Morphology and dynamics of tumor cell colonies propagating in epidermal growth factor supplemented media.

The epidermal growth factor (EGF) plays a key role in physiological and pathological processes. This work reports on the influence of EGF concentration (c EGF) on the modulation of individual cell phenotype and cell colony kinetics with the aim of perturbing the colony front roughness fluctuations. For this purpose, HeLa cell colonies that remain confluent along the whole expansion process with initial quasi-radial geometry and different initial cell populations, as well as colonies with initial quasi-linear geometry and large cell population, are employed. Cell size and morphology as well as its adhesive characteristics depend on c EGF. Quasi-radial colonies (QRC) expansion kinetics in EGF-containing medium exhibits a complex behavior. Namely, at the first stages of growth, the average QRC radius evolution can be described by a t 1/2 diffusion term coupled with exponential growth kinetics up to a critical time, and afterwards a growth regime approaching constant velocity. The extension of each regime depends on c EGF and colony history. In the presence of EGF, the initial expansion of quasi-linear colonies (QLCs) also exhibits morphological changes at both the cell and the colony levels. In these cases, the cell density at the colony border region becomes smaller than in the absence of EGF and consequently, the extension of the effective rim where cell duplication and motility contribute to the colony expansion increases. QLC front displacement velocity increases with c EGF up to a maximum value in the 2-10 ng ml-1 range. Individual cell velocity is increased by EGF, and an enhancement in both the persistence and the ballistic characteristics of cell trajectories can be distinguished. For an intermediate c EGF, collective cell displacements contribute to the roughening of the colony contours. This global dynamics becomes compatible with the standard Kardar-Parisi-Zhang growth model, although a faster colony roughness saturation in EGF-containing medium than in the control medium is observed.

Spatio-temporal morphology changes in and quenching effects on the 2D spreading dynamics of cell colonies in both plain and methylcellulose-containing culture media.

To deal with complex systems, microscopic and global approaches become of particular interest. Our previous results from the dynamics of large cell colonies indicated that their 2D front roughness dynamics is compatible with the standard Kardar-Parisi-Zhang (KPZ) or the quenched KPZ equations either in plain or methylcellulose (MC)-containing gel culture media, respectively. In both cases, the influence of a non-uniform distribution of the colony constituents was significant. These results encouraged us to investigate the overall dynamics of those systems considering the morphology and size, the duplication rate, and the motility of single cells. For this purpose, colonies with different cell populations (N) exhibiting quasi-circular and quasi-linear growth fronts in plain and MC-containing culture media are investigated. For small N, the average radial front velocity and its change with time depend on MC concentration. MC in the medium interferes with cell mitosis, contributes to the local enlargement of cells, and increases the distribution of spatio-temporal cell density heterogeneities. Colony spreading in MC-containing media proceeds under two main quenching effects, I and II; the former mainly depending on the culture medium composition and structure and the latter caused by the distribution of enlarged local cell domains. For large N, colony spreading occurs at constant velocity. The characteristics of cell motility, assessed by measuring their trajectories and the corresponding velocity field, reflect the effect of enlarged, slow-moving cells and the structure of the medium. Local average cell size distribution and individual cell motility data from plain and MC-containing media are qualitatively consistent with the predictions of both the extended cellular Potts models and the observed transition of the front roughness dynamics from a standard KPZ to a quenched KPZ. In this case, quenching effects I and II cooperate and give rise to the quenched-KPZ equation. Seemingly, these results show a possible way of linking the cellular Potts models and the 2D colony front roughness dynamics.

Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: a biological system approaching a quenched Kardar-Parisi-Zhang universality.

The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α=0.63±0.04,ß=0.75±0.05, and z=0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

Influence of individual cell motility on the 2D front roughness dynamics of tumour cell colonies.

The dynamics of in situ 2D HeLa cell quasi-linear and quasi-radial colony fronts in a standard culture medium is investigated. For quasi-radial colonies, as the cell population increased, a kinetic transition from an exponential to a constant front average velocity regime was observed. Special attention was paid to individual cell motility evolution under constant average colony front velocity looking for its impact on the dynamics of the 2D colony front roughness. From the directionalities and velocity components of cell trajectories in colonies with different cell populations, the influence of both local cell density and cell crowding effects on individual cell motility was determined. The average dynamic behaviour of individual cells in the colony and its dependence on both local spatio-temporal heterogeneities and growth geometry suggested that cell motion undergoes under a concerted cell migration mechanism, in which both a limiting random walk-like and a limiting ballistic-like contribution were involved. These results were interesting to infer how biased cell trajectories influenced both the 2D colony spreading dynamics and the front roughness characteristics by local biased contributions to individual cell motion. These data are consistent with previous experimental and theoretical cell colony spreading data and provide additional evidence of the validity of the Kardar-Parisi-Zhang equation, within a certain range of time and colony front size, for describing the dynamics of 2D colony front roughness.

