A Process-based Model with Temperature, Water, and Lab-derived Data Improves Predictions of Daily Culex pipiens/restuans Mosquito Density.

While the number of human cases of mosquito-borne diseases has increased in North America in the last decade, accurate modeling of mosquito population density has remained a challenge. Longitudinal mosquito trap data over the many years needed for model calibration, and validation is relatively rare. In particular, capturing the relative changes in mosquito abundance across seasons is necessary for predicting the risk of disease spread as it varies from year to year. We developed a discrete, semi-stochastic, mechanistic process-based mosquito population model that captures life-cycle egg, larva, pupa, adult stages, and diapause for Culex pipiens (Diptera, Culicidae) and Culex restuans (Diptera, Culicidae) mosquito populations. This model combines known models for development and survival into a fully connected age-structured model that can reproduce mosquito population dynamics. Mosquito development through these stages is a function of time, temperature, daylight hours, and aquatic habitat availability. The time-dependent parameters are informed by both laboratory studies and mosquito trap data from the Greater Toronto Area. The model incorporates city-wide water-body gauge and precipitation data as a proxy for aquatic habitat. This approach accounts for the nonlinear interaction of temperature and aquatic habitat variability on the mosquito life stages. We demonstrate that the full model predicts the yearly variations in mosquito populations better than a statistical model using the same data sources. This improvement in modeling mosquito abundance can help guide interventions for reducing mosquito abundance in mitigating mosquito-borne diseases like West Nile virus.

Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell development.

An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.

HIV-induced T-cell syncytia release a two component T-helper cell chemoattractant composed of Nef and Tat.

Using a newly developed gradient chamber to provide independent measurements of chemokinesis (stimulated motility) and chemotaxis (stimulated motility up a concentration gradient) of individual T-helper cells, it was recently demonstrated that HIV-induced T-cell syncytia release two distinct chemotactic activities that are separable by their rates of diffusion. The molecular masses of the two chemoattractant activities were estimated to be 30 and 120 kDa. The higher molecular mass activity was demonstrated to be the viral glycoprotein gp120. In an attempt to identify the lower molecular mass activity, chemotaxis and chemokinesis of T-helper cells were analyzed in individual concentration gradients of the virally encoded proteins Rev, p24, Tat and Nef. None functioned alone as a chemoattractant, but both Tat and Nef alone functioned as chemokinetic stimulants. When Tat and Nef were used together to generate parallel gradients, they stimulated chemotaxis. Antibody to either Tat or Nef neutralized the lower molecular mass chemotactic activity released by syncytia. The addition of antibody to the CD4 receptor or the addition of soluble CD4 inhibited high molecular mass chemotactic activity but not the low molecular mass chemotactic activity in HIV-induced syncytium-conditioned medium, demonstrating that the former but not the latter activity is mediated through the CD4 receptor. These results identify the combination of Nef and Tat as the lower molecular mass T cell chemoattractant released by HIV-induced syncytia, and provide the first evidence suggesting that parallel concentration gradients of two proteins are necessary for chemotaxis.

Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis.

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassembly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and chemotaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-assisted methods in buffer, in a spatial gradient of cAMP, and after the rapid addition of cAMP. 3XALA cells crawling in buffer exhibit distinct abnormalities in cellular shape, the maintenance of polarity and the complexity of the pseudopod perimeter. 3XALA cells crawling in buffer also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which the shape of each cell changes abnormally from elongate to extremely wide with lateral, opposing pseudopods. At the end of each cycle, 3XALA cells turn 90 degrees into the left or right lateral pseudopod, resulting in a dramatic depression in chemotactic efficiency, even though 3XALA cells are chemotactically responsive to cAMP. These results demonstrate that the phosphorylation of myosin II heavy chain plays a critical role in the maintenance of cell shape and in persistent translocation in a spatial gradient of chemoattractant.

T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber.

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.

HIV-induced T cell syncytia are self-perpetuating and the primary cause of T cell death in culture.

