Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated.B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β.B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant  However, a variety of practical problems limits the use of biotechnological protein drugs. Stability of the active proteins, need for parenteral administration, and high costs of the final purified materials are among the most significant drawbacks. A way of circumventing these issues is represented by the direct administration of recombinant bacteria, acting simultaneously as cell factory and delivery system for pharmacologically active proteins. This approach has been already extensively experimented for the mucosal delivery of vaccine antigens ,2. In reversus-host disease, and multiple sclerosis  has received vast attention. IL-1 is a family of cytokines highly active in the modulation of immune amplification and inflammation. The IL-1 family includes two agonist proteins, IL-1α and IL-1β, and one antagonist protein, IL-1Ra. IL-1β is a very potent immunostimulatory and inflammatory cytokine, responsible for initiating and amplifying the host response to invasion. If not properly controlled, IL-1 can cause fever, acute inflammation, tissue destruction, organ failure, and eventually shock and death (reviewed in ). IL-1Raiewed in ,12,16). iewed in ,12,16,18iewed in ,12,19-21iewed in ,23. The in vivo cell factory, here we describe a novel system of local delivery of IL-1Ra, able to achieve systemic effects. The system exploits the ability of certain bacteria (such as Bacillus subtilis) to undergo autolysis in non-permissive conditions  thereby releasing the cytoplasmic proteins. Intra-colon instillation of B. subtilis expressing recombinant human mature IL-1Ra induces significant serum levels of the recombinant protein in rats and rabbits, and prevents the inflammatory effects of systemic IL-1. Intra-peritoneal administration of recombinant B. subtilis in the mouse could inhibit LPS-induced shock and death. Further experimental evidence with a B. subtilis strain producing human IL-1β demonstrates that this delivery system can be generalised to other recombinant proteins.Based on previous experience of using recombinant bacteria as B. subtilis to generate spores by autolysis of the cell wall, thereby releasing cytoplasmic proteins, was exploited as system for delivery of proteins in vivo. Following a sporulation signal (e.g., nutrient depletion) bacteria undergo an autolytic process with release of most of their cellular components and formation of a highly resistant spore containing DNA and few essential proteins  caused the release of large amounts of intact and active recombinant protein within a few hours after the sporulation signal  and Spo- bacteria, i.e. genetically modified cells which, in response to the sporulation signal, start the autolysis process but are unable to form a complete spore thus undergoing complete cell destruction /mg vs. 0.9 × 106 IU/mg of reference standard IL-1Ra [in vivo by engineered B. subtilis, the bacterial strain engineered with IL-1Ra was administered intra-peritoneally in the mouse, in the small and large intestine of rats, and in the rabbit distal colon. The presence of human IL-1Ra was assessed at different times after administration, both locally and in the serum of treated animals, by Western blotting, ELISA, and BIAcore analysis  or in the colon (5/9 rats, 27/31 rabbits), but not when bacteria were administered in the small intestine (0/5 rats).The IL-1Ra recovered after d IL-1Ra . To asseB. subtilis vegetative life [i.e. B. subtilis cells transformed with the β-lactamase-expressing control plasmid pSM214. Data in Figure B. subtilis strain pSM539, whereas serum of animals receiving B. subtilis pSM214 remained negative. As a control, pSM539 bacteria delivered in the small intestine released detectable amounts of IL-1Ra locally, but no IL-1Ra could be found at the serum level , but it was still highly significant when fever was induced by 100 ng/kg IL-1β (>60% reduction of peak fever) (data not shown). To confirm these data, the effect of intra-colonic treatment with IL-1Ra-producing pSM539 was evaluated on other inflammation-related parameters induced by IL-1β, i.e., granulocytosis and decrease of blood glucose and iron concentrations. As shown in Figure in vivo by intra-colonic administration of engineered B. subtilis is biologically active and able to counteract the systemic inflammatory effects of IL-1.The red IL-1 . As showB. subtilis can have an anti-inflammatory protective effect in vivo was shown in a model of shock and death induced by bacterial endotoxin (LPS) in the mouse  and administered in vivo to rabbits. As shown in Figure 9 live cells of B. subtilis pSM261 induced a significant increase in body temperature, superimposable to that caused by intra-colonic instillation of purified recombinant IL-1β. Furthermore, in agreement with the systemic effects of massive doses of IL-1β administered parenterally [B. subtilis in the large intestine is a suitable system for attaining significant blood levels of bioactive recombinant proteins and systemic effectiveness.To validate the concept of delivery of bioactive recombinant proteins via the colonic mucosa by means of recombinant nterally ,28, intrSalmonella, Listeria monocytogenes, Mycobacterium tuberculosis, Vibrio cholerae are being developed and used as vaccine carriers [e.g., food-grade lactic acid bacteria), with the possibility of including T- and B-stimulating epitopes from different antigens, and also to engineering into the same carrier adjuvant sequences derived for instance from an immunostimulating cytokine [The use of live bacteria is very common in particular in vaccinology, where attenuated or mutant bacteria have been employed for decades as antigen carriers. The advantage of live bacteria relies on their capacity of colonising the host and enter the host organs/tissues with the same modalities as their virulent counterparts, thus eliciting the relevant immune response and immune memory, at variance with killed bacteria or purified bacterial components. Thus, attenuated strains of carriers -31. A fucytokine -34.Lactococcus lactis and of Streptococcus gordonii, which have been engineered to produce recombinant antibodies, adjuvant and anti-inflammatory cytokines, and used to deliver these proteins locally at the mucosal surface after oral administration [Among bacterial systems developed for antigen delivery in vaccination, some strains of non-pathogenic, food-grade or GRAS  bacteria have been examined for the topical delivery of pharmacologically active protein drugs, after cell engineering with the DNA coding for the protein of interest. This is the case of stration -9. The gstration ,4, cytokstration ,6, inhibstration -9. Althostration , the proB. subtilis cells engineered to produce human IL-1Ra were able to release the recombinant protein  following a sporulation signal in vitro [in vivo, when recombinant B. subtilis cells were inoculated in the intestine of rats or rabbits . The recombinant protein could be detected locally shortly after administration of bacteria and persisted at measurable levels for several hours. Release and recovery of recombinant IL-1Ra was much more abundant and consistent in the large intestine as compared to the small intestine. Most interestingly, the recombinant protein released from sporulating bacteria delivered in the large intestine was absorbed in the bloodstream at detectable levels, whereas no circulating IL-1Ra could be found after bacterial delivery in the small intestine. IL-1Ra present in the blood was intact, as judged by its molecular mass in Western blotting, and retained full IL-1-inhibiting activity, as judged by its capacity of dose-dependent neutralisation of IL-1β in vitro. The passage of an intact protein from the intestinal lumen to the bloodstream is not a new concept. Indeed, transcytosis has been extensively described in intestinal epithelial cells, and allows transport of intact proteins and macromolecules from the intestinal lumen to the circulation through an endocytic non-degradative pathway in physiological conditions of integrity of the intestinal mucosal barrier [The delivery system proposed here is not based on the permanence/colonisation capacity of bacteria in the host mucosal surfaces, but it relies on the capacity of sporulating bacteria of releasing intracellular proteins in non-permissive environments. in vitro . This ob barrier -39. Thismax at 60 min), IL-1Ra released from intra-colonically administered B. subtilis had a much slower kinetics of absorption (Tmax 200 min), as expected by the fact that the protein must be released from bacteria before being absorbed. Furthermore, although the Cmax was decreased for B. subtilis IL-1Ra , the AUC/dose were almost identical. Thus, IL-1Ra delivered intra-colonically by B. subtilis is absorbed into the bloodstream at a slower and more constant rate than the purified protein delivered in the same site, which is absorbed quickly into the bloodstream and rapidly disappears thereafter. Thus, it appears that bacteria do not undergo sporulation all at the same time (which would result in a rapidly appearing and disappearing peak of protein), but release the protein constantly from the moment of administration for about 8 h. This would allow a controlled and sustained circulating level of the protein, thus a more favourable pharmacodynamic profile, with a single administration.Delivery of IL-1Ra through engineered sporulating bacteria apparently had some pharmacokinetics advantages as compared to the purified protein. Whereas the absorption into the bloodstream was quick after administration of the purified protein (Tin vivo delivery with B. subtilis is the IL-1 receptor antagonist IL-1Ra, a competitive non-activating ligand of the IL-1 receptor with IL-1 inhibitory activity [e.g., rheumatoid arthritis) [The protein selected for activity ,12. IL-1activity . Anti-ILactivity ,12. The activity ,12,18. Athritis) ,41. To athritis) -23. Howethritis) .Streptococcus gordonii engineered to release human IL-1Ra resulted in a prolonged local delivery of the protein, consequent to the capacity of S. gordonii to colonise the mucosal surfaces [S. gordonii) also had a local therapeutic effect in a model of ulcerative colitis [S. gordonii, as it causes rapid local release of the recombinant protein (e.g. in the large intestine, where IL-1Ra peaks at 4 h and decreases towards background at 24 h), followed by absorption into the bloodstream. In preliminary experiments in the mouse, IL-1Ra-expressing bacteria were also administered intragastrically or subcutaneously. This achieved appearance of human IL-1Ra in the serum, and systemic effects of inhibition of LPS-induced shock and death (data not shown). This is a new finding, that opens the possibility of exploiting localised bacterial administration  for systemic drug delivery. The amount of protein released at the mucosal site directly correlates with the number of administered bacteria, since the internal body environment does not sustain bacterial replication but induces sporulation. This allows an exact control of the dose of drug delivered and, based on the pharmacokinetics parameters, of the blood levels that can be reached. The same result could not be easily obtained with S. gordonii, as amount and timing of protein release may be influenced by variation of the colonisation capacity depending on variations