An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.

An ultra scale-down method is described to determine the response of cells to recovery by dead-end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (~2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity.

Use of adenovirus as a model system to illustrate a simple method using standard equipment and inexpensive excipients to remove live virus dependence on the cold-chain.

Thermolability of complex biological molecules is a major consideration for the long-term maintenance of titer during periods of storage. The development of a simple, cost effective method for long term storage of virus samples, which maintains viral titer would prove useful for a wide variety of applications including the preservation of viral vaccines, and is paramount for alleviating the reliance upon the cold chain. We have investigated the potential use of a method adapted for this purpose originating from a natural mechanism used by plants which helps to maintain the integrity of seeds, enabling them to overcome extensive periods of temperature elevation and desiccation. As maturation of a seed progresses, many complex biological macromolecules are laid down which maintain the germination potential. Sucrose and raffinose (in addition to other oligosaccharides) are commonly found to accumulate. In addition highly charged protein molecules accumulate, Late Embryogenesis Abundant (LEA) proteins, reaching their maximal level when the seed is most desiccation and thermally tolerant, and indeed are among the first molecules to be lost when germination is initiated. We have examined the potential use of sucrose and raffinose in concert with chemical replacements for the LEA, which when dried with the active product forms an amorphous solid able to maintain the titer of infectious Adenovirus at elevated temperatures for extended periods, in the case of lyophilized presentations several months at 37 °C, or as liquid, stability for several weeks at 37 °C was achieved. We demonstrate that after embedding the active product in the matrix, the viral titer is preserved even at temperatures for relatively extended periods at temperatures significantly greater than ambient. In addition we believe that these results could open the way for a new type of vaccine which we refer to as a hybrid stability vaccine, whereby for the first time the same excipient components are used to confer stability in both liquid and solid forms (albeit at different concentrations) which may ultimately result in a stable vaccine which has a very high stability index whilst dry, whereas upon reconstitution using nothing more than WFI at proscribed volumes, the vaccine would benefit from having much improved stability during the administration procedures. This paper describes the use of Adenovirus (itself fast becoming a vector of choice for a new generation of vaccines) as a model system, and identifies non-toxic, inexpensive excipients which are compatible with current manufacturing processes which could be instrumental in removing the dependence upon the cold chain.

CB1-independent mechanisms of Δ9-THCV, AM251 and SR141716 (rimonabant).

WHAT IS KNOWN AND OBJECTIVE: The potential beneficial therapeutic effects of cannabinoid CB1 receptor antagonists or partial agonists have driven drug discovery and development efforts and have led to clinical candidates. It is generally assumed that these compounds are CB1 'selective' and produce their effects exclusively via CB1 receptors. METHODS: A literature search was conducted of preclinical publications containing information about non-CB1 receptor pharmacology of these agents. The information was summarized and evaluated from the perspective of contribution to a fuller understanding of this aspect of these compounds. RESULTS AND DISCUSSION: A number of recent studies have revealed that these compounds have CB1-independent pharmacological actions. We highlight the evidence regarding effects produced in cells lacking CB1 receptors, effects on neuronal membranes from CB1 receptor-deficient mutant KO 'knockout' mice and affinity for µ-opioid receptors. WHAT IS NEW AND CONCLUSION: CB1 'selective' antagonists and partial agonists have been studied for their anorexigenic and other potential therapeutic uses. An awareness of CB1-independent mechanism(s) of these agents might contribute to a better understanding of the pharmacologic and toxicologic profiles of these agents.

The endocannabinoid system and rimonabant: a new drug with a novel mechanism of action involving cannabinoid CB1 receptor antagonism--or inverse agonism--as potential obesity treatment and other therapeutic use.

There is considerable evidence that the endocannabinoid (endogenous cannabinoid) system plays a significant role in appetitive drive and associated behaviours. It is therefore reasonable to hypothesize that the attenuation of the activity of this system would have therapeutic benefit in treating disorders that might have a component of excess appetitive drive or over-activity of the endocannabinoid system, such as obesity, ethanol and other drug abuse, and a variety of central nervous system and other disorders. Towards this end, antagonists of cannabinoid receptors have been designed through rational drug discovery efforts. Devoid of the abuse concerns that confound and impede the use of cannabinoid receptor agonists for legitimate medical purposes, investigation of the use of cannabinoid receptor antagonists as possible pharmacotherapeutic agents is currently being actively investigated. The compound furthest along this pathway is rimonabant, a selective CB(1) (cannabinoid receptor subtype 1) antagonist, or inverse agonist, approved in the European Union and under regulatory review in the United States for the treatment of obesity. This article summarizes the basic science of the endocannabinoid system and the therapeutic potential of cannabinoid receptor antagonists, with emphasis on the treatment of obesity.

