Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control

Background Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. Methods Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. Results After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. Conclusion We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

Systematic Review: Comparison of triple drug therapy versus double drug therapy for Lymphatic Filariasis

Background: Lymphatic filariasis is a parasitic infection caused by nematodes such as filaria Wuchereria bancrofti, Brugia malayi, and Brugia timori. These parasites can be transmitted through mosquito bites such as several species of mosquitoes, particularly Anopheles, Aedes, Culex, and Mansonia with geographical variations in the dominant vector identity. The main strategy used consists of community-wide mass drug administration (MDA) for the entire population at risk to stop disease transmission and prevent infectious morbidity. WHO recommends the use of annual medication in combination with the triple drug ivermectin therapy. Objective: To compare DEC and albendazole (IDA) versus the two drugs albendazole and diethycarbamazine or albendazole and ivermectin therapy. Methods: The literature search was carried out independently by the researcher using the Sciencedirect, Pubmed, and Cochrane online databases without limiting the type of study or the year of publication. The keywords used in this study were combined with the Boolean operator, namely "AND" namely ((((Lymphatic filariasis) AND (albendazole)) AND (diethylcarbamazine)) AND (ivermectin)) AND (compare). Results: Where triple drug therapy was significantly better in reducing and clearing microfilariae and worm nests in patients with lymphatic filariasis compared to two drug therapy alone. However, side effects occur more frequently in the combination of three therapies. The average side effects were low, such as headaches, joint pain, fatigue, and nausea. Conclusion: although it has relatively low side effects that occur in three drug combinations rather than two drug combination therapy, triple therapy combination therapy is more effective than two drug therapy in treating lymphatic filariasis disease.

Microbial abundance, composition, and function in nectar are shaped by flower visitor identity

ABSTRACT Microbial dispersal is essential for establishment in new habitats, but the role of vector identity is poorly understood in community assembly and function. Here, we compared microbial assembly and function in floral nectar visited by legitimate pollinators (hummingbirds) and nectar robbers (carpenter bees). We assessed effects of visitation on the abundance and composition of culturable bacteria and fungi and their taxonomy and function using shotgun metagenomics and nectar chemistry. We also compared metagenome-assembled genomes (MAGs) of Acinetobacter, a common and highly abundant nectar bacterium, among visitor treatments. Visitation increased microbial abundance, but robbing resulted in 10× higher microbial abundance than pollination. Microbial communities differed among visitor treatments: robbed flowers were characterized by predominant nectar specialists within Acetobacteraceae and Metschnikowiaceae, with a concurrent loss of rare taxa, and these resulting communities harbored genes relating to osmotic stress, saccharide metabolism and specialized transporters. Gene differences were mirrored in function: robbed nectar contained a higher percentage of monosaccharides. Draft genomes of Acinetobacter revealed distinct amino acid and saccharide utilization pathways in strains isolated from robbed versus pollinated flowers. Our results suggest an unrecognized cost of nectar robbing for pollination and distinct effects of visitor type on interactions between plants and pollinators. Overall, these results suggest vector identity is an underappreciated factor structuring microbial community assembly and function.

The life-cycle of the avian haemosporidian parasite Haemoproteus majoris, with emphasis on the exoerythrocytic and sporogonic development

Background Haemoproteus parasites (Haemosporida, Haemoproteidae) are cosmopolitan in birds and recent molecular studies indicate enormous genetic diversity of these pathogens, which cause diseases in non-adapted avian hosts. However, life-cycles remain unknown for the majority of Haemoproteus species. Information on their exoerythrocytic development is particularly fragmental and controversial. This study aimed to gain new knowledge on life-cycle of the widespread blood parasite Haemoproteus majoris. Methods Turdus pilaris and Parus major naturally infected with lineages hPHYBOR04 and hPARUS1 of H. majoris, respectively, were wild-caught and the parasites were identified using microscopic examination of gametocytes and PCR-based testing. Bayesian phylogeny was used to determine relationships between H. majoris lineages. Exoerythrocytic stages (megalomeronts) were reported using histological examination and laser microdissection was applied to isolate single megalomeronts for genetic analysis. Culicoides impunctatus biting midges were experimentally exposed in order to follow sporogonic development of the lineage hPHYBOR04. Results Gametocytes of the lineage hPHYBOR04 are indistinguishable from those of the widespread lineage hPARUS1 of H. majoris, indicating that both of these lineages belong to the H. majoris group. Phylogenetic analysis supported this conclusion. Sporogony of the lineage hPHYBOR04 was completed in C. impunctatus biting midges. Morphologically similar megalomeronts were reported in internal organs of both avian hosts. These were big roundish bodies (up to 360 μm in diameter) surrounded by a thick capsule-like wall and containing irregularly shaped cytomeres, in which numerous merozoites developed. DNA sequences obtained from single isolated megalomeronts confirmed the identification of H. majoris. Conclusions Phylogenetic analysis identified a group of closely related H. majoris lineages, two of which are characterized not only by morphologically identical blood stages, but also complete sporogonic development in C. impunctatus and development of morphologically similar megalomeronts. It is probable that other lineages belonging to the same group would bear the same characters and phylogenies based on partial cytb gene could be used to predict life-cycle features in avian haemoproteids including vector identity and patterns of exoerythrocytic merogony. This study reports morphologically unique megalomeronts in naturally infected birds and calls for research on exoerythrocytic development of haemoproteids to better understand pathologies caused in avian hosts.