of environmental conditions of the host tissues.The drug delivery strategy here described merges the well-known approach of vaccination with live bacteria with that of gene therapy. The delivery of pharmacologically active proteins by live sporulating bacteria, as described here, presents a series of advantages over other similar approaches. At variance with conventional gene therapy, the gene coding for the drug protein is introduced in a bacterial carrier rather than in host cells, a situation that would allow a complete control of its permanence in the body. In a previous study, intragastric or vaginal administration of surfaces . Mucosal colitis . The delin vivo for therapy or vaccination is that of safety and contained release of genetically modified organisms (GMO). The use of suicidal genes or the deletion of genes vital for survival outside the host organism have been explored with very promising results [B. subtilis pSM539 strain was engineered in order to inactivate a gene involved in sporulation control. As a consequence, in response to in vitro sporulation signals  the mutated Spo- strain could regularly initiate the sporulation process, undergoing cell autolysis and release of the cytoplasmic proteins (including the recombinant IL-1Ra), but it was incapable of eventual spore formation and further survival. Likewise, release of the recombinant protein from Spo- in vivo was comparable to that of Spo+ bacteria, but spores could never be recovered from intestinal lavage and faeces (data not shown). This suggests that the system can be optimised to full biological containment and environmental safety without altering its delivery properties.A problem that should be faced when using recombinant bacteria  results ,43. The i.e., avoiding the risk of release of genetically modified bacteria in the environment).The novel system of protein drug delivery here proposed links some of the advantages of gene therapy (endogenous production of the relevant protein, targeted delivery) to the possibility of controlled release in terms of timing and protein amount. Exploitation of the mechanism of bacterial autolysis in non-permissive environments allows release of intracellular proteins, including the known amount of the pharmacologically active recombinant protein drug. The release is persistent for several hours, allowing to maintain more constant protein levels in the bloodstream. The system is simple, cheap, and can be developed to full environmental safety  opens promising avenues to the use of local delivery for the therapy of systemic diseases.B. subtilis strains were constructed as previously described in detail [EcoRI and HindIII in pSM214, a B. subtilis plasmid which promotes the synthesis of recombinant products intracellularly, to obtain recombinant plasmids pSM539 (carrying the cDNA for IL-1Ra) and pSM261 (carrying the cDNA for IL-1β). Plasmids were used to transform the B. subtilis SMS118 strain. The pSM539-harbouring B. subtilis strain SMS118(pSM539) could produce 1.0–2.0 mg IL-1Ra/109 cells/0.35–0.49 g (wet weight), after conventional culture overnight in 1 liter flasks. The SMS118(pSM261) strain in the same culture conditions produced 0.15–0.25 mg IL-1β/109 cells/0.35–0.49 g. As negative control, B. subtilis strain SMS118 was transformed with the pSM214 plasmid, which contains the gene of β-lactamase (conferring resistance to penicillin). All strains were leu-, pyrDI, npr-, apr-. Sporulation-defective (Spo-) strains were constructed by mutation in the srfA gene, as previously described [Engineered n detail ,45. Brieescribed  and were9 cells) were suspended in Difco sporulating medium (bacto beef extract 3 g/l, peptone 5 g/l, NaOH 0.25 mM, MgSO4 10 mM, KCl 0.1%, MnCl2 0.1 mM, Ca(NO3)2 1 mM, FeSO4 1 mM, pH 6.8) without chloramphenicol and incubated at 35°C with shaking. Aliquots of sporulation supernatant were harvested by centrifugation (14,000 × g, 5 min) at different time points. Following sporulation signals, both Spo+ and Spo- bacteria initiate the autolysis process, which ends in cell autolysis with release of cytoplasmic content. However, whereas in Spo+ bacteria there is formation of a spore with preservation of strain survival, Spo- bacteria are unable to form a spore thus undergoing complete cell destruction . For sporulation supernatant preparation, 0.25 g wet weight of bacteria Protein samples were run on 13.5% mini SDS-PAGE according to Lämmli  and staiin vivo experiments), female C3H/HeJ mice of 5–8 weeks of age (for thymocyte proliferation), female Sprague-Dawley rats (around 300 g), and female New Zealand rabbits (1.9–2.5 kg). All animals were purchased from Charles River Italia  and were housed in standard cages at 22 ± 1°C with 12 h light-12 h dark cycle. Animals received standard diet and tap water ad libitum.Experimental animals were: female C3H/HeOuJ mice of 10–12 weeks of age (20–25 g)  were instilled in the small intestine (duodenum) with a 22 1/2 G needle, in a volume of 1–10 ml. Two surgical ligatures were applied, one at the beginning of the duodenum immediately below the needle entry puncture (to avoid exit of instilled bacteria), and another one near the ileocecal valve, to limit to the small intestine the transit of bacteria. Intra-colon instillation was performed again with a 22 1/2 G needle in the ceacum immediately below the ileo-caecal valve, in a volume of 5–10 ml. Two surgical ligature were applied just below the needle entry point and at the colon terminal region, to avoid loss of bacteria. Animals were sacrificed by exanguination at different times after treatment, to collect blood and intestinal washings.B. subtilis suspension were administered. Serum samples were prepared from blood collected from the rabbit marginal ear vein at different times (0–8 h) after intra-colonic administration of bacteria. In some experiments, animals were sacrificed, to collect the large intestine content .For intra-colonic administration of bacteria in rabbits, animals were fasted overnight prior to treatment, then lightly restrained in conventional stocks and maintained conscious throughout the experiment. A rounded-tip urethral catheter (Rüsh, Germany) was carefully inserted 10 cm into the distal colon via the anal route and 2 ml of Protocols of animal experimentation were reviewed by the institutional ethical board for adherence to ethical guidelines for animal research conduct , and previously authorised by the Italian Ministry of Health.Western blotting: samples were subjected to reducing 15% mini SDS-PAGE and analysed by Western blotting using a polyclonal rabbit serum anti-human IL-1Ra and a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase, as described in detail elsewhere . Serum sELISA measurement: samples were subjected to quantitative determination of human IL-1Ra using a specific ELISA , following the manufacturer's instructions. The lower detection limit was 20 pg/ml. Purified human recombinant IL-1Ra was used as standard. Serum samples were filtered on Microcon 100 (Amicon) before analysis.Biosensor measurement: detection of IL-1Ra in serum samples and intestinal washings was confirmed with the biosensor BIAcore™ system , which allows real time biospecific interaction analysis by means of the optical phenomenon of surface plasmon resonance, as previously described in detail . The low5 cells/well of Cluster96 plates (Costar, Cambridge, MA) in 0.2 ml of RPMI-1640 medium (Life Technologies, Paisley, Scotland) supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, 50 μg/ml gentamycin sulfate, 1.25 × 10-5 M 2-ME , 5% fetal bovine serum (Hyclone, Logan, UT) for 72 h in moist air with 5% CO2 [3H]TdR  and their proliferation was measured as radiolabel incorporation with a β-counter.The classical assay of co-stimulation of murine thymocyte proliferation was used to evaluate the bioactivity of IL-1 and IL-1Ra. Briefly, thymocytes from 5–8 week-old C3H/HeJ mice  were cultured at 6 × 10h 5% CO2  and a suThe biological activity of IL-1Ra was evaluated as inhibition of IL-1β-dependent thymocyte proliferation. To this end, cells were stimulated to proliferate (with IL-1β and PHA) in the presence of increasing concentrations of human recombinant IL-1Ra  or seriai.e. the dilution previously found to inhibit 50% of the activity of 10 ng/ml IL-1Ra in the thymocyte assay (not shown).To assess that the effect of IL-1Ra-containing serum was indeed due to IL-1Ra, a polyclonal rabbit antibody against human IL-1Ra  was addeB. subtilis suspensions (2 × 109 live cells/rabbit) were instilled in the distal colon 1 h before i.v. administration of 50–100 ng/ml highly purified LPS-free human recombinant IL-1β in pyrogen-free saline through the marginal ear vein. Temperature was recorded every 20 min for 3 h starting from IL-1β administration. In experiments with IL-1β-producing strain pSM261, rabbits received an intra-colonic administration of 1 × 109 pSM214 (control) or pSM261 (IL-1β) bacteria, or 250 μg purified human IL-1β. Temperature was recorded up to 22 h after treatment.Rabbits were lightly restrained in conventional stocks throughout the experiment, and accustomed to the stocks over a period of 2 h, to minimise variations in body temperature. Body temperature was measured by means of a cutaneous thermistor probe  placed between the left posterior paw and the abdomen and allowed to stabilise for 2 min. B. subtilis strains pSM214 and pSM539 were instilled in the distal colon (1 × 109 cells/kg), 2 h before administration of IL-1β. Blood samples were drawn 2, 4, 6, 8 and 24 h after intra-peritoneal inoculum of 0.1 μg/kg human recombinant IL-1β. The number of circulating neutrophils was assessed by flow cytometry. The plasma iron concentration was determined colorimetrically with a commercially available kit . Hypoferremia (60–75% decrease of plasma iron level) was evident from 4 to 24 h after IL-1β inoculum. The blood glucose concentration was measured in serum samples by the glucose/glucose oxidase/peroxidase method with commercially available kits  or by biosensor detection with devices for diagnostic monitoring . Overlapping results were obtained in rats and rabbits.Live cells of 6 bacteria/mouse), 24 h before i.p. administration of 15–20 mg/kg of LPS . LPS inoculum was delayed to 24 h after bacteria administration to avoid interference of pre-inoculum. In fact, preliminary experiments showed that intra-peritoneal inoculum of PBS decreased significantly LPS toxicity when administered at shorter times before LPS (data not shown). Mice were observed for 7 days after LPS administration and deaths recorded.LPS-sensitive C3H/HeOuJ mice received an intra-peritoneal inoculum of 0.5 ml PBS alone or containing bacterial suspensions .Results are presented as mean ± SEM. Statistical significance was assesed by two-tailed Student's IL, interleukin; IL-1, interleukin-1; IL-1Ra, interleukin-1 receptor antagonist; IBD, inflammatory bowel disease; LPS, bacterial lipopolysaccharide, AUC, area under the curve; PMN, polymorphonuclear leukocytes; GRAS, generally regarded as safe; GMO, genetically modified organisms.in vivo and pharmacokinetics studies in rats and rabbits, and performed the statistical analysis.SP carried out the PB designed and performed the bioactivity studies.PR designed and performed the microbiological and biochemical work.DB coordinated the bioactivity studies, organised the data, and wrote the manuscript.AT designed and coordinated the entire study.All authors read and approved the final manuscript.