Growth dynamics of cancer cell colonies and their comparison with noncancerous cells.

The two-dimensional (2D) growth dynamics of HeLa (cervix cancer) cell colonies was studied following both their growth front and the pattern morphology evolutions utilizing large population colonies exhibiting linearly and radially spreading fronts. In both cases, the colony profile fractal dimension was d(f)=1.20±0.05 and the growth fronts displaced at the constant velocity 0.90±0.05 µm min(-1). Colonies showed changes in both cell morphology and average size. As time increased, the formation of large cells at the colony front was observed. Accordingly, the heterogeneity of the colony increased and local driving forces that set in began to influence the dynamics of the colony front. The dynamic scaling analysis of rough colony fronts resulted in a roughness exponent α = 0.50±0.05, a growth exponent ß = 0.32±0.04, and a dynamic exponent z=1.5±0.2. The validity of this set of scaling exponents extended from a lower cutoff l(c)≈60 µm upward, and the exponents agreed with those predicted by the standard Kardar-Parisi-Zhang continuous equation. HeLa data were compared with those previously reported for Vero cell colonies. The value of d(f) and the Kardar-Parisi-Zhang-type 2D front growth dynamics were similar for colonies of both cell lines. This indicates that the cell colony growth dynamics is independent of the genetic background and the tumorigenic nature of the cells. However, one can distinguish some differences between both cell lines during the growth of colonies that may result from specific cooperative effects and the nature of each biosystem.

Dynamics and morphology characteristics of cell colonies with radially spreading growth fronts.

The dynamics of two-dimensional (2D) radially spreading growth fronts of Vero cell colonies was investigated utilizing two types of colonies, namely type I starting from clusters with a small number of cells, which initially exhibited arbitrary-shaped rough growth fronts and progressively approached quasicircular ones as the cell population increased; and type II colonies, starting from a relatively large circular three-dimensional (3D) cell cluster. For large cell population colonies, the fractal dimension of the fronts was D(F) = 1.20±0.05. For low cell populations, the mean colony radius increased exponentially with time, but for large ones the constant radial front velocity 0.20±0.02 µm min(-1) was reached. Colony spreading was accompanied by changes in both cell morphology and average size, and by the formation of very large cells, some of them multinuclear. Therefore the heterogeneity of colonies increased and local driving forces that set in began to influence the 2D growth front kinetics. The retardation effect related to the exponential to constant radial front velocity transition was assigned to a number of possible interferences including the cell duplication and 3D growth in the bulk of the colony. The dynamic scaling analysis of overhang-corrected rough colony fronts, after arc-radius coordinate system transformation, resulted in roughness exponent α = 0.50±0.05 and growth exponent ß = 0.32±0.04, for arc lengths greater than 100 µm. This set of scaling exponents agreed with that predicted by the Kardar, Parisi, and Zhang continuous equation. For arc lengths shorter than 2-3 cell diameters, the value α = 0.85±0.05 would be related to a cell front roughening caused by temporarily membrane deformations occasionally interfered by cell proliferation.

Morphology and dynamic scaling analysis of cell colonies with linear growth fronts.

The growth of linear cell colony fronts is investigated from the morphology of cell monolayer colonies, the cell size and shape distribution, the front displacement velocity, and the dynamic scaling analysis of front roughness fluctuations. At the early growth stages, colony patterns consist of rather ordered compact domains of small cells, whereas at advanced stages, an uneven distribution of cells sets in, and some large cells and cells exhibiting large filopodia are produced. Colony front profiles exhibit overhangs and behave as fractals with the dimension D(F)=1.25±0.05. The colony fronts shift at 0.22±0.02 µm min(-1) average constant linear velocity and their roughness (w) increases with time (t). Dynamic scaling analysis of experimental and overhang-corrected growth profile data shows that w versus system width l log-log plots collapse to a single curve when l exceeds a certain threshold value l(o), a width corresponding to the average diameter of few cells. Then, the influence of overhangs on the roughness dynamics becomes negligible, and a growth exponent ß=0.33±0.02 is derived. From the structure factor analysis of overhang-corrected profiles, a global roughness exponent α(s)=0.50±0.05 is obtained. For l>200 µm, this set of exponents fulfills the Family-Vicsek relationship. It is consistent with the predictions of the continuous Kardar-Parisi-Zhang model.

First Report of Iris yellow spot virus on Onion in Uruguay.

