Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BACKGROUND: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed. METHODS: Microorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry. RESULTS: The BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h). CONCLUSIONS: When a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Two year follow-up of immunological response in mite-allergic children treated with sublingual immunotherapy. Comparison with subcutaneous administration.

Although the efficacy of allergen-specific sublingual immunotherapy (SLIT) is now accepted, the underlying mechanisms remain elusive. Such mechanisms are better documented in the case of subcutaneous immunotherapy (SCIT). In order to understand the T-lymphocyte response in patients receiving SLIT, we compared children with respiratory disease monosensitized to Dermatophagoides pteronyssinus receiving SLIT or SCIT over a 2-yr period. Peripheral blood was obtained before beginning immunotherapy, and after 3 months, 1 yr and 2 yr. Total IgE, specific IgE and IgG4 to D. pteronyssinus were determined in serum. T-cell markers (CD3, CD4, CD8, CD25) and intracellular cytokine production (TNF-alpha, IL-2, IL-4 and IFN-gamma) were determined in peripheral blood mononuclear cells (PBMC) by flow cytometry. No differences between SCIT and SLIT were detected in the clinical variables or in the subjective evaluation. Although an increase in specific IgE and IgG4 was only detected in SCIT, a significant decrease in the specific IgE/IgG4 ratio was found in both groups. SCIT and SLIT experienced an increase in the CD4/CD8 ratio over time, but an increase in the CD4(+)CD25(+) and a decrease in the CD8(+)CD25(+) subsets were only found with SCIT. A slight shift from a Th2 to a Th1 pattern, measured by the IFN-gamma/IL-4 ratio, was only detected in the CD4 T cells with SCIT. A decrease in both groups was found in TNF-alpha and IL-2 production over time. Children with respiratory allergic diseases receiving SCIT or SLIT had a different immunologic response in peripheral blood during treatment, though the clinical improvement was similar. Whether SLIT induces a mucosal protective response should be studied.

Local IgE production and positive nasal provocation test in patients with persistent nonallergic rhinitis.

BACKGROUND: Allergic rhinitis is an IgE-mediated inflammatory disease of the nasal mucosa, which is usually diagnosed by typical symptoms, positive skin tests, and/or serum specific IgE antibodies to allergens. Despite suggestive symptoms of allergic rhinitis, some patients have a negative diagnostic test for atopy. OBJECTIVE: To evaluate in the nose the inflammatory response, specific IgE to Dermatophagoides pteronyssinus (DP), and the response to a nasal allergen provocation test with DP (NAPT-DP), in patients with persistent nonallergic rhinitis (PNAR) compared with patients with persistent allergic rhinitis (PAR) and healthy controls. METHODS: Fifty patients with PNAR, 30 with PAR to DP, and 30 healthy controls were studied by determining the nasal leukocyte-lymphocyte phenotype by flow cytometry (CD16, CD8, CD4, CD33, CD3, and CD45), nasal eosinophil cationic protein (ECP), albumin, total and specific IgE to DP, and NAPT-DP. RESULTS: The PNAR patients showed a similar leukocyte-lymphocyte phenotype in nasal lavage to the PAR patients and was different to the healthy controls. Within the PNAR group, 54% showed a positive NAPT-DP, with 22% of these having nasal specific IgE to DP. CONCLUSION: These data support the hypothesis that in persistent nonallergic rhinitis some patients may have local inflammation, nasal IgE production, and a positive response to a nasal allergen provocation test despite no evidence of systemic atopy. Further research is needed to evaluate the influence of other perennial allergens and/or immunologic mechanisms. CLINICAL IMPLICATIONS: The local production of IgE antibodies without systemic detection is a condition that should be considered in patients with PNAR.

Potential involvement of dendritic cells in delayed-type hypersensitivity reactions to beta-lactams.

BACKGROUND: Although the involvement of T cells in delayed reactions to drugs has been studied, little is known about the interaction between the drug and the antigen-presenting cells. Dendritic cells (DCs) are professional antigen-presenting cells essential for initiating T-cell responses. Their ability is regulated in a process known as maturation, by which they modulate the effector immune response. OBJECTIVES: We studied the role of DCs in subjects who had a delayed-type hypersensitivity reaction to amoxicillin to assess the effect on the pattern of maturation and determine the capacity of DCs to activate T lymphocytes. METHODS: We examined the consequences of the interaction between monocyte-derived DCs, lymphocytes, and amoxicillin by means of phenotypic and functional studies, including endocytosis, proliferation, and cytokine production. RESULTS: Amoxicillin drove DCs from hypersensitive subjects to a phenotypic and functional semimature status, inducing a T-cell proliferation response. CONCLUSIONS: In delayed reactions to amoxicillin, DCs play a relevant role in inducing the T-cell responses. These results are useful not only to understand the mechanism but potentially as a possible approach to diagnosis. CLINICAL IMPLICATIONS: A better understanding of T-cell and DC involvement in delayed-type hypersensitivity reactions is needed. This in vitro assay might provide clues to the diagnostic evaluation of patients allergic to penicillins.