We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4+ T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4+ and higher levels of activated T-cells have greatly reduced levels of FoxP3+CD4+CD25hi T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.A T-cell subset, defined as CD4 Human regulatory T-cells cells (Treg) -- or conventional T cells that have been genetically modified to generate Tregs -- express the HIV-coreceptor CCR5 and are susceptible to HIV infection There is now compelling evidence that a subset of T-cells with regulatory activity suppresses T-cell activation in both mice and humans . Regulat+ T-cells that also express the α subunit of the IL-2 receptor (CD25), which is currently the best marker for identifying these cells   stimulation and by their ability to suppress activation of conventional T-cells , 2002. Tse cells . Howeveress CD25 . In adulNFRSF18) . Upon acNFRSF18) . InteresNFRSF18) .FOXP3 gene carried by the scurfy mouse strain or a knockout of this gene causes a CD4+ T-cell-mediated lymphoproliferative disease characterized by cachexia and multiorgan lymphocytic infiltrates  express CD25 (+ T-cells within the memory subset (CD45RO+) express high levels of CD25 (CD25hi) (+CD25hi subset (+CD25hi (referred to as Treg), CD45RO+CD25low/neg (memory T) and CD45RO−CD25neg (naïve T) cells. Treg cells expressed higher levels of GITR and HLA-DR  A. Previoi subset . We firsd HLA-DR B, consisd HLA-DR . Treg ced HLA-DR B.low/neg memory CD4+ T-cells were labeled with CFSE and stimulated under suboptimal T-cell activation conditions in the presence of unlabeled purified autologous Treg, naïve, or memory T-cells. Coculture with Treg cells significantly slowed the proliferation of TCR-stimulated resting naïve and memory CD4+ T-cells as compared to the coculture with either unlabeled naïve or memory T-cells , or replication-defective viruses pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G.HIV) that encode green fluorescent protein (GFP) as a marker of infection . Treg anFoxP3 cDNA into a HIV-derived vector (HDV) that encodes murine CD24 (mCD24) as a marker  and adult blood (AB), activated through the TCR and transduced with FoxP3-expressing HDV (HDV.FoxP3) or control HDV as described previously .Treg cells constitute less than 1%–2% of total human T-cells see . Althouga marker . CD4+ T-eviously . Express+ cells as described previously  and 24 HIV-infected individuals . Total RNA was then isolated from each subset and FoxP3 mRNA expression was quantified using real-time PCR analysis. In order to normalize for experimental variability, FoxP3 expression of the CD4+CD25hi cells was normalized to GAPDH levels from the same samples and compared to the naïve T-cell subset isolated from the same donor. We found that within HIV-negative subjects there was on average a 49-fold higher level of expression of FoxP3 in CD4+CD25hi cells as compared to naïve T-cells (FoxP3 expression as compared to memory T-cells (data not shown). In HIV-positive subjects FoxP3 expression was only increased a mean of 25-fold in CD4+CD25hi cells. In contrast to healthy donors, we also observed that in about half of the HIV-positive subjects, CD4+CD25hi cells expressed very low to undetectable levels of FoxP3 . Because a portion of HIV-positive individuals contain high levels of activated T-cells, simple cell surface analysis would not be sufficiently reliable to quantify Treg cells in these donors. Therefore, we utilized FoxP3 expression as the most reliable marker that defines Treg cells. To accomplish this, we sorted CD4 T-cells A. The lo T-cells A. There of FoxP3 A. FoxP3 subjects A.+ T-cell percentages and increased levels of activated T-cells. We hypothesized that this hyperactivation may be due to a loss of Treg cells. Therefore, to further evaluate relationships between FoxP3 expression and these parameters in HIV-infected individuals, samples were divided into low FoxP3 expressors (less than 10-fold higher expression in CD4+CD25hi T-cells compared to naïve T-cells, designated FoxP3-low) versus high FoxP3 expressors (greater than 10-fold higher expression compared to naïve T-cells, designated FoxP3-high). HIV-positive subjects with a FoxP3-low profile had significantly lower CD4+ T-cell percentages, while FoxP3-high HIV-positive subjects had CD4+ T-cell percentages comparable to HIV-seronegative subjects (+T-cells (CD4+HLA-DR+) than either HIV-positive subjects with FoxP3-high profiles or HIV-seronegative subjects . These findings demonstrate that a decrease in Treg cells is associated with HIV disease progression and suggest that loss of Treg cells may contribute to increased T-cell hyperactivation.Progressive HIV disease is associated with decreased CD4subjects B. Similasubjects C. Interepressors D. These pressors C. Among +CD25hi T-cells are greatly decreased in a portion of HIV-infected individuals with low CD4 and high activated T-cells, suggesting a loss of Treg cells. We therefore propose that infection and disruption of Treg cells during HIV infection could have a major influence on T-cell homeostasis and immune regulation.In this study we demonstrated that human Treg cells are highly susceptible to HIV infection and that ectopic expression of FoxP3 genetically reprograms conventional naïve T-cells, phenotypically and functionally, into Treg cells. Remarkably, overexpression of FoxP3 also greatly enhances the susceptibility of activated T-cells to HIV infection. Although Treg cells constitute a small subset of the total T-cells in humans (less than 1%–2%) and thus may not be a significant target population for HIV, they appear to have very potent suppressive activity against activation of T-cells . Here wehi subset of CD4+ T-cells. However, it is not clear if Treg cells are the only population represented in the CD25hi subset since they share this phenotype with recently activated T-cells. Indeed, a portion of purified CD25hi cells proliferated and their suppressive function was less efficient as compared to FoxP3-expressing cells (see + memory T-cells), it is conceivable that there is sizable contamination of non-Treg cells in these sorted preparations. In addition, the differences seen in the infection susceptibility of Tregs as compared to FoxP3-expressing cells may also be partly due to presence of non-Treg activated T-cells within the purified cells. Identification of the Treg subset in disease conditions with chronic T-cell activation, such as HIV, is even more problematic because a large proportion of CD4+CD25hi cells possibly represent recently activated T-cells. Availability of large numbers of genetically reprogrammed Treg cells should facilitate the identification of novel markers that can reliably detect human Treg cells.Similar to previous reports, our findings demonstrate that human Treg cells appear to be enriched within the CD25ells see . BecauseOur results clearly demonstrate that, similar to the mouse system, ectopic expression of FoxP3 is sufficient to recapitulate all of the characteristics of Treg cells, including lower cytokine secretion, higher expression of CD25 and GITR, and their suppressive functions. This system has allowed us to generate large numbers of Treg cells, which will be invaluable in characterizing their suppressive function as well as mechanisms of enhanced HIV susceptibility. The ability to genetically manipulate primary T-cells to reprogram them into the Treg phenotype also could have profound implications for preventing graft-versus-host disease, a serious clinical condition that can be manifested following hematopoietic cell transplantation .It is interesting to note that naïve T-cells are more prone to reprogramming into a Treg phenotype than memory T-cells are. This loss in flexibility of reprogramming with a master transcription factor is reminiscent of Th1- and Th2-type T-cell reprogramming with ectopic expression of lineage-specific transcription factors T-bet and GATA-3, respectively . The los+ cells and the reason for high T-cell activation or turnover during HIV infection remains controversial . Indeed, preactivated T-cells require reactivation to render them susceptible to infection after about 1–2 weeks in culture (unpublished results). We do not yet know the mechanisms by which FoxP3 renders activated T-cells more susceptible to infection; however, two possibilities can be considered: (1) FoxP3 may be overcoming innate resistance factor(s) that accumulate in activated T-cells that block HIV infection, or (2) FoxP3 expression may be inducing critical host factors that are required for efficient completion of the HIV life cycle in primary T-cells. It is important to note that since FoxP3 expression enhances VSV-G.HIV single-round infections, it likely affects an early, postentry step in viral replication. Determining the mechanisms by which FoxP3 enhances HIV infection could reveal host factors involved in this process.+CD25+ T-cells are greatly reduced in HIV patients with low CD4+ T-cell percentages and increased T-cell activation suggests that loss of Treg cells may contribute to HIV disease progression. Further prospective studies will be required to unravel the role of this important subset in HIV infection. The ability to genetically reprogram conventional human T-cells to generate Treg cells will also lead the way to identifying unique markers expressed on this population in order to further investigate their status in HIV-infected individuals. Moreover, understanding how FoxP3 enhances HIV infection and programs T-cells into the Treg subset could help in the identification of novel host factors that mediate HIV infection in primary T-cells and decoding the molecular mechanisms by which Treg cells mediate their suppressive function.In summary, our results indicate that both naturally occurring and genetically reprogrammed Treg cells are susceptible to HIV infection and that ectopic FoxP3 expression greatly increases the susceptibility of T-cells to HIV infection. Our finding that FoxP3-expressing CD4n = 11) were adults who were HIV-negative and with no history of chronic viral infections such as Hepatitis B or C. Blood samples from adults with HIV infection (n = 24) were obtained during routine primary care visits at the Comprehensive Care Center, Vanderbilt University Medical School, Nashville, Tennessee, United States. There were no selection criteria based on race or sex. All subjects provided written consent, and the study was approved by the Vanderbilt Institutional Review Board. Continuous variables were compared by a Mann–Whitney U test, and categorical variables by an χ2 test. All significance levels were based on two-tailed tests. Statistical analyses were performed using SPSS, version 12.0 (SPSS, Chicago, Illinois, United States).Healthy