A nonsynonymous substitution of cystatin A, a cysteine protease inhibitor of house dust mite protease, leads to decreased mRNA stability and shows a significant association with atopic dermatitis.

BACKGROUND: Cystatin A (CSTA) is a strong candidate for atopic dermatitis (AD) because it maps to AD susceptibility locus on chromosome 3q21 and it does inhibit Der p 1 and Der f 1, major house dust mite cysteine proteases and environmental triggers for AD and asthma. OBJECTIVE: To examine any association between polymorphisms in CSTA and AD and study the effect on the CSTA mRNA expression level. METHODS: We identified three polymorphisms and characterized the linkage disequilibrium mapping of the CSTA gene. All three CSTA polymorphisms were genotyped in 100 AD patients and 203 matched controls. Subsequently, we performed transfection-based RNA stability assays. RESULTS: We found a significant association between the CSTA +344C variant and AD [odds ratio (OR) = 1.91; P = 0.024]. When further 61 control samples were genotyped. The association with CSTA +344C allele was enhanced OR = 2.13; P = 0.006. To test whether the CSTA +344 affected the CSTA transcriptional activity, the decay rates of RNAs transcribed from the CSTA +344C and CSTA +344T variants were investigated. COS-7 cells were transfected with a pcDNA3.1-CSTA+344C or a pcDNA3.1-CSTA+344T construct and cultured in the presence or absence of actinomycin D. Real-time RT-PCR revealed that CSTA +344C mRNA is more than two times less stable than the CSTA +344T mRNA (P < 0.001). CONCLUSION: These results suggest that the CSTA +344C allele associated with unstable mRNA could result in failing to protect the skin barrier in AD patients from both exogenous and endogenous proteases.

The role of CB1 receptors in sweet versus fat reinforcement: effect of CB1 receptor deletion, CB1 receptor antagonism (SR141716A) and CB1 receptor agonism (CP-55940).

It is well established that Cannabis sativa can increase appetite, particularly for sweet and palatable foods. In laboratory animals, cannabinoid CB1 receptor antagonism decreases motivation for palatable foods, and most recently, the CB1 receptor antagonist SR141716A, or rimonabant (Acomplia), was reported to produce weight loss in obese human subjects. Indeed, the endocannabinoid system plays a select role in the rewarding properties of palatable foods, and this is well characterized in laboratory animals with sweet sucrose solutions. In the present study, CB1 knockout mice (CB1 KO) and wild-type littermate mice (WT) were trained to respond for a complex sweet as well as a pure fat reinforcer under a progressive ratio (PR) schedule, to determine whether motivation to consume different palatable foods is tonically regulated by CB1 receptors. To assess sweet reinforcement, several concentrations of the liquid nutritional drink, Ensure, were presented under the PR schedule. For fat reinforcement, several concentrations of corn oil (emulsified in 3% xanthan gum) were made available. Additionally, to compare the result of genetic invalidation of the CB1 receptor to antagonism of the CB1 receptor system, the effect of SR141716A (3.0 mg/kg) on responding for Ensure and corn oil were also assessed using the PR schedule. We also assessed the effect of the CB1 agonist CP-55940 (30 microg/kg) on responding for Ensure and corn oil. CB1 KOs took significantly longer to acquire operant responding maintained by Ensure, and responding for Ensure under the PR schedule was significantly reduced in CB1 KOs as well as in WTs pretreated with SR141716A, as compared to WT controls. Additionally, pretreatment with the CB1 agonist CP-55940 increased responding for Ensure. In contrast, responding for corn oil during acquisition and under the PR schedule was not significantly different in CB1 KOs versus wild-type mice. However, SR141716A did reduce responding for corn oil in WTs, and CP-55940 significantly increased responding for corn oil. Taken together, these results suggest that CB1 receptors are preferentially involved in the reinforcing effects of a complex sweet, as compared to a pure fat, reinforcer. These data also suggest, however, that antagonism of CB1 receptors with SR141716A is sufficient to attenuate the reinforcing effect of Ensure and corn oil, while activation of the central CB1 system is sufficient to enhance Ensure and corn oil reinforcement.

The effects of habitat fragmentation via forestry plantation establishment on spatial genotypic structure in the small marsupial carnivore, Antechinus agilis.