Prior studies suggested that induced fusion in T cell cultures infected with syncytium-inducing HIV was not the primary cause of T cell death. However, these studies failed to assess the contribution of fusion in terms of syncytium volume, rather than syncytium frequency. This question has been reassessed by monitoring the frequency, volume, rate of growth, longevity, p24 production, viral budding, and self-propagating ability of syncytia in HIV-infected SUP-T1 cell cultures and individually isolated syncytia seeded in uninfected SUP-T1 cell cultures. The results demonstrate that in these cultures syncytium formation is the principal mechanism of T cell death, syncytia are the main source of virus production, and both virus production and syncytium longevity are independent of syncytium size, but dependent on syncytium age, suggesting that both are programmed events.

Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis.

Previous studies have demonstrated that overexpression of the carboxyl-terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359-368]. To assess the consequences of having CaD39-stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39-expressing lines. We here found that CaD39-expressing cells adhered faster to plastic, glass, fibronectin-coated glass, and collagen-coated glass than control cells. Moreover, the CaD39-expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39-expressing cells were more elongate and encompassed less area than non-expressing cells during migration in a wound-healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin-enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39-induced advantage for attachment and spreading was lost during mitosis. In CaD39-expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39-expressing lines.

Ponticulin plays a role in the positional stabilization of pseudopods.

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

HIV-induced syncytia in peripheral blood cell cultures crawl by extending giant pseudopods.

It was previously demonstrated that HIV-induced syncytia of the immortalized T cell line SupT1 reorganize their cytoskeleton and form a spherical supernuclear complex, thus mimicking the organization, polarity, and morphology of a single SupT1 cell. Then, through extension of a single, giant pseudopod, these syncytia, which grow to more than 100 times the volume of a single SupT1 cell, translocate along a substratum. To verify that syncytium motility is not peculiar to the SupT1 cell line, we have analyzed the cytoskeletal organization and motile capabilities of HIV-induced syncytia formed in peripheral blood cell cultures containing more than 90% CD4-positive cells. The results demonstrate that although peripheral blood T cells differ from SupT1 cells in size and morphology, they are continuously motile and translocate along a substratum in a manner quite similar to that of SupT1 cells, and peripheral blood T cell syncytia induced by HIV-1LAI as well as two additional clinical isolates translocate by the extension of a giant anterior pseudopod in a fashion indistinguishable from that of HIV-induced SupT1 syncytia. Together, these results support the generalization that HIV-induced T cell syncytia are motile.

T cells and HIV-induced T cell syncytia exhibit the same motility cycle.

Ameboid cells ranging in complexity from Dictyostelium amebas to human polymorphonuclear leukocytes (PMNs) translocate in a cyclical fashion. Using computer-assisted motion analysis, we have analyzed the motility of human lymphocytes of the immortal SupT1 cell line and of a peripheral blood mononuclear cell population highly enriched for CD4-positive cells (CD4-enriched PBMCs) on four substrates--plastic, dehydrated rat tail collagen, hydrated rat tail collagen, and bovine aortic endothelium. In addition, we have analyzed the motility on these substrates of syncytia induced by human immunodeficiency virus (HIV) in cultures of both cell types. It is demonstrated that both SupT1 cells and CD4-enriched PBMCs exhibit a motility cycle with a period of 1.6 min that is independent of substrate, independent of average cell velocity, and similar to the periods of translocating Dictyostelium amebas and PMNs. More surprisingly, it is demonstrated that HIV-induced SupT1 and PBMC syncytia with volumes 10 to 100 times those of single cells exhibit the same motility cycle as their single-cell progenitors. These observations support the generality of the motility cycle in animal cells and, for the first time, demonstrate that the cycle is independent of cell size.

A comparison of stress in surgically and non-surgically mulesed sheep.

