Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance

Background

We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs).

Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.     

Fetomaternal hemorrhages and their importance in perinatal pathology]

Transplacental haemorrhage is usually studied as an aspect of Rh immunisation prevention. In this paper the authors emphasize importance of this syndrome in noe-natology, as massive transplacental blood loss may result in severe foetal and neo-natal anemia or even lead to intra uterine death. Different technics for evidencing the presence of fetal cells in the mother's circulation are first discussed, the acid elution method appearing to be the easiest and fastest one. Results of nearly 40.000 Kleihauer's tests screening routinely performed in Paris at the time of delivery, are reported. The much higher frequency of very large transplacental haemorrhage is pointed out in cases of stillbirth. On a practical point of view, routine testing for transplacental haemorrhage finds its major interest in Rh prevention. A formula is proposed by one of the authors to calculate the most accurate dose of passive anti-D antibody in relation with quantitation of fetal haemorrhage. At last the autors attempt a new approach to the problem of neonatal unexplained anemias. Two different types of fetal bleeding are postulated, either chronic associated with haematologic signs of regeneration, or massive at the time of delivery without haematologic symptomatology. These condtions could lead to two different clinical pictures, either hydropsfetalis when chronic, or hypovolemic schock when massive and immediate.     

Associations between antimicrobial resistance genes in fecal generic Escherichia coli isolates from cow-calf herds in western Canada

The objective of this study was to examine associations among the genetic determinants of antimicrobial resistance (AMR) in 207 fecal generic Escherichia coli isolates obtained from 77 cow-calf herds in western Canada. Twenty-three resistance genes corresponding to six different antimicrobial families were assessed using DNA hybridization and PCR. The most common resistance genes in the study sample (207 isolates) were sul2 (48.3%), tet(B) (45.4%), and ant(3')-Ia (aadA1) (19.3%). Several statistically significant associations between the examined resistance genes were detected. The strongest associations observed were those between genes for resistance to chloramphenicol (catI) and trimethoprim (dhfrI) (odds ratio [OR] = 214; P = 0.0001), sulfonamide (sul1) and chloramphenicol (catI) (OR = 96.9; P = 0.0001), streptomycin [ant(3')-Ia (aadA1)] and trimethoprim (dhfrI) (OR = 96.2; P = 0.0001), sulfonamide (sul1) and streptomycin [ant(3')-Ia (aadA1)] (OR = 79.3; P = 0.0001), and tetracycline [tet(B)] and sulfonamides (sul2) (OR = 25.7; P = 0.0001). At least one of the resistance genes corresponding to each nonaminoglycoside family of antimicrobials examined in this study was associated with the two aminoglycoside resistance genes ant(3')-Ia (aadA1) and aph(3')-Ia. The multiple, strong associations between genes and the diverse nature of the associations described in this study demonstrate the complexity of resistance gene selection in cow-calf herds and should be considered in the planning of AMR control practices for cow-calf operations.     

Alveolar metabolism of natural vs. synthetic surfactants in preterm newborn rabbits

We compared the recoveries of foursurfactant preparations: two natural [term fetal rabbit surfactant(FRS) and adult rabbit surfactant (ARS)] and two commerciallyavailable preparations [apoprotein-based Survanta (S) and syntheticExosurf (E)] from 27-day gestation rabbit pups treated at birth andventilated up to 120 min. At 5, 60, and 120 min, we measured therecovery of the heavy-aggregate, metabolically active form (H) and thelight-aggregate, nonsurface active metabolic breakdown form (L) ofalveolar surfactant and determined the phospholipid content andcomposition of the intracellularly stored lamellar body (LB) pool. Pupstreated with FRS had <15% loss of H by 2 h. ARS-treated pups hada >50% loss of H by 1 h, and E- and S-treated pups had ~50%loss by 5 min, with a slower rate of continuing loss of up to 80% by2 h. The major losses of H phospholipid were not explained by theL-form recovery. LB phospholipid significantly increased only in the E-treated pups and only at 2 h. FRS provides a biologically active form (H) of surfactant that appeared to remain in the airway for asignificantly longer time than the other surfactant preparations. Theunique properties of FRS merit further study.

