as N-ethylmaleimide and 4-(chloro-mercuri)-benzenesulfonic acid, that down-modulate HNE entrance and localisation inside the cell, prevent both phenomena. The possibility to control fibrogenic cytokine levels by means of antioxidant or dietetic treatments opens new potential pharmacological and nutritional horizons in the treatment of many chronic diseases characterised by excessive fibrosis.

Resveratrol inhibits cell cycle progression in U937 cells.

Resveratrol, a naturally occurring stylbene present in grapes, is proposed to be responsible for the positive effects of red wine consumption on cardiovascular diseases (French paradox). In recent years this molecule has also been proposed as a cancer chemopreventive agent. The aim of the present study was to investigate the mechanisms by which resveratrol inhibits tumor growth. For this purpose, U937 cells were exposed to resveratrol at concentrations usually present in red wine, and the effects on proliferation, death and cell cycle machinery were assessed. The U937 cell growth was impaired due to reduced cell proliferation, without significant induction of apoptosis. This anti-proliferative effect is associated with modulations in the pattern of DNA distribution, with a reduction of G0/G1, particularly G2-M peaks and accumulation in the S phase of the cell cycle. This result was confirmed by the observation that the rate of [3H]-thymidine incorporation into DNA was significantly reduced in resveratrol-treated U937 cells. Consistent with this observation, in the same experimental conditions, the activity of ribonucleotide reductase, an enzyme critically involved in the process of DNA duplication, decreased. The altered progression in the cell cycle could depend on modulations in the activity of cyclin dependent kinases and their inhibitors. After exposure of U937 cultures to resveratrol, the expression of cyclins A and E, as well as that of CDK2 increased, while that of p21CIP was significantly reduced. The data shown in this report suggests that the prevention of cancer development exerted by resveratrol may result from modulations of molecules involved in the regulation of cell cycle progression, which block the cells at the S phase checkpoint.