From October to December 2005, onion (Allium cepa) plants in seed-production fields in south Uruguay (Canelones) had symptoms suggestive of those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae). Symptoms included diamond-shaped lesions on seed stalks (scapes), each 1 to 5 cm long with a necrotic border, green center, and sometimes a second necrotic area in the center of the diamond (2,3). Necrotic lesions with more irregular shape were also associated with diseased plants. In 2006, scape samples with these symptoms were collected from four onion seed crops and assayed for IYSV using an IYSV-specific antiserum (Agdia Inc., Elkhart, IN) in a double-antibody sandwich-ELISA. IYSV was detected in all four onion seed crops monitored in 2006. IYSV incidence, expressed as the number of plants with symptoms, ranged from <1% (1 of 120 plants evaluated) to 20% (24 of 120 plants). Two fields were monitored in 2007, in which IYSV incidence increased from 2 and 3% in October to 7% (198 of 2,768 plants) and 40% (253 of 638 plants) in December, respectively. The highest incidence was observed in the same farm in 2006 and 2007. Scapes were sampled from the field with the highest incidence of symptoms in 2007 and tested for IYSV with IYSV-specific primers (3). Total RNA was extracted from 100 mg of symptomatic tissue, with green tissue adjacent to typical lesions, following a Trizol-based protocol (1). A reverse transcriptase-PCR assay with nucleocapsid gene-specific primers was used (3). A PCR product of approximately 26 bp was obtained, coincident with the expected length for IYSV. The PCR product was cloned and sequenced. The tospovirus N sequence of the isolate in Uruguay (Accession No. GU550518) had maximum identity (97%) with an Australian IYSV isolate (Accession No. AY345227), and >87% identity only with IYSV N protein sequences in GenBank. Because of the presence of IYSV in Brazil, Chile, and Peru, this first documentation, to our knowledge, of IYSV in onion crops in Uruguay suggests that the threat of IYSV to onion is increasing in South America. References: (1) P. Chomczynski and K. Mackey. Biotechniques 19:942, 1995. (2) D. H. Gent et al. Plant Dis. 90:1468, 2006. (3) H. R. Pappu et al. Plant Dis. 92:588, 2008.

Increase of murine splenic natural antibody-secreting cells after cyclophosphamide treatment.

Administration of a low sub-immunosuppressive dose of cyclophosphamide (CY) to naive mice induced a marked increase in the number of splenic cells forming natural antibodies against unrelated antigens such as foot-and-mouth disease virus, keyhole limpet hemocyanin, horseradish peroxidase, or bovine serum albumin, as determined by an enzyme-linked immunosorbent assay spot technique. These results suggest that in mice there exists a repertoire of B cells forming natural antibodies which is restrained by an unknown mechanism and that CY is able to interfere with that restriction.

The use of cyclophosphamide as an enhancer of the vaccine against foot-and-mouth disease.

The immunization of biungulate animals with killed foot-and-mouth disease virus (FMDV) requires periodic vaccinations due to a low vaccine immunogenicity. Therefore, FMDV antigens need to be combined with adjuvants such as aluminium hydroxide, saponin or oil emulsions. Animal handling for periodic inoculations, and the repeated doses of vaccines that have to be administered increase the commercialization costs. Moreover, the use of adjuvants may induce adverse effects. In the present work we show that it is possible to increase the life span of neutralizing antibodies in serum when a single dose of cyclophosphamide (Cy) is administered four days before vaccination with aluminium hydroxidesaponin FMDV vaccine.

Protective effect of lidocaine in the experimental foot-and-mouth disease pancreatitis.

Experimental infection of mice with foot-and-mouth disease virus (FMDV) induces a necrotizing pancreatitis of the exocrinar portion of the organ. The lesions are characterized by vascular congestion, edema and interstitial polymorphonuclear leukocyte (PMN) infiltrates. When infected mice were treated with different amounts of lidocaine (a local anesthetic, chemically defined as a tertiary amide compound), reduction in intensity of the pancreatic necrosis and in the number of PMN were observed. Even though lidocaine could interfere with FMDV post-replicative cytolytic mechanisms, it appears that protection against pancreatic necrosis is by attenuation of PMN presentation in the infected tissue.

Cytotoxicity and cytoadherence of human and murine polymorphonuclear leukocytes against Junin virus infected targets in the absence of anti-viral antibody and complement.

Human and murine polymorphonuclear leukocytes (PMN) lysed L cells infected with Junin Virus (JV) in an in vitro system free of antiviral antibody and complement. Infected VERO cells proved to be resistant to the cytolytic effect. Murine PMN showed an increased adherence on JV-infected L cells but did not attach to VERO cells. The opposite was observed with human PMN, which did adhere to infected VERO cells but not to infected L cells. These results indicate that PMN activity may be an early mechanism of defense in JV infection by lysing virus-infected cells when no immune response has been established, and that cytoadherence is not a necessary step for target lysis.