Cytokine production, activation marker, and skin homing receptor in children with atopic dermatitis and bronchial asthma.

T cells are known to develop a critical role in the pathogenesis of atopic dermatitis (AD) and bronchial asthma. T cells involved in AD express the skin homing receptor CLA, but no lung homing receptor has been identified in bronchial asthma. We compared different cell markers and the cytokine production in T cells from children with AD or bronchial asthma. We studied the involvement of CLA+ and CLA- T-cell subpopulations in these diseases. We studied 20 children with acute AD lesions, 15 with mild persistent asthma, and 15 non-atopic controls. All patients were sensitized to house dust mite (DP) and evaluated during the acute phase. Total and specific IgE were measured by immunoassay and the expression of different cell markers and the cytokine production was analyzed by flow cytometry in peripheral blood mononuclear cells. Total IgE was significantly higher in AD children and IgE to DP in the asthmatic children. There was a significant increase in CD25+ CD4+ cells in asthmatic children and in HLA-DR+ CD4+ and HLA-DR+ CD8+ cells in AD. In the CD4+ subsets, there was an increase in IL-13, IL-5 and TNF-alpha in AD compared to controls, a decrease in IFN-gamma in asthmatic children compared to controls, and an increase in IL-13, IL5, IL2, TNF-alpha, and IFN-gamma in the AD compared to asthmatic children. Changes in cytokine production were mainly detected in CLA+ cells in AD and in CLA- cells in asthma. Differences exist in total and specific IgE, activation markers, and cytokine patterns between AD children and children with asthma, with the former expressing a Th2 pattern whereas in asthmatic children we only detected a decrease in IFN-gamma. Moreover, the subpopulations (CLA+ vs. CLA-) expressing these changes were different, indicating that the underlying mechanisms in the two diseases are not exactly the same.

Immediate allergic reactions to cephalosporins: evaluation of cross-reactivity with a panel of penicillins and cephalosporins.

BACKGROUND: Allergy to cephalosporins has mainly been evaluated in the context of patients with confirmed penicillin allergy. The problem of studying cross-reactivity in subjects primarily sensitized to cephalosporins and potentially allergic to penicillins has not been sufficiently addressed. OBJECTIVE: To evaluate the in vitro IgE response and cross-reactivity to betalactams in patients with immediate allergic reactions to cephalosporins. METHODS: The study included 24 patients with immediate allergic reactions to cephalosporins and RAST-positive to at least 1 cephalosporin. Skin testing and RAST were performed with a panel of penicillins and cephalosporins. RAST inhibition assay with different monomeric conjugates of penicillin and cephalosporin was performed to establish cross-reactivity. RESULTS: The culprit cephalosporins were cefaclor (N = 7), cefonicid (N = 1), cefotaxime (N = 2), ceftazidime (N = 2), ceftriaxone (N = 3), and cefuroxime (N = 9). Two patients had a positive skin test result to penicillin determinants, and 22 patients had a negative result to penicillin determinants and tolerated benzylpenicillin administration. Of these 22, 19 had a positive skin test result to cephalosporins and divided into patients reacting only to the culprit cephalosporin (63.2%) and those reacting to more than 1 cephalosporin (36.8%). RAST and RAST inhibition studies confirmed that the side chain at the R1 position is crucial for recognition. CONCLUSION: The R1 side chain rather than the betalactam structure, shared by penicillins and cephalosporins, seems to play a dominant role in determining the specificity of immunologic reactions to cephalosporins. Thus, penicillin can be administered safely to patients allergic to cephalosporins and with a negative skin test result to penicillin determinants.

Allergic reactions to beta-lactams.

Allergy to beta-lactam antibiotics is the most frequent cause of drug-induced immunological reactions, although the prevalence is not exactly known. IgE- and T-cell-dependent responses are the main mechanisms involved, although other immunological mechanisms can also participate, especially in haematological abnormalities, such as immune haemolytic anaemia or thrombocytopoenia. Aside from their frequency, the clinical entities reported nowadays have changed little since penicillin was first used. The variation in beta-lactams consumption through the year has modified the pattern and specificities of allergic reactions for IgE and T cell responses. Benzylpenicillin is no longer the beta-lactams most frequently prescribed and other chemical structures, with new or modified haptens, have progressively replaced it. This is relevant for the diagnostic evaluation and management of beta-lactam hypersensitivity.