subjects , and CD4+CD45RO+CD25hi and CD4+CD45RO−CD25low/neg cells were sorted using flow cytometry . For some experiments, adult CD4+ T-cells were sorted into CD45RO+ (memory) and CD45RO− (naïve) T-cells with anti-CD45RO conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) using AutoMACS (Miltenyi Biotec). Purified resting T-cells were activated by cross-linking with plate-bound anti-CD3 antibody  and soluble anti-CD28 antibody (1 μg/ml, BD Biosciences Pharmingen). The plates were first coated with goat antimouse IgG  followed by either 3 μg/ml anti-CD3 for optimal TCR stimulation or 100 ng/ml anti-CD3 for suboptimal stimulation. The culture medium used in all experiments was RPMI  and was prepared as previously described  were separated from buffy coats of healthy and HIV-positive donors through Ficoll–Hypaque separation . Resting CD4escribed . This puescribed . All cytescribed . Superanescribed .nef gene as previously described (6 ifu/ml for replication-competent viruses and 10–30 × 106 for VSV-G.HIV. T-cells were infected at varying multiplicities of infection (MOI), and infection was quantified by GFP expression using flow cytometry. In some experiments, cells inoculated with virus were centrifuged for 1 h at 2,000 rpm to enhance infectivity, as described by VSV-G.HIV particles were generated as previously described . R5-HIV escribed . TypicalCell division was measured by labeling the T-cells with CFSE (Molecular Probes, Eugene, Oregon, United States). Purified cells were first washed and resuspended in PBS. While vortexing the cells, CFSE was added at a final concentration of 5 μM. The mixture was vortexed for an additional 15 s and incubated at 37 °C for 3 min. Labeling was quenched by addition of 50% fetal calf serum in PBS. Cells were washed one more time with 50% serum PBS, followed by two washes with RPMI-supplemented medium. All CFSE labeling and culturing were performed under dark conditions.T-cells were stained with the relevant antibody on ice for 30 min in PBS buffer containing 2% fetal calf serum and 0.1% sodium azide. Cells were then washed twice, fixed with 1% paraformaldehyde, and analyzed with a FACSCalibur four-color cytometer. Live cells were gated based on forward- and side-scatter properties, and analysis was performed using FlowJo software (Tree Star, Ashland, Oregon, United States). The following antihuman antibodies were used for staining: CD3, CD4, CD45RO, CD45RA, CD25, GITR, HLA-DR, CCR5, CCR4, CCR7, CXCR4, CXCR3, and antimouse CD24, all obtained from PharMingen . Cytokines (IL-2, IL-4, IL-5, and IFNγ) in the supernatants were assayed using a commercially available cytometric bead array (CBA) (BD Biosciences Pharmingen)  and analFoxP3 was PCR amplified from T-cell cDNA with the following primers: FoxP3 forward, 5′-AGATATCCCAGCCATGCCCAACCCCAGGCCTGGCAAG-3′; FoxP3 reverse, 5′-TCAGGGGCCAGGTGTAGGGTTGGAACACCT-3′. The forward primer included an EcoRV restriction site to facilitate cloning. The FoxP3 PCR product was subcloned into a TOPO shuttle vector . Following an EcoRV digest, FoxP3 was ligated into an HDV-encoding mCD24 down stream of an internal ribosome entry site . To synthesize cDNA 100 ng RNA was used . try site . The FoxGAPDH primer mix assay ID Hs99999905_m1; FoxP3 primer mix assay ID Hs00203958_m1.RNA was extracted, as described above, from cells transduced with HDV or HDV.FoxP3 or sorted Treg, naïve, and memory T-cells from HIV-positive and HIV-negative subjects. RNA (100 ng) was used to synthesize cDNA (as described above). Taqman Assays-on-Demand Gene Expression Primers  were used in real-time PCR analyses: FoxP3 or GAPDH primer mix. The reactions were amplified as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min and 65 °C for 1 min. Expression of FoxP3 was normalized to GAPDH expression in each sample.Real-time PCR was performed using the ABI 7700 apparatus (PE Applied Biosystems, Weiterstadt, Germany). The reaction mixtures  contained 2 μl of serially diluted cDNA, 10 μl of Taqman Universal PCR Master Mix (PE Applied Biosystems), and 1 μl of either http://www.ncbi.nlm.nih.gov/) accession number for the gene FoxP3 discussed in this paper is AF277993.The GenBank (


A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle.We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules.We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle. Microtubules undergo striking reorganization during anaphase and telophase. During anaphase, antiparallel, interdigitating microtubules and many associated proteins become organized into discrete bundles in the spindle midzone , the regRecent progress suggested that, in addition to cytokinesis, the spindle midzone might be involved in additional functions. For example, midzone and midbody microtubules are associated with many regulatory proteins apparently unrelated to cortical contraction, such as the DNA replication initiator Orc6 , the inhTo address this possibility, we severed cells at anaphase or telophase by microsurgery, to bypass the normal mechanism of cytokinesis. The functional role of the spindle midzone or midbody in the following cell cycle was then tested by manipulating the position of the microsurgery or by applying pharmacological agents. Using extended time-lapse microscopy, we found that anaphase midzone microtubules play an important role in the progression of the subsequent interphase. However, there was no evidence of the involvement of telophase midbody in cell cycle progression.To investigate if anaphase spindle midzone affects interphase progression in daughter cells, we cut NRK52E cells at early anaphase, as soon as sister chromosomes have separated completely, with a fine glass fiber to form daughter cells with or without spindle midzone structures. Staining of severed daughter cells with antibodies against aurora B Figure , which iA total of eight manipulated cells were followed by extended time-lapse imaging through the following interphase. All the daughter cell with spindle midzone showed cytokinesis-like cortical contraction activities, while the daughter cell without spindle midzone showed no cortical contraction Figure . The dauThe inhibition of interphase progression was affected by the position of severing. When cells were severed near the equator to divide midzone structures between the two daughter cells, both cells showed cytokinesis-like cortical contractions and no effect on the progression of interphase. This observation was repeated with 4 cells. To test the possibility that the duration of interphase was affected by the size of the daughter cell, we took pairs of normally divided daughter cells and cut away a lateral region near the pole from one of the daughter cells. In all 5 cases, both daughter cells entered the subsequent mitosis with a timing similar to that of neighboring unmanipulated cells Figure . TogetheTo test if cortical contraction is the primary determining factor of the rate of interphase progression, we treated cells with cytochalasin D at early anaphase for 10 min then severed them at various positions in the spindle midzone, at a time when midbody started to form and ingression started to appear in control cells. Cytochalasin D was removed 20 min later to ensure that there was no lingering cytokinesis activity upon removal of the drug  see Meth, but remWe then asked if midzone microtubules, or proteins associated with the anaphase spindle midzone, are involved in interphase progression. Treatment of cells with nocodazole for 6–13 min at early anaphase before severing, with or without additional incubation with the drug, caused a significant delay  in the progression of interphase for both daughter cells, as compared to neighboring cells  during anaphase . DegradaOur results suggest that anaphase midzone not only play a role in the stimulation of cytokinesis in cultured cells, but also provide a scaffold for the activation/deactivation of factors essential for the progression of subsequent cell cycle.Normal Rat Kidney epithelial cells  were cultured in Kaighn's modified F12 (F12K) medium supplemented with 10% FBS (JRH Bioscience, Lenexa, KS), 50 U/ml penicillin, and 50 μg/ml streptomycin, on glass chamber dishes as previously described . The celGlass needles for microsurgery were prepared with a David-Kopf Model 700 vertical puller. The tip of the needle was melted and elongated into a fine fiber with a Narishige microforge (Model MF900). Microsurgery of the cells was achieved by carefully lowering a fiber onto the target cell followed by slow dragging with a micromanipulator (Leica, Deerfield, IL).Cytochalasin D (Sigma, St. Louis, MO) were stored at -20°C as 2.5 mM stock in DMSO, and diluted into warm medium before application to cells. We found that treatment of early anaphase cells with cytochalasin D for 30 min completely inhibited cytokinesis even upon removal of the drug, similar to what was reported with dihydrocytochalasin B . Thus, eAurora B-GFP was constructed and transfected into NRK cells as described previously . ImmunofMMH carried out all the experimental work and drafted the manuscript. GS and YLW participated in its design and coordination of the research and edited the manuscript. All authors read and approved the final manuscript.Interphase progression of the daughter cells with and without spindle midzone. An NRK cell was severed at early anaphase to form daughter cells with and without spindle midzone. Subsequently, time-lapse imaging was performed to investigate if anaphase spindle midzone was involved in interphase progression. The daughter cell with spindle midzone showed a cytokinsis-like cortical contraction and entered into the subsequent mitosis 11 h 23 min after microsurgery, while the daughter cell without spindle midzone showed no cortical contraction and entered into mitosis 23 h 8 min after microsurgery.Click here for fileInterphase progression of the daughter cells with and without midbody. An NRK cell was severed at telophase to form daughter cells with and without midbody. Subsequently, time-lapse imaging was performed to investigate if midbody was involved in interphase progression. The daughter cell with midbody entered into the subsequent mitosis 7 h 35 min after microsurgery, while the daughter cell without midbody entered into mitosis 9 h 25 min after microsurgery.Click here for file