Dispersal is an important influence on species' distributions, patch colonization and population persistence in fragmented habitat. We studied the impacts of habitat fragmentation resulting from establishment of an exotic pine plantation on dispersal of the marsupial carnivore, Antechinus agilis. We applied spatial analyses of individual multilocus microsatellite genotypes and mitochondrial haplotypes to study patterns of gene flow in fragmented habitat and natural habitat 'control' areas, and how this is affected by the spatial dispersion of habitat patches, the presence of corridors and a 'mainland' source of migrants. Spatial analysis of molecular variance and partial Mantel tests confirmed the absence of cryptic barriers to gene flow in continuous habitat, which if present would confound the comparison of genetic structures in fragmented vs. unfragmented habitats. Spatial genotypic structure suggested that although dispersal was male-biased in both habitat types, fragmentation restricted dispersal of males more than that of females and the degree of restriction of male dispersal was dependent on the geographical isolation of the patch. The scale of positive genotypic structure in fragmented habitat was restricted to the two closest patches for females and the three closest patches for males. Our results provide evidence for significantly increased gene flow through habitat corridors relative to that across the matrix and for significantly lower gene flow between 'mainland' unfragmented habitat and habitat patches relative to that within either habitat type, suggesting a behavioural barrier to crossing habitat interfaces.

The effects of habitat fragmentation on the social kin structure and mating system of the agile antechinus, Antechinus agilis.

Habitat fragmentation is one of the major contributors to the loss of biodiversity worldwide. However, relatively little is known about its more immediate impacts on within-patch population processes such as social structure and mating systems, whose alteration may play an important role in extinction risk. We investigated the impacts of habitat fragmentation due to the establishment of an exotic softwood plantation on the social kin structure and breeding system of the Australian marsupial carnivore, Antechinus agilis. Restricted dispersal by males in fragmented habitat resulted in elevated relatedness among potential mates in populations in fragments, potentially increasing the risk of inbreeding. Antechinus agilis nests communally in tree hollows; these nests are important points for social contact between males and females in the mating season. In response to elevated relatedness among potential mates in fragmented habitat, A. agilis significantly avoided sharing nests with opposite-sex relatives in large fragment sites (but not in small ones, possibly due to limited nest locations and small population sizes). Because opposite-sex individuals shared nests randomly with respect to relatedness in unfragmented habitat, we interpreted the phenomenon in fragmented habitat as a precursor to inbreeding avoidance via mate choice. Despite evidence that female A. agilis at high inbreeding risk selected relatively unrelated mates, there was no overall increased avoidance of related mates by females in fragmented habitats compared to unfragmented habitats. Simulations indicated that only dispersal, and not nonrandom mating, contributed to inbreeding avoidance in either habitat context. However, habitat fragmentation did influence the mating system in that the degree of multiple paternity was reduced due to the reduction in population sizes and population connectivity. This, in turn, reduced the number of males available to females in the breeding season. This suggests that in addition to the obvious impacts of reduced recruitment, patch recolonization and increased genetic drift, the isolation of populations in habitat patches may cause changes in breeding behaviour that contribute to the negative impacts of habitat fragmentation.

Genetic association between an AACC insertion in the 3'UTR of the stratum corneum chymotryptic enzyme gene and atopic dermatitis.

Atopic dermatitis is a disease with an impaired skin barrier that affects 15%-20% of children. In the normal epidermis, the stratum corneum chymotryptic enzyme (SCCE) thought to play a central role in desquamation by cleaving proteins of the stratum corneum (e.g., corneodesmosin and plakoglobin). Genetic variations within the SCCE gene could be associated with dysregulation of SCCE activity leading to an abnormal skin barrier. We screened the SCCE gene for variations and performed a case-control study on 103 atopic dermatitis patients and 261 matched controls. 16 synonymous single nucleotide polymorphisms (SNPs) have been identified and a 4 bp (AACC) insertion has been found in the 3'UTR. We performed an association study of the SCCE AACC insertion in the 3'UTR, and found a significant trend between the AACC allele with the two insertions and disease in the overall data set [odds ratio (OR)=2.31; p=0.0007]. The AACC insertion in the SCCE gene may result in a change to SCCE activity within the skin barrier. These findings suggest that SCCE could have an important role in the development of atopic dermatitis.

A cluster randomised, controlled trial of the value of dental health educators in general dental practice.