A comparison has been made in 9- to 10-month-old castrated male Merino sheep of the changes in plasma total cortisol concentration and behaviour after being treated by either the modified Mules operation or by topical application of a quaternary ammonium compound to achieve non-surgical mulesing. After surgical mulesing, plasma total cortisol concentration increased immediately and rapidly and reached a peak value in 15 minutes, whereas after non-surgical treatment an immediate rise did not occur, but a similar peak value was observed in blood samples collected 24 hours after treatment. The concentrations were lower in both groups at 48 hours. Likewise postural changes indicative of discomfort were immediately apparent in the surgically treated sheep, but not until 3 to 4 hours later in those treated non-surgically. Arena testing revealed that a lasting aversion to the person who restrained them during treatment developed in the surgically mulesed sheep, but not in those treated non-surgically. The non-surgical procedure did not create large open wounds, as did the surgical operation, but still achieved similar enlargement of the bare area on the breech, and healing was quicker in the non-surgically treated sheep.

HIV-induced syncytia of a T cell line form single giant pseudopods and are motile.

The human immunodeficiency virus, HIV, induces syncytium formation in cultures of many T cell lines. These syncytia have previously been viewed as disorganized fusion products in the throes of death. Evidence is presented that in HIV1-infected SupT1 cultures, syncytia five times to over one hundred times larger than single cells organize their many nuclei into blastula-like balls, reorganize their cytoskeleton to mimic that of a single cell, and extend single, giant pseudopods in a polar fashion. Medium-sized syncytia are capable of translocation through extension of these giant pseudopods. The rate of translocation of syncytia is comparable to that of single cells. Single cell motility, syncytium motility and pseudopod extension also appear to play roles in the recruitment of cells into syncytia. Finally, condensation of F-actin at cell-syncytium and syncytium-syncytium adhesion sites suggests the involvement of the cytoskeleton in the adhesion and/or subsequent fusion event. These results suggest that the fusion events involved in HIV-induced syncytia formation involve both cell motility and reorganization of the cytoskeleton, and demonstrate that syncytia are highly organized, motile entities.

Salivary and plasma cortisol as an index of stress in goats.

Total assayable cortisol in plasma was highly correlated (r = 0.97) with physiologically active free cortisol in plasma after routine management procedures in 1- to 3-weeks-old goats. Transport of adult goats caused significant increases (P less than 0.001) in free cortisol in saliva and in free and total cortisol in plasma. No difference (P greater than 0.05) between concentrations of free cortisol in saliva and in plasma was apparent before or after transport. The results demonstrated that the salivary cortisol method is a useful measure of stress in adult goats, and that the relationship between free and total cortisol in plasma, and the adrenocortical response to transport, appear to be similar in sheep and goats.

Effects of ovine corticotropin-releasing factor and vasopressin on plasma beta-endorphin, cortisol and behavior after minor surgery in sheep.

The evidence for an analgesic effect arising from increased peripheral concentrations of beta-endorphin (beta-EP) in various animal species is controversial, and has not been fully evaluated in the sheep. To stimulate beta-EP release, ovine corticotropin-releasing factor (oCRF) and arginine vasopressin (AVP) were injected intravenously (iv) into a group of 12 out of 24 sheep, 15 min prior to minor surgery on all sheep. This brought about significant increases (P less than 0.01) in plasma beta-endorphin (beta-EP) and cortisol concentrations, relative to the non-injected control sheep, 15-30 min after injection. Ultrafiltration indicated that less than 30% of the released beta-EP immunoreactivity was present as higher molecular weight forms (mol. wt greater than 10,000) and that the majority (about 75%) of the beta-EP was probably bound to plasma proteins. By 75 min after injection there was no significant difference in plasma beta-EP or cortisol concentrations between the two groups of sheep. Consistent with previous observations the sheep showed a characteristic aversive behavior to the human handler following surgery, lasting several days. This behavior appeared to be unaffected by a pre-operative increase in peripheral plasma beta-EP, and may indicate that this increase in beta-EP was not sufficiently analgesic to block the cognitive response to the operation, or long-lasting enough to prevent the perception of post-operative soreness.