Many three-dimensional (3D) images are routinely collected in biomedical research and a number of digital atlases with associated anatomical and other information have been published. A number of tools are available for viewing this data ranging from commercial visualization packages to freely available, typically system architecture dependent, solutions. Here we discuss an atlas viewer implemented to run on any workstation using the architecture neutral Java programming language.We report the development of a freely available Java based viewer for 3D image data, descibe the structure and functionality of the viewer and how automated tools can be developed to manage the Java Native Interface code. The viewer allows arbitrary re-sectioning of the data and interactive browsing through the volume. With appropriately formatted data, for example as provided for the Electronic Atlas of the Developing Human Brain, a 3D surface view and anatomical browsing is available. The interface is developed in Java with Java3D providing the 3D rendering. For efficiency the image data is manipulated using the Woolz image-processing library provided as a dynamically linked module for each machine architecture.We conclude that Java provides an appropriate environment for efficient development of these tools and techniques exist to allow computationally efficient image-processing libraries to be integrated relatively easily. Three-dimensional 3D) images are now commonplace in biomedical research. Techniques for direct capture of 3D data are widespread and new techniques are becoming available, ,2 to comD images For the expert viewer, digital re-sectioning is sufficient for data-analysis but for others, panning through the volume at non-standard angles leads to disorientation. In addition if used in conjunction with atlas information in which the histology images are segmented in terms of the recognisable tissues, the building of a 3D view of the tissue/anatomical components is very difficult, particularly when learning the anatomy. This orientation and structural visualisation problem is solved by using 3D visualisation of the underlying tissue coupled with interactive feedback of the section location within the volume.The basic structure of the JAtlasView is therefore a combination of a 3D feedback window with a number of section views. Each section view is independent and feedback is provided by displaying the position of the section within the 3D volume either as a simple polygon indicating the plane of section or as a full grey-level image, displayed appropriately in 3D. In addition each section will display the intersection with all other sections currently being viewed.In this short note we describe the structure of the software and the functionality of the interface. This application is directed to the use of the EADHB and EMAP atlases and for browsing 3D grey-level data. In the first instance the data is formatted as a Woolz image structure , tools aThe software design has been developed to meet a number of code requirements:• portability to all major architectures – Unix/X11, Microsoft Windows and Macintosh,• fast and efficient image processing, compatible with existing formats and interfaces,• freely available code and modular design so display elements and functionality can be easily included in other applications and• the user-interface should be mappable to the "look and feel" of the specific machine window system.The portability and user-interface behaviour requirements are satisfied by using Java as the language and environment for the user interface level. For image processing we have adopted the ANSI standard C image processing library Woolz. This already includes the required functionality for calculating and manipulating section views through 3D voxel images and is open-source software.A potential problem with Java is that it can be very inefficient for heavy numerical work (such as image processing) and the effort required to port existing libraries (for example Woolz is 185 K lines of code) to Java is too high. To solve this we use the tools within Java for accessing "native" code so that the computational work is undertaken in C. The management and coding of the interface is potentially time-consuming and prone to error with any small change in the C code requiring complementary effort to modify the native interface code. We have addressed this problem by implementing an automated method which will build the interface directly from the C-library header files. By adopting a standard convention for function prototyping it is possible to use a parser generator, javacc to buildTwo other key choices have been made in the design of the code structure. The first is that the 3D visualisation and feedback should be developed at a level independent of the underlying hardware within an environment that allows a high level of abstraction of the 3D view. The java 3D extension to the core Java environment provides such a model and we have adopted this as standard. Java 3D is available for all Java 2 platforms.CVS[gmake for compilation. The interfaces are developed using Borland JBuilder[The second key choice is that the software will be delivered using Java Webstart. This isCVS for vers JBuilder or a sta JBuilder.Help is provided in two ways, the first is a simple popup "balloon" help on mouse-over and as a series of help files arranged using JavaHelp which prmodel-view-controller (MVC) design pattern [java-bean, that components should meet to guarantee the MVC behaviour and enable easy re-use in other applications using CASE tools. We have adopted this standard for the JAtlasViewer application so that individual interface elements, e.g. the section panel or even our extended view of the slider, can be used simply and conveniently in other code.Java is now widely used and the first choice for new applications that require portability across machine architectures. It is a strict object-oriented language and interfaces adhere to the  pattern . Java alThe user interface, shown in figure With appropriate data, the JAtlasViewer will import a mapped "anatomy". This is in two parts, a hierarchy of terms and a set of "domains" linked to specific terms in the hierarchy. The domains are 3D binary images which identify the region of space or set of voxels within the grey-level image associated with that term. The anatomy will then be used to provide feedback within the section views. These anatomy options, the controls for the section views and the main window options are discussed below.When the application is invoked a top-level dialog is presented to the user. Before anything can be displayed the user must select a grey-level 3D image. Currently this must be formatted as a Woolz image, but converters for many 3D formats are available from the EMAP web site. Once read in, the bounding box of the 3D voxel image will be displayed in the main window along with a surface representation of the data if available. This 3D view can be manipulated interactively using the cursor to provide views from arbitrary orientations and positions.The menu options of the primary window are:Commands to open image data, save views, save and restore settings, recent file-list and quit.Select a section view through the voxel model. A new window will display one of the pre-set sections which are transverse, frontal and sagittal planes if the image model is appropriately aligned. Each of these can be set to display views at arbitrary orientations and locations within the 3D volume image.If the voxel model data is configured with a set of anatomical regions, these can be selected from the menu and displayed in the section views. For the EADHB and EMAP atlases the menu hierarchy corresponds to the HUMAT and EMAP anatomy ontologies.Options to control the 3D visualisation in the main window, toggle the visibility of 3D surface, bounding box and intersection lines, display the focus section and selected anatomy.Preset 3D orientations to provide standard viewing directions.On-line help menu.The 3D view window displays the bounding box of the opened volume and a transparent view of the embryo surface. This surface is pre-determined and stored in the visualization toolkit (VTK) format.If an anatomy hierarchy and associated data files are provided then an additional window will allow browsing through the ontology and selection of components for display both in the section views and the 3D view window. As for the embryo surface, the surface models are pre-calculated and stored in VTK format. The data layout recognized by the JAtlasViewer is described in detail on the EMAP web-site.pitch, yaw and roll respectively. These viewing controls are hidden by default.Each Section View is displayed in its own Section Viewer, either inside the main window (Microsoft Windows style) or in an independent external window. Section Viewers are Java components that can be easily imported into other applications. The primary viewing control is to move the view plane-parallel through the image volume as a form of "digital microtome" with section thickness determined by the underlying resolution of the 3D image, i.e. moving the microtome by a single step will move to the next voxel in the stack. The assumption is that once the section orientation has been determined the typical use will be to explore the volume in this fashion. The section position is determined by the "distance" parameter which is the voxel distance from the fixed point (by default in the centre of the bounding-box). Section orientation is selected by setting a number of view-angles. These control the view-direction which is perpendicular to the view-plane. We use the standard viewing angles defined by  which arView-mode: options for automatic roll determination in terms of the pitch and yaw values.In addition to the primary view-direction controls there are options to assist navigation. These are Fixed-point: select the fixed point used as the centre of rotation. The effect of setting this is to keep that voxel in view for all view-directions provided the "distance" is zero.Fixed-line: set a second fixed-point and constrain the view so that both fixed points remain in the section. The effect of this is to reduce the degrees of freedom to a single parameter of rotation around the line between the two points.The remaining controls for each section view are to set the feedback options including between section views, between the section view and the 3D view and to allow saving of the view and its settings (view parameters). The within view and between views feedback options are provided by the "Show" menu. This provides toggle controls to enable:• cursor position in the reference image coordinate space and image grey-value to be displayed,• line of intersection with section views. If two views intersect then the line of intersection is displayed in the appropriate colour,• anatomy feedback – shows the domain and name of the anatomical component under the current cursor position,• visible fixed point,• visible fixed line.The 3D view can provide feedback of the viewed sections in terms of the 3D volume. For most users these are important aids to understanding the position and direction of