     

Functional antagonism of IL-1α induced gene expression profiles define the cAMP/PKA pathway as a unique regulator of IL-1α signaling networks

Interleukin-1 (IL-1α) induced inflammatory and pro-fibrotic responses in human lung fibroblasts are mediated by activation of MAPK and NFκB pathways. The purpose of the present study was to broadly profile the activity of a variety of compounds which function as inhibitors of these key signaling pathways that may affect IL-1α mediated gene changes. A reference set of genes was derived from microarray analysis of IL-1α stimulated cells. The genes were chosen to provide a range of expression profiles which serve to represent the actions of the underlying signaling network. We show that G_s-coupled receptor agonists have a unique pattern of activity as represented by their impact on IL-1α dependent gene changes. These effects were not mimicked by direct inhibitors of p38, JNK, MEK or IKK but were mimicked by forskolin and cAMP analogs. These findings indicate that cAMP/PKA serves as a point of convergence for regulation of IL-1α responses by multiple G_s-coupled receptors and regulates IL-1α responses by a distinct mechanism that does not solely involve direct inhibition of p38, JNK, MEK or IKK. The data also point to a potentially useful paradigm wherein monitoring of a small subset of genes is sufficient to identify pathway activity of novel compounds.     

Immunity of dogs against Babesia canis, its vector tick Dermacentor reticulatus, and Ixodes ricinus in endemic area

Previous epidemiological studies allowed us to accurately define endemic areas of canine babesiosis and tick distribution in southeastern France (Martinod, 1983). Using a micro-ELISA test 100 dogs sera were tested with 3 antigens: Babesia canis, Dermacentor reticulatus and Ixodes ricinus. Antibodies against B. canis and its vector D. reticulatus were detected in an endemic area, sometimes with high levels (optical density 1.38 and 0.80 respectively). A correlation factor and regression lines were found between ELISA activity of B. canis and vector tick antigens, even for dogs which never showed any babesiosis symptoms. These results were compared with those of an area without any babesiosis. Furthermore I. ricinus antigens detected ELISA activity in sera of dogs; some cross reactions were observed between I. ricinus and D. reticulatus antigen.     

Characterization of the c-Myb-responsive region and regulation of the human type I collagen alpha 2 chain gene by c-Myb

We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen alpha2 chain gene expression in fibroblastic cells. We have identified four Myb-binding sites (MBSs) in the promoter. Transactivation assays on wild type and mutant promoter-reporter constructs demonstrated that c-Myb, but not B-Myb, can transactivate the human type I collagen alpha 2 chain gene promoter via the MBS-containing region. Electrophoretic mobility shift assay experiments showed that c-Myb specifically binds to each of the four MBS; however, the mutagenesis of site MBS-4 completely inhibited transactivation by c-Myb, at least in the full-length promoter. In agreement with these results, c-myb(-/-) mouse embryo fibroblasts (MEFs) showed a selective lack of expression of type I collagen alpha 2 chain gene but maintained the expression of fibronectin and type III collagen. Furthermore, transforming growth factor-beta induced type I collagen alpha 2 chain gene expression in c-myb(-/-) MEFs, implying that the transforming growth factor-beta signaling pathway is maintained and that the absence of COL1A2 gene expression in c-myb(-/-) MEFs is a direct consequence of the lack of c-Myb. The demonstration of the importance of c-Myb in the regulation of the type I collagen alpha 2 chain gene suggests that uncontrolled expression of c-Myb could be an underlying mechanism in the pathogenesis of several fibrotic disorders.     

Infection of red foxes with Echinococcus multilocularis in western Switzerland

In the Jura mountains, Plateau and Alps of western Switzerland important variations in the prevalence of Echinococcus multilocularis infection in red foxes were observed between geographical areas from 1990 to 1995. The Jura mountains and the Plateau had higher mean prevalence levels than the Alps with 30.6, 32.4 and 18.8%, respectively. The highest rate was recorded in the Plateau in the canton of Fribourg with a prevalence of 52.3%. The prevalence of E. multilocularis infection in foxes in the alpine canton of Valais was the lowest (7.1%). Juvenile foxes were found to be more susceptible to E. multilocularis than adults. Adult foxes were less heavily infected in summer and autumn, while the prevalence in juveniles (less than 1 year old) increased between the spring and winter, when they are more than 6 months old. The retrospective data relate to the beginning of the 1990s, since when a drastic prevalence increase of E. multilocularis infection in foxes has occurred in several regions of Europe. Nevertheless, the study is a major contribution to the epidemiological situation of E. multilocularis in central Europe, in that it contains valuable information on spatial distribution and seasonal differences in different age groups of foxes.