Stress responses in lambs docked and castrated surgically or by the application of rubber rings.

A comparative study has been made in lambs 3 to 6 weeks of age of the behavioural responses and changes in plasma immunoreactive beta-endorphin (ir beta-endorphin) and cortisol after docking or docking plus castration by the application of rubber rings or by surgery. The use of rubber rings on lambs at this age was characterised by very agitated behaviour indicative of considerable distress for a period of up to 1 h. In contrast, surgery was accompanied by some post-operative shock exhibited by reduced exploratory and social behaviour, at least in the lambs exposed to docking plus castration. In the latter group there were highly significant increases in both plasma ir beta-endorphin and cortisol concentrations that may be consistent with the induction of stress-induced analgesia. We conclude that surgery caused less distress than the rubber rings, and is therefore preferable for docking and castration of lambs 3 to 6 weeks of age.

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep.

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.

Stress-induced changes in plasma concentrations of immunoreactive beta-endorphin and cortisol in response to routine surgical procedures in lambs.

Following four different surgical procedures in lambs 3-5 weeks old, plasma immunoreactive beta-endorphin (beta-EP) and cortisol were assayed at 15 min and 24 h as determinants of post-operative stress. A threefold increase in mean plasma beta-EP levels occurred 15 min after tail docking, and a maximal eight- to tenfold increase occurred in response to castration and/or mulesing with tail docking. Significant increments in mean plasma cortisol levels followed these surgical procedures with the maximal response 15 min after mulesing plus castration with tail docking. The physiologically active 'free' cortisol in plasma represents about 25% of the cortisol, as measured, and the two are highly correlated. At 24 h, beta-EP levels in all treated groups were similar to controls, although a small elevation in cortisol levels was still present in the lambs subjected to mulesing. Ultrafiltration of plasma extracts showed that peak beta-EP levels contained about 40% immunoreactivity from low molecular weight species (mol. wt less than 10,000). By specific radioimmunoassay and reverse-phase high-performance liquid chromatography this comprised about 75% beta-EP1-31, the most potent analgesic endorphin, 10% beta-EP1-27, and 15% alpha-N-acetyl-beta-EP. Increased beta-EP1-31 levels may modulate post-operative pain in lambs.

Stress in calves castrated surgically or by the application of rubber rings.

The effects of castration were studied in calves 4 to 11 weeks of age, using increases in salivary cortisol as an indicator of stress. Groups were castrated surgically or by rubber ring application or were non-castrated (control) females. The surgically castrated group showed more agitation during the operation, but both castrated groups resumed normal behaviour soon after the operation was completed. The short-term salivary cortisol response was significantly higher after surgical castration than after the application of rubber rings, where, in turn, it was significantly higher than in the control group. Salivary cortisol was elevated over a period from 15 min to 3 h following the castration, but at 4, 24 h and 6 days post-treatment there were no significant differences between treated groups and controls.

Development of a salivary cortisol method for detecting changes in plasma "free" cortisol arising from acute stress in sheep.

A simple device for collecting saliva (mainly parotid) from sheep is described. The collection of saliva, and the assay of "free" cortisol in saliva appears to offer certain advantages over the collection of blood, and the assay of serum cortisol, for the assessment of stress in sheep. With a little experience, it is easier to collect saliva than take blood samples when sheep are passing through a race. The "free" cortisol can be measured directly in saliva, whereas in serum, it is first necessary to separate "free" from protein-bound cortisol. Basal levels of "free" cortisol of less than 10 nmol/l were recorded in saliva and blood plasma or serum in unstressed sheep which had previous experience of being handled in a race, Significant increases in salivary cortisol and "free" and total ("free" plus protein-bound) cortisol in serum were found in sheep following adrenal stimulation with synacthen, or after 30 min of stressful transport. This indicates that the salivary cortisol technique is applicable to studies of stress in sheep, and should also be useful for other ruminants.