the viewed section. Most publications adhere to a convention for displaying section images, with this interface it is possible to view section data at any orientation and direction, i.e. depending on the view-direction the section may appear "reversed", so positional and directional feedback is critical. The positional feedback is provided in a number of forms but all indicate the intersection of the viewed plane with the bounding-box of the reference image. The most informative choice is to use texture mapping to render the grey-level image of the section into the 3D view. This is computationally expensive and so two other options are provided. These use the intersection polygon between the section plane and bounding box, either as-is, or filled with solid colour. The directional feedback is optional and provided by an arrow displayed in the 3D view.atlases. These comprise a grey-level  reference image and a set of domains or regions which are associated with terms in a text hierarchy. For a geographic atlas these would correspond to the physical geography and the areas associated with individual countries. The hierarchy would then list the country names, perhaps under continents and split into counties. For EADHB and EMAP the reference image is the voxel reconstruction of the embryo and the domains are delineated anatomical components. The hierarchy of terms are the corresponding anatomy ontologies [Anatomy Manager  and the final part of the name is capitalised. An asterisk following a name indicates that this is an atomic component, referred to in the anatomy menu as a (domain). Anatomical components are selected from the anatomy menu using a left mouse click. Higher level components or structures may also be selected from the anatomy menu using a right mouse click or a combination of the shift key and (left) mouse click. The anatomy name may be scrolled by dragging the mouse left or right inside the text box.The delete is a toggle control which removes an anatomy component from the table.The visibility toggles select whether a component is displayed in the section views and in the 3D feedback window. This fine control helps the process of analysis and allows the user to build up a visualization showing all or parts of the anatomy. The 3D images are in widespread use in medical and biological research and there are a large number of options to view this data, but many of these are commercial and expensive, and are architecture and operating system dependent. More recently atlases and spatially mapped databases in biomedicine have been developed and whilst these packages can provide solutions for browsing this data we believe a simple, free-to-use, open-source and architecture-neutral solution provides a useful tool for biological research and teaching. The JAtlasViewer is intended to fill this requirement. The viewer provides the browsing functionality to locate and display arbitrary sections through the data with simultaneous 3D display. The JAtlasVIewer can also read and display a full anatomy atlas.The JAtlasViewer is programmed in Java. The 3D programming technology is Java3D, which is a wrapper to the OpenGL or DirectX libraries. The Java and Java3D runtime environment are freely available from the Sun Microsystems web site and in most systems Java is pre-installed. These techniques minimize the coding work and developing time. The file size of the JAtlasViewer is less than 1.5 MB. Java WebStart manages the deployment, installation, upgrade and launch via a simple click on a html page link or an icon in the WebStart application. It is portable to any operating system to which Java has been ported and is currently available for Windows, Linux, Solaris and Mac OS.The JAtlasViewer design is of reusable and extensible components. Based on the viewer a 3D tie-point collector for capturing 3D to 3D correspondences, and an atlas viewer that can also import gene expression data, have been developed.• Project name: The Mouse Atlas Project• Project home page: • Application download: • Operating system(s): Solaris, Linux, Mac OSX, MS Windows.• Programming language: Java, ANSI C.• Other requirements: Java 1.4, JavaDoc, Java 3D.• License: GNU GPL• Any restrictions to use by non-academics: NoneAuthors GF and NB undertook the main Java development and implementation, BH and RB develop and maintain the Woolz image processing library and BH implemented the automatic generation of the JNI. DD, JK, MS and SL all contributed to the design and testing of the interface and the preparation of the Atlas data for use with the tool.


Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking.Septin2 filaments are associated with actin fibres, but Septin2 is not associated with actin at the leading edge of moving cells or in ruffles where actin is highly dynamic. Rather, Septin2 is spatially segregated from these active areas and forms O- and C-shaped structures, similar to those previously observed after latrunculin treatment. FRAP experiments showed that all assemblies formed by Septin2 are highly dynamic with a constant exchange of Septin2 in and out of these structures, and that this property is independent of actin. A combination of RNAi experiments and expression of truncated forms of Septin2 showed that Septin2 plays a significant role in stabilising or maintaining actin bundles.We show that Septin2 can form dynamic structures with differing morphologies in living cells, and that these morphologies are dependent on the functional state of the actin cytoskeleton. Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and are consistent with the model proposed by Kinoshita and colleagues, that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover. Septin-requiring processes include cytokinesis, polarity establishment, cell cycle checkpoints and formation of a diffusion barrier in yeast , as wellin vitro ,7. How tin vitro [in vivo [Septin2 (formerly known as Nedd5) is the best-characterised member of the septin family so far. It is ubiquitously expressed and belongs to the acidic subgroup of the septin family consisting of a short N-terminus, a conserved GTPase domain and a coiled coil structure at the C-terminus . Septin2in vitro  and also[in vivo . Kinoshi[in vivo . Microin[in vivo ,13.in vitro bundling assay showed that the interaction between actin bundles and recombinant Septin2/Septin6/Septin7 can be mediated by the bundling protein anillin [in vitro results suggest a mechanism to account for the recruitment of Septin2 to the actin contractile ring during cytokinesis, it is unlikely that anillin has the same function in interphase cells. Anillin is sequestered in the nucleus in interphase cells [In interphase cells Septin2 co-localises with actin bundles, and disruption of the actin cytoskeleton by latrunculin or cytochalasin perturbs Septin2 distribution, inducing curved or circular Septin2-containing assemblies ,14. Reduse cells  and the in vivo approaches. Both knock down of Septin2 expression and a specific Septin2 truncation mutant resulted in loss of visible actin fibres or bundles. Expression of dominant-negative mutants of the actin-regulating Rho-GTPases RhoA, Rac1 and CDC42 caused re-organisation of both cortical actin and Septin2. Significantly, in ruffling and migrating cells we observed wholesale redistribution of Septin2 into ring-like structures with morphology and dynamics highly similar to those previously observed upon depolymerisation of actin filaments with latrunculin. We propose that Septin2 is required for actin bundling, and that global re-organisation of the actin cytoskeleton in migrating or ruffling cells triggers concomitant re-organisation of Septin2 into a distinct functional state.Here we characterise the mutual inter-dependence of the actin cytoskeleton and Septin2 using a range of in vivo. As a first step, indirect immunofluorescence was used to characterise the distribution of these molecules in NRK cells. In interphase cells Septin2 had a filamentous and granular appearance, and co-localised with actin bundles [We sought to understand the functional relationship between actin cytoskeleton and Septin2  bundles . The int bundles . Septin2 bundles , disrupt bundles . This de bundles . For SepIn order to test directly the requirement for Septin2 function in maintaining the actin cytoskeleton, we performed RNAi to knock down Septin2 expression in cells. NRK and Hela cells were transfected separately with two different siRNAs based on the Septin2 sequence. Both siRNAs produced equivalent effects, and control siRNAs produced none of the effects described below. 48 hrs after transfection cell morphology was severely compromised and the overall amount of cells was reduced to 43 ± 11.7% of control levels. 70–90% of the remaining cells showed a decreased Septin2 staining by immunofluorescence. There was no indication of selective arrest of the cell cycle at mitosis or cytokinesis, implying that Septin2 is required for correct cell morphology and adhesion or viability during interphase. Western blot analysis of whole cell homogenate revealed a 60% reduction of Septin2 level  resulted in the formation of large polymeric structures Fig. . UntaggeAnother way of determining the extent of interdependence between actin and Septin2 is to perturb actin organisation by interfering with regulators of actin organization. Rho GTPases are such regulators and have diverse effects on the actin filament system ,19. RhoABoth actin depolymerisation and expression of Rho-family-GTPase mutants are non-physiological perturbations and functional significance of resultant changes in Septin2 distribution was unclear. We therefore sought a more physiological situation where parallel re-organisation of actin and Septin2 could be assayed. In NRK cells migrating into an experimentally wounded monolayer actin is concentrated in the leading-edge lamellipodia and ruffles characteristic of many moving cells Fig.  21]. Se. Se21]. To gain more insight into Septin2 dynamics we performed fluorescence recovery after photobleaching (FRAP). In a FRAP experiment fluorescent molecules are irreversibly bleached in a small area of the cell (= region of interest, ROI). Subsequent diffusion of surrounding non-bleached molecules into the bleached area leads to recovery and is monitored over time. We used full length YFP-Septin2 for bleaching experiments and investigated the dynamics of GFP-actin in parallel (Clontech). YFP-Septin2 had the same distribution as endogenous Septin2 in interphase and dividing cells and it did not alter the organisation of endogenous Septin2 . First wNext we looked at the dynamics of the Septin2 positive ring-like structures formed in the cell body of ruffling cells. Time-lapse movies of these cells revealed a very active