Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.
Summary PCR fingerprinting technique was applied to subtype 44 Pasteurella multocida subspecies multocida (P.m.sp.m.) isolates from the respiratory system of pigs. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 (core sequence of the M13 phage) grouped the 44 P.m.sp.m. strains into five distinct fingerprinting profiles, while primer 2 ((GACA)4) grouped them into seven profiles. The results suggest that PCR fingerprinting is an efficient technique to detect DNA polymorphism in the species P.m.sp.m. This technique could be used to differentiate P.m.sp.m. strains of the same capsular serotype.
Summary Electron‐microscopic Examination of Spirochaetes in Dermatitis digitales lesions in Cows In typical Dermatitis digitalis (D.d. lesions, spirochaete‐like bacteria with variations in spiralization were revealed by electron microscopy. While, in the early stages of the disease, these are found to be associated with fibrillar material of keratocytes, they occur massively in vacuoles at more advanced stages. The spirochaetes carry one pair of endoflagella, originating with a hook from the poles of the bacteria. These flagellas are composed of coiled flagelling fibrils in the pole region, merging towards the centre of the bacterium. A coat of fibrils was found in association with the cytoplasmatic membrane. The winding of this coat follows and may influence the coiling of the protoplast, and is probably involved in the rapid motility of this spirochaetes, together with the flagella. Immuno‐electronmicroscopy revealed an antigenic relationship with Borrelia burgdorferi, at least with regard to the regions of flagella and undulating membrane. The paper discusses: 1. The possible classification of these spirochaetes with the genus Treponema; 2. The layer of peptidoglycan occuring on the outer membrane; and 3. The keratolytic activity of spirochaetes in D.d. Zusammenfassung In typischen Läsionen einer Dermatitis digitalis des Rindes werden spirochätenartige Keime elektronenmikroskopisch aufgezeigt, die im Frühstadium an faserigem Material von Keratozyten, in fortgeschrittenen Stadien in Vakuolen im Interzellularspalt massenhaft zu finden sind. Diese Spirochäten weisen je Pol ein Paar Endoflagellen auf, die polar hakenförmig entspringen, sich in der Keimmitte treffen und von verdrillten Flagellinfibrillen gebildet werden. Darunter findet sich assoziiert mit der Cytoplasmamembran ein Fibrillenmantel, der den variablen Keimwindungen folgt, vom Keimpol ausgeht und mit den Geißeln die starke Keimbewegung bewirken dürfte. Eine antigene Verwandtschaft zu Borrelia burgdorferi, zumindest im Bereich von Geißeln und ondulierender Membran zeigt sich bei der Immunelektronenmikroskopie. Diskutiert werden die Zuordnung der Spirochäten zum Genus Treponema, eine peptidoglykanhaltige Schicht auf der Außenmembran und eine keratolytische Aktivität der Spirochäten bei der D.d.
Running title: NK cell motility and cytotoxicity in a 3-D environmentSynopsis: Cryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors. AbstractNatural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.
Vortrag anlal3lich des 4. Leipziger Biotechnologie-Symposiums 12.-16. Dezember 1988 SummaryThree wild type strains of B. bronchiseptica were mutagenized with the transposon T n 5. Since the transponson Tn 5 encodes resistance to kanamycin, a selection for resistance to these antibiotics can be used as an indicator for Tn 5 transposition.9 mutants were identified that are not able t o grow on minimal medium. Subsequent auxotrophic tests on these mutants revealed 7 vsline, 1 cysteine and 1 valine and Ieucine auxotrophic mutants.It was not possible for us to find B. bronchisepticu tmnsconjugants in the virulent phase I. All in vivo and in vitro tested parameters showed that the isolated mutants were avirulent in phase 111.Die Rhinitis atrophicans suum ist als chronisch progressive Infektionskrankheit wachsender Schweine weltweit verbreitet. Sie ist das Produkt einer schweren persistierenden Entziindungsreaktion in der Nasenhohle, die zu Umbaustorungen und Deformationen der unter der Nasenschleimhaut liegenden Knochen fiihrt. Die Umbaustorungen werden primar durch Antigenkomponenten von Bordetella bronchiseptica ausgelost. Im Gefolge dieses Geschehenv werden iiber die entziindlich veranderte Nasenschleimhaut Toxine aufgenommen, die uber ihre stoffwechseldepressive Wirkung zu verringerten Lebenstagszunahmen, Abwehrschwache und Atrophie der Conchae nasales fiihren. Um die Adharenz und die Kolonisation von Wildstammen zu reduzieren bzw. unmoglich zu machen, mu13 eine lokale Immunitat durch das sekretorische IgA in der Nasenschleimhaut aufgebaut werden. Nach intranasaler Applikation von inaktivierten Ganzzellvakzinen konnte keine ausreichende lokale Immunitat erzielt werden. Der Einsatz von schwach virulenten bzw. avirulenten, jedoch noch immunogenen Impfstammen, fuhrt nach intranasaler Inokulation zu einer lokalen Immunitat, die mehrere Wochen anhalten sollte. Bei der Konstruktion von Lebendimpfstoffmutanten miissen die Pathogenesemechanismen des Erregers strikte Beachtung finden. Erfahrungen mit bereits eingesetzten Lebendimpfstoffen zeigen, daB die Auslosung einer Immunitat gegen die Adhasionsprinzipien des betreffenden Erregers bereits im Sinne einer Infektionsverhiitung wirkSam sein kann [l, 21. Wir gehen davon aus, daB es fur die Virulenzabschwachung eines Erregers schon ausreichend ist, einen Virulenzfaktor zu eleminieren. Das lieBe sich uber eine Mutante realisieren, bei welcher die entsprechende Genfunktion fehlt bzw. verandert ist. 6 Acta Biotechnol. 10 (IQW) 2
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