Effect of polymorphonuclear depletion on experimental Argentine hemorrhagic fever in guinea pigs.

The role that polymorphonuclear leukocytes (PMN) may play in Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junín virus (JV), was investigated in experimentally infected guinea pigs depleted of PMN by means of specific antiserum. In leucopenic animals the evolution of the infection with a highly pathogenic strain of JV was more severe, with earlier mortality and higher virus yields in blood and viscera. The pathological study showed similar lesions in both the control and PMN-depleted animals with the exception of the lung, which showed the pathological picture of the human "pulmonary distress syndrome of the adult" in nontreated guinea pigs and appeared histologically unaltered in the PMN-depleted animals. On the basis of these results it is suggested that in AHF, PMN play a dual role. In the first stage of infection they display a defensive antiviral action, but later on they participate in the pathogenesis of tissue damage.

In vitro induction of human polymorphonuclear leuckocyte damage by junin virus

Diferentes modelos experimentales, utilizando cepas patógenas de virus Junín (VJ), han reproducido la característica leucopenia con que cursa la enfermedad humana. Los mecanismos responsables de esta leucopenia son poco conocidos. Con el fin de indagar si el VJ posee algún efecto citopatógeno directo sobre leucocitos polimorfonucleares (PMN), se incubó una suspensión de PMN humanos con VJ, cepa XJCl3. Las células se obtuvieron de doadores sanos de banco de sangre y fueron separados en gradientes de Ficoll Hypaque, lográndose 98% de pureza. Se obtuvieron muestras secuenciales del sobrenadante y de las células para determinación de actividad enzimática (peroxidasa, betaglucuronidasa y lisozina), titulación de VJ y estudios morfológicos con microscopía de luz y electrónica. Se observó una pérdida gradual y constante de las granulaciones citoplásmicas entre las 2 y 10 horas post-infección (pi) disminución del número de células viables según prueba de exclusión de azul tripan, entre las 10 y 20 horas pi y un aumento de la concentración de enzimas lisosomales en el sobrenadante a partir de las 10h pi. Aparentemente no hubo replicación viral ya que luego de las 11h pi no pudo rescatarse infectividad por medio de técnicas convencionales. Para la producción de estos efectos, el virus necesita poseer su capacidad infectante intacta ya que el tratamiento previo con irradiación ultravioleta o con suero inmune anti-VJ previene cambios. Estas observaciones indican que el virus Junín infectante induce alteraciones tempranas irreversibles sobre los PMN (AU)

In vitro induction of human polymorphonuclear leuckocyte damage by junin virus

Diferentes modelos experimentales, utilizando cepas patógenas de virus Junín (VJ), han reproducido la característica leucopenia con que cursa la enfermedad humana. Los mecanismos responsables de esta leucopenia son poco conocidos. Con el fin de indagar si el VJ posee algún efecto citopatógeno directo sobre leucocitos polimorfonucleares (PMN), se incubó una suspensión de PMN humanos con VJ, cepa XJCl3. Las células se obtuvieron de doadores sanos de banco de sangre y fueron separados en gradientes de Ficoll Hypaque, lográndose 98% de pureza. Se obtuvieron muestras secuenciales del sobrenadante y de las células para determinación de actividad enzimática (peroxidasa, betaglucuronidasa y lisozina), titulación de VJ y estudios morfológicos con microscopía de luz y electrónica. Se observó una pérdida gradual y constante de las granulaciones citoplásmicas entre las 2 y 10 horas post-infección (pi) disminución del número de células viables según prueba de exclusión de azul tripan, entre las 10 y 20 horas pi y un aumento de la concentración de enzimas lisosomales en el sobrenadante a partir de las 10h pi. Aparentemente no hubo replicación viral ya que luego de las 11h pi no pudo rescatarse infectividad por medio de técnicas convencionales. Para la producción de estos efectos, el virus necesita poseer su capacidad infectante intacta ya que el tratamiento previo con irradiación ultravioleta o con suero inmune anti-VJ previene cambios. Estas observaciones indican que el virus Junín infectante induce alteraciones tempranas irreversibles sobre los PMN

Junin virus infection of Callithrix jacchus: pathologic features.

Infection of Callithrix jacchus, a New World primate, with the prototype strain of Junin virus produced a severe disease. The animals developed multifocal hemorrhages and characteristic microscopic lesions such as meningoencephalitis, interstitial pneumonia, lymphocytic depletion of lymphatic tissue, hepatocytic necrosis, and a variable decrease in bone marrow cellularity. High virus concentrations correlated with lesions, and with the presence of viral antigenic determinants as revealed by immunofluorescent methods. With the exception of central nervous system damage, the morphological features and immunohistochemical and viral findings were similar to those recorded in human Argentine hemorrhagic fever.