behaviour of Septin2 and a relationship between Septin2 filaments and rings. For example, as shown in Fig. Next we investigated whether Septin2 rings induced by latrunculin have equivalent properties to those seen in ruffling or migrating cells. Septin2 rings formed upon latrunculin treatment also recovered after photobleaching, with comparable dynamics : 2. That Septin2 when not associated with actin forms rings and ring-like structures instead of filaments . Bleaching experiments clearly showed the constant exchange of Septin2 in and out of the assemblies as well as in and out of actin-dependent Septin2 filaments. The function of actin-independent Septin2 assemblies, however, is still elusive. Since they seem to be freely localised in the cytosol and are not associated with membrane structures (unpublished observations), they might represent storage containers of Septin2. The dynamic behaviour of Septin2 explains how cells can adjust Septin2 function to different needs. The identification of further proteins involved in this process (e.g. GAPs, GEFs, bundling proteins) is necessary for molecular characterisation of the interplay between Septin2 and actin.It has been shown  fashion . Here, wnts Figs , 6. This spirals . In contOur data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and confirm the physiological relevance of the model proposed by Kinoshita and colleagues , that Se®FL phallacidin and Alexa Fluor ® 568 phalloidin for immunofluoresence were obtained from Molecular Probes. Anti-Endoplasmic Reticulum Protein 72 (Anti-Erp72) was from Calbiochem. Myc-constructs of GDP-locked RhoA, Rac1 and CDC42 and the anti-rac monoclonal antibody were kindly provided by H. Mellor. pEGFP-actin was obtained from Clontech. Secondary antibodies used were as follows: Donkey Anti-Rabbit IgG Cy™3 conjugated (Jackson Immuno Research Lab.), Donkey Anti-Mouse IgG Cy™5 conjugated (Jackson Immuno Research Lab.) and Alexa Fluor ® 488 goat anti-mouse IgG1 (Molecular Probes) for immunofluorescence. IRDye 800 donkey anti-rabbit IgG (Rockland) and Alexa Fluor ® 680 goat anti-mouse IgG (Molecular Probes) for immunoblotting.Anti-Septin2 polyclonal antibody was a gift from M. Kinoshita. Anti-human vinculin monoclonal antibody (clone hVIN-1), anti-c-myc monoclonal antibody (clone 9E10) and anti-α-Tubulin monoclonal antibody (clone DM1a) were all from Sigma. Anti-beta actin monoclonal antibody (AC-15) for immunoblotting was from abcam, BODIPYTo generate full length and truncated YFP-Septin2-constructs, PCRs of Septin2 image clone (Clone Id: 548005, from HGMP, Hinxton/UK) were performed using the following primers: 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) for full length YFP-Septin2-PB/G/CC (nucleotides 1–1081); 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTAGCCTCTCTTGAGTCTCTC (antisense) for YFP-Septin2-PB/G (nucleotides 1–922); 5'-GCGCTCGAGTGTCTAA GCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTACACCATCAGTGTGAACTC (antisense) for YFP-Septin2-PB (nucleotides 1–127); 5'-GCGCTCGAGAGTTCACACTGATGGTGGT (sense) and 5'-ATCCCGGGTTACACGTGGTGCCCGAGAGC (antisense) for YFP-Septin2-G/CC (nucleotides 113–1081). The fragments were cloned in pEYFP-C1 digested with Xho1 and Xma1. To generate untagged full length Septin2 5'-GCGCTCGAGATGTCTAAGCAACA GCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) were used in the PCR reaction. The fragment was cloned in SNAG4M cut with Xho1 and Xma1. All clones were confirmed by DNA sequencing.2. To induce the formation of ruffles in NRK cells coverslips were coated with 0.01% poly-D-lysine for 5 min, washed with PBS and air dried before cells were seeded. A wound assay was used to investigate distribution of Septin2 in moving cells. Briefly, NRK cells were grown until confluency and a wound was scratched in the monolayer using a tip. Two hours later cells were fixed and processed for immunofluorescence. To disrupt the actin cytoskeleton or microtubules cells were incubated for 30 min in DMEM containing 150 nM Latrunculin B (Molecular Probes) or 10 μM nocodazole (Sigma), respectively. Plasmid transfections were carried out with Fugene6 (Roche).NRK cells were grown using standard techniques in DMEM, 10% FCS, 1% Penicillin at 10%CO2, 4% PEG 8000) for 20 min, permeabilised with 0.2% saponin/10%FCS in either PBS or microtubule-stabilisation buffer for 10 min and subsequently incubated with primary and secondary antibodies.For immunofluorescence cells were fixed with 2% Formaldehyd in either PBS or microtubule-stabilisation buffer (0.1 M Pipes, pH 6.9, 2 mM EGTA, 2 mM MgClTwo siRNAs with the following sequences: 5'-AAAGGACATGAATAAAGACCA (sense), and 5'-AAGTGAATATTGTGCCTGTCA (sense) were chosen to target nucleotides 939–957 and 521–539 of Septin2, respectively, and were supplied by Dharmacon Research Inc. siRNAs were annealed to make siRNA duplexes according to the manufacture's protocol and transfections were carried out using Oligofectamine (Invitrogen). To check for specificity of knock down we used siRNA duplexes targeting caveolin 1  and rhomD, which indicates the time at which half of the fluorescence has recovered.All images and movies were obtained using BioRad Radiance and Zeiss LSM510 confocal microscopes equipped with standard filter sets and laser lines for the detection of YFP, Cy2, Alexa Fluor 488, BODIPY, Cy3 and Cy5. Live cell microscopy was carried out at 35°C in imaging medium (DMEM without phenol red, 10%FCS, 50 mM HEPES pH 7.2). Photobleaching experiments were performed with the confocal zoom set to 3 and the confocal pinhole set to 2–4 Airy units. Bleaching of actin or Septin2 filaments was carried out with a 40X 1.3 NA objective lens. A box of interest was bleached using 35 scans with the 488 and 514 laser line at full laser power. For photobleaching of ring structures a 63X 1.4 objective lens was used and a ring of interest was bleached using 25 scans with the 514 laser line at full laser power. Pre- and post-bleach images were monitored at low laser intensity. Fluorescence recovery in the bleached region and the overall fluorescence in the whole cell during the time series were quantified using the Zeiss LSM software. After subtracting the background (= mean fluorescence intensity in the bleached region after bleach) the ratio between mean fluorescence intensity of the bleached region and the mean fluorescence of the whole cell was expressed as a percentage of the pre-bleach ratio of these values. These normalised data were fitted to a single exponential curve using the PRISM software (GraphPad Software Inc., San Diego) to derive amount of recovery and characteristic diffusion time tGFP, green fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interference RNA; RNAi, RNA interference; ERP72, endoplasmic reticulum protein 72; FRAP, fluorescence recovery after photobleaching; ROI, region of interest; GAP, GTPase activating protein; GEF, GTP exchange factor.KS carried out all the experimental work and drafted the manuscript. BJN conceived of the study, participated in its design and edited the manuscript.Photobleaching of YFP-Septin2 ring-like structures formed in the cell body of ruffling cells. Ruffling NRK cells were transfected with YFP-Septin2. The YFP-Septin2 ring in the middle was photobleached and recovery monitored over time. Movie covers 200 s (also see Fig. Click here for filePhotobleaching of YFP-Septin2 rings upon latrunculin treatment. NRK cells transfected with YFP-Septin2 were treated with Latrunculin B for 20–25 min before photobleaching. A ring in the upper right part of the cell was photobleached and recovery monitored over time. Note recovery seems to occur from a structure in the background. Movie covers 200 s.Click here for file


In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.It has recently been suggested that RhoA plays an important role in the enhancement of the CaOvalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.+-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca2+ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca2+ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca2+ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K2+ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.These findings suggest that the augmentation of Ca Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive diseases, including bronchial asthma. This abnormality is an important symptom of the disease, although the pathophysiological variations leading to the hyperresponsiveness are unclear now. Several mechanisms have been suggested to explain the airway hyperresponsiveness (AHR), such as alterations in the neural control of airway smooth muscle , increas2+ concentration in myocytes. Recently, additional mechanisms have been suggested in agonist-induced smooth muscle contraction by studies in which the simultaneous measurements of force development and intracellular Ca2+ concentration, and chemically permeabilized preparations in various types of smooth muscles were used. It has been demonstrated that agonist stimulation increases myofilament Ca2+ sensitivity in permeabilized smooth muscles of the rat coronary artery [2+ sensitization has been suggested by many investigators [2+ sensitization in smooth muscle contraction has been reported in experimental animal models of diseases such as hypertension [Smooth muscle contraction including airways is mainly regulated by an increase in cytosolic Cay artery , guinea y artery , canine y artery  and rat y artery . Althougtigators . Moreovertension -13, corortension -16 and crtension -19 vasos2+ sensitization in the augmented ACh-induced contraction of BSM was demonstrated in this animal model of AHR.Here, we show an increased acetylcholine (ACh)-induced contraction of bronchial smooth muscle (BSM) isolated from repeatedly ovalbumin (OA)-challenged BALB/c mice, which have been reported to have in vivo AHR . A partiet al. [Male BALB/c mice  were used. All experiments were approved by the Animal Care Committee at the Hoshi University (Tokyo, Japan). Preparation of a murine model of allergic bronchial asthma, which has in vivo airway hyperresponsiveness (AHR), was performed as described by Kato et al. . In briei.p.) anesthesia. Then the airway tissues under the larynx to lungs were immediately removed. About 3 mm length of the left main bronchus (about 0.5 mm diameter) was isolated and epithelium was removed by gently rubbing with keen-edged tweezers [2 2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2 and glucose 10.0. The buffer solution was maintained at 37°C and oxygenated with 95% O2-5% CO2. The BSM responsiveness to exogenously applied Ca2+ in acetylcholine (ACh)-stimulated or high K+-depolarized muscle was determined as previously [2+-free solution containing 10-6 M nicardipine with the following