First Report of Lethal Yellowing Group (16Sr IV) of Phytoplasmas in Vernonia cinerea in Jamaica

Coconut lethal yellowing disease (CLY) has had a devastating effect on the coconut (Cocos nucifera L.) industry in Jamaica and Latin America. A study was conducted in Jamaica during 2005 to identify alternate hosts of the CLY phytoplasma. Since weeds are known to act as reservoir hosts of numerous pathogens, Vernonia cinerea (L.) (Asteraceae), a prevalent weed species on coconut farms island-wide, was collected from coconut farms in areas of high and low levels of CLY incidence, although none of the plants displayed disease symptoms. DNA was extracted from plant samples by the method of Dellaporta et al. (1) and analyzed by nested PCR assay employing phytoplasma universal rRNA operon primers P1/P7 (2,4) and LY16Sf/LY16-23Sr (3). DNA derived from CLY-diseased or healthy coconut palm served as positive and negative controls, respectively, in each assay. Amplification of an rDNA product of the expected size (1.7 kb) confirmed phytoplasma infections in 53 of 118 (44.9%) V. cinerea test plants. Twenty-seven of the rDNA PCR products were analyzed by digestion with restriction enducleases RsaI, MspI, MseI, TaqI, HinfI, and HhaI. The restriction fragment length polymorphism profiles obtained were similar to that observed in the CLY-infected coconut palm. V. cinerea rDNA amplicons were cloned and sequenced (in both directions) and a representative sequence was deposited in GenBank (Accession No. EU057983). Blast analysis determined this sequence to be most similar (99%) to that of CLY phytoplasma in Jamaica (Accession No. AF49807) and Florida (Accession No. AF498309). To our knowledge, this is the first report of the 16Sr IV group of phytoplasmas infecting V. cinerea. Presence of the lethal yellowing phytoplasmas in dicotyledonous plant species has important epidemiological implications concerning vector identity and ecology. Futhermore, it is now evident that weed control on coconut farms could assist in the management of CLY disease in Jamaica. References: (1) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1993. (2) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (3) N. A. Harrison et al. Ann. Appl. Biol. 141:183, 2002. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

On: “New prospects in shallow depth electrical surveying for archeological and pedological applications” by A. Hesse, A. Jolivet and A. Tabbagh (GEOPHYSICS, 51, 585–594, March 1986).

The forward solution given by Hesse et al. is incorrect. The error is a result of the erroneous governing differential equation [their equation (2)], which for the only nonzero component of the magnetic field has the following form: [Formula: see text]Unfortunately, the authors did not show how they arrived at this equation, but the mistake is so frequently encountered that its origin can be reconstructed quite easily. Indeed, by neglecting displacement currents in the fourth Maxwell equation and by applying the vector operator ∇× to both parts of the equation, we obtain [Formula: see text]Making use of the well known vector identity [Formula: see text]and of the first and third Maxwell equations, we obtain [Formula: see text]

A vector identity for the Dirichlet tessellation

SummaryA vector identity associated with the Dirichlet tessellation is proved as a corollary of a more general result. The identity has applications in interpolation and smoothing problems in data analysis, and may be of interest in other areas.

A Two-Point Boundary Problem for Ordinary Self-Adjoint Differential Equations of Fourth Order

The purpose of this note is to establish Theorem A below for the two-point homogeneous vector boundary problemwhere the Pi(x) are given real m × m symmetric matrix functions of x with P0(x) positive definite and Pi(x) of class C2−i on an infinite interval [a, ∞), and where by a solution of (1.1) — (1.2) for a ≤ x1 < x2 < ∞ we understand a real m-dimensional column vector u = u(x) of class C2 on [a, ∞) which is such that Pi(x)u(2−i) is of class C2−i on [a, ∞) and which satisfies (1.1) — (1.2) with the former a vector identity on [a, ∞).