composition (mM); NaCl 122.4, KCl 4.7, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, glucose 10.0 and EGTA 0.05. Fifteen min later, 1 mM ACh was added and, after attainment of a plateau  response to ACh, a cumulative concentration-response curve for Ca2+ (0.1–6.0 mM) was made. A higher concentration of Ca2+ was added after the response to the previous concentration reached a plateau. In another series of experiments, bronchial smooth muscles were depolarized with 60 mM K+, instead of ACh, in the presence of 10-6 M atropine and in the absence of nicardipine in the Ca2+-free solution. All these functional studies were performed in the presence of 10-6 M indomethacin. The concentration of indomethacin had no effect both on baseline tension and on the ACh- and high K+-induced constrictions of BSMs (data not shown).Twenty-four h after the last antigen challenge, the mice were sacrificed by exsanguination from abdominal aorta under urethane (1.6 g/kg, tweezers . The reseviously . In brie2+ sensitization of BSM contraction, permeabilized BSMs were prepared as described previously [2+ ionophore A23187 (10 μM, Sigma) at room temperature in relaxing solution. Relaxing solution contained: 20 mM PIPES, 7.1 mM Mg2+-dimethanesulfonate, 108 mM K+-methanesulfonate, 2 mM EGTA, 5.875 mM Na2ATP, 2 mM creatine phosphate, 4 U/ml creatine phosphokinase, 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 1 μg/ml E-64 (pH 6.8) containing 10 μM A23187. Free Ca2+ concentration was changed by adding an appropriate amount of CaCl2. The apparent binding constant of EGTA for Ca2+ was considered to be 106 M-1 [2+ sensitization, the α-toxin-permeabilized muscle strips were treated with Clostridium botulinum C3 exoenzyme  in the presence of 100 μM NAD for 20 min at room temperature.To determine the change in Caeviously  with min2, 2 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 1% Triton X-100 and 1% sodium cholate. The tissue homogenate was then centrifuged (3,000 g, 4°C for 15 min) and the resultant supernatant was stored at -85°C until use. To determine the level of RhoA protein in BSMs, the samples  were subjected to 15% SDS-PAGE and the proteins were then electrophoretically transferred to a PVDF membrane. After blocking with 3% gelatin, the PVDF membrane was incubated with polyclonal rabbit anti-RhoA antibody . Then the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG , detected by an enhanced chemiluminescent system (Amersham Biosciences, Co.) and analyzed by a densitometry system. Thereafter, the primary and secondary antibodies were stripped and the membrane was reprobed by using monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase  to confirm the same amount of proteins loaded.Protein samples of BSMs were prepared as previously . In brei-3 M for 10 min) and were quickly frozen with liquid nitrogen. The tissues were then lysed in lysis buffer with the following composition (mM); HEPES 25.0 (pH 7.5), NaCl 150, IGEPAL CA-630 1%, MgCl2 10.0, EDTA 1.0, glycerol 10%, NaF 25.0, sodium orthovanadate 1.0 and peptidase inhibitors. Active RhoA in tissue lysates (200 μg protein) was precipitated with 25 μg GST-tagged Rho binding domain , which was expressed in Escherichia coli and bound to glutathione-agarose beads. The precipitates were washed three times in lysis buffer, and after adding the SDS loading buffer and boiling for 5 min, the bound proteins were resolved in 15% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-RhoA antibody as described above.The active form of RhoA, GTP-bound RhoA, in BSMs was measured by RhoA pull down assay. In brief, bronchial tissues containing the main and intrapulmonary bronchi were isolated as described above. The isolated bronchial tissues were equilibrated in oxygenated Krebs-Henseleit solution at 37°C for 1 hr. After the equilibration period, the tissues were stimulated by ACh (10Phosphorylated proteins were detected by using the fluorescent Pro-Q-Diamond dye (Molecular Probes, Eugene, OR, USA), which can directly detect phosphate groups attached to tyrosine, serine or threonine residues in gels . In briet-test, Bonferroni/Dunn's test or two-way analysis of variance (ANOVA).All the data are expressed as the mean ± S.E. Statistical significance of difference was determined by unpaired Student's 2+-free condition (in the presence of 10-6 M nicardipine and 0.05 mM EGTA), ACh (10-3 M) generated a transient phasic contraction in all BSM preparations used. The generated tension of BSM from the repeatedly OA-challenged mice (69 ± 12 mg, N = 6) was significantly greater than that from the sensitized control animals . The concentration of nicardipine used in the present study completely blocked high K+ (10–90 mM)-induced BSM contraction in Ca2+ (2.5 mM) containing normal Krebs-Henseleit solution (data not shown), indicating that voltage-dependent Ca2+ channels were completely blocked in this condition.Under Ca2+ was measured. Figure 2+ of murine BSMs that were preincubated with nicardipine (10-6 M) and ACh (10-3 M) under Ca2+-free (0.05 mM EGTA) condition. Addition of Ca2+ induced a concentration-dependent BSM contraction in both the sensitized control and OA-challenged groups. The contractile response to Ca2+ of the ACh-stimulated BSMs from the repeatedly OA-challenged mice was markedly augmented as compared to that from the sensitized control animals. By contrast, no significant difference in the response to Ca2+ of BSMs depolarized with 60 mM K+ (in the absence of nicardipine and presence of 10-6 M atropine) was observed between groups  concentration-response curve determined in normal Krebs-Henseleit solution (2.5 mM Ca2+) was significantly shifted upward in BSMs from the OA-challenged mice as compared with that from the sensitized control animals, whereas no significant difference in the contractile response induced by isotonic high K+ (10–90 mM) was observed between groups (data not shown).The tension returned to baseline level within 5 min after the ACh application, and then the contraction induced by cumulatively administered Caups Fig. . Likewis2+ (pCa = 6.5, 6.3, 6.0, 5.5 and 5.0) induced a concentration-dependent reproducible contractile response, indicating successful permeabilization. In the α-toxin-permeabilized BSM, no significant difference in the Ca2+ responsiveness or the maximal contractile response induced by pCa 5.0 (Emax) was observed between the sensitized control (pEC50[Ca2+ (M)] = 5.67 ± 0.04, Emax = 26.7 ± 1.2 mg; N = 6) and repeatedly OA-challenged (pEC50[Ca2+ (M)] = 5.78 ± 0.15, Emax = 22.8 ± 5.9 mg; N = 6) groups. In both groups, when the Ca2+ concentration was clamped at pCa 6.0, application of ACh (10-5–10-3 M) in the presence of GTP (10-4 M) caused a further contraction, i.e., ACh-induced Ca2+ sensitization, in an ACh concentration-dependent manner . However, the ACh (10-3 M, in the presence of 10-4 M GTP)-induced Ca2+ sensitizing effect was inhibited by treatment with C3 in both the sensitized control and OA-challenged groups . In the Pro-Q Diamond dye-stained gels, there were several positive bands, i.e., phosphorylated proteins [bottom panel). The ACh-induced phosphorylation of MLC in BSMs of OA-challenged mice was markedly augmented as compared with those of control animals. A Pro-Q Diamond dye-positive 140 kD band probably corresponding to MYPT1, i.e., phosphorylated MYPT1, was also found in the ACh-stimulated BSM samples and was increased in the OA-challenged group (data not shown).Figure proteins , in the in vivo AHR accompanied by increased IgE production and pulmonary eosinophilia has been demonstrated in the actively sensitized and repeatedly OA-challenged BALB/c strain of mice [+-depolarized (without receptors stimulation), intact muscle strips of the repeatedly OA-challenged mice . These findings suggest that the C3-sensitive, RhoA-mediated Ca2+ sensitization might be augmented in BSMs of the OA-challenged AHR mice. Indeed, the current study also demonstrated a marked increase in the expression and activation of RhoA protein in BSMs of the AHR mice  of α-toxin-permeabilized BSMs was observed between groups (see Result section), indicating that the contents of typical contractile elements such as calmodulin, myosin light chain  BSMs induced by high K+ depolarization was not changed after OA challenge also support our speculation. Thus, the baseline Ca2+ sensitivity of contractile elements themselves in BSM cells is unlikely to change in AHR.In the present study, no significant difference in the Ca2+ itself, the ACh-stimulated contraction of intact BSM strips from the OA-challenged mice was significantly augmented as compared to that from the sensitized control animals  and receptor-operated (nicardipine-insensitive) Ca2+ channels [2+ mobilization in BSM cells. However, the possibility might be denied by the current result that the ACh-induced contraction of α-toxin-permeabilized BSMs from the OA-challenged mice was significantly augmented as compared with that from the control animals even at a constant Ca2+ concentration  of up-regulation of RhoA in OA-challenged BSMs is not known here, inflammatory cytokines such as tumor necrosis factor-α [One of the important findings in the present study is that the C3-sensitive, RhoA-mediated Cace Figs. . Thus, tfactor-α , which ifactor-α ,35. It i2+ level was within normal level even in the hyperresponsive BSMs [2+ sensitization, although the mechanism(s) for activation of RhoA by ACh is still unclear. If RhoA proteins are activated by receptors other than muscarinic receptor, it might account for the 'non-specific' AHR, which is a common feature of allergic asthmatics. Indeed, the BSMs of the OA-challenged mice also have hyperresponsiveness to endothelin-1 [An increase in responsiveness to muscarinic agonists of airway smooth muscle has been demonstrated in animal models of AHR ,22,36,37ive BSMs ,30. Takethelin-1 , which hthelin-1 .An upregulation of RhoA/Rho-kinase associated with the augmented smooth muscle contractility has also been reported in rat myomertium during pregnancy ,43, arte2+ sensitization in murine BSM contraction. An augmentation of the Ca2+ sensitizing effect, probably by the upregulation of RhoA protein, might be involved in the enhanced BSM contraction observed in the antigen-induced AHR in mice.In conclusion, the current study demonstrated an ACh-induced, RhoA-mediated CaYC conceived of the study, participated in its design and coordination, and drafted the manuscript. AU carried out the intact smooth muscle studies. KS, HT and HS carried out the skinned fiber studies and immunoblot analysis. SN carried out the analysis of active RhoA. MM participated in the direction of the study as well as writing and preparing the manuscript. All authors read and approved the final manuscript.