AIM: To test the effectiveness of dental health educators in general dental practice. OBJECTIVE: To evaluate the effectiveness and cost of primary care trusts seconding dental health educators free of charge to suitable general dental practices to provide dental health counselling to mothers of regularly attending pre-school children at risk to caries. METHOD: Two-cell, parallel group, cluster randomised, controlled clinical trial of two years' duration. CLINICAL SETTING: 30 general dental practices in North-West England. PARTICIPANTS: 269 mothers of 334 pre-school children. INTERVENTIONS: Those in the test group were given visits to a dental health educator over a 2-year period to counsel mothers of at-risk, pre-school children. The rest were held as a control. MAIN OUTCOME MEASURES: Caries prevalence of the children and dental health knowledge, attitudes and toothbrushing skills of the parents. The full costs of the exercise were kept throughout. The statistical analysis controlled for the clustering of children within practices. RESULTS: After 2 years, 271 (81%) children and 248 (92%) mothers remained in the study. There was an 18% difference in mean dmft between the groups in favour of the test group children but this was not statistically significant. At the end of the study there was an 18% difference in mean dmft between the groups in favour of the test group children but this was not statistically significant. No difference in plaque levels was found. The mothers in the test group were more knowledgeable, had better attitudes towards the dental health of their offspring and better toothbrushing skills than those in the control. Each 2-hour session to counsel ten parents cost pound 40. CONCLUSION: Primary care trusts should carefully consider the cost value of seconding dental health educators to counsel parents of regularly attending, at-risk, pre-school children when considering such an option.

Paternity success and the direction of sexual selection in a field population of a semelparous marsupial, Antechinus agilis.

Antechinus agilis is a small sexually size dimorphic marsupial with a brief annual mating period of 2-3 weeks. All males die after this period, and females give birth to up to 10 young. Mating is thought to be promiscuous, however, there is no field data to confirm this. Using microsatellites, we investigated paternity patterns over two seasons in a wild population. Male weight was significantly positively related to the number of females fertilized and with the number of offspring sired, in both years. Furthermore, selection gradients indicated selection for larger males. Both results suggest that size dimorphism in A. agilis can be explained by sexual selection for larger males. The proportion of offspring sired within litters, did not relate to male size. Therefore, larger males are more successful through higher mating access, not through their sperm outcompeting that of smaller males. As expected from their known ranging behaviour, the number of offspring within litters left unassigned to a father did not depend on the grid location of the mother. Female size did not differ between successful reproducing and unsuccessful females. However, females that weaned offspring had larger heads than females that did not wean offspring. Males did not 'prefer' mating with larger females, nor did assortative mating occur. From our results, the mating system of A. agilis is clearly promiscuous. Selection for larger males occurred in both years, even though in one year the operational sex ratio was highly female biased, suggesting that the potential reproductive rate is a better predictor of the direction of sexual selection in A. agilis.

Association analysis of IL1A and IL1B variants in alopecia areata.

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The disease is characterized by focal inflammatory lesions with perifollicular T-cell infiltrates, reflecting the role of local cytokine production in the development of patchy hair loss. IL-1 alpha and IL-1 beta are important inhibitors of hair growth in vitro. Their effect is opposed by the interleukin-1 receptor antagonist, IL-1ra. Genes of the IL-1 cluster are candidate genes in the pathogenesis of alopecia areata. To investigate the role of the IL-1 system in alopecia areata we examined three biallelic polymorphisms within the IL-1 gene cluster (IL1A+4845, IL1B+3954 and IL1B-511) in 165 patients and a large number of matched controls (n=1150). There was no significant association of IL1B-511 or IL1B+3954 genotypes with the overall dataset, or with disease severity or age at onset, in contrast with a previous report. The results suggested the possibility of an association with IL1A+4845 in the overall dataset [OR 1.39 (95% CI 1.00, 1.93)] although this was not statistically significant. This was due mainly to the contribution from mild cases of alopecia areata [OR 1.48 (0.96, 2.29)], suggesting that IL-1 alpha may have a particular role in the pathogenesis of this subgroup.

Use of transgenic mice model for understanding the placentation: towards clinical applications in human obstetrical pathologies?

The mammalian embryo and fetus are unable to develop without a well-established, functional placenta. This transitory yet indispensable structure attaches the conceptus to the uterus and establishes the vascular connections necessary for nutrient and gaseous exchange between maternal and fetal compartments. Genetic targeting strategy allows the generation of mice lacking a specific gene. Such approaches reveal: (i) the high incidence of mutant embryonic or fetal death in utero, and (ii) the extraembryonic (placental) causes of these deaths. Due to the similarities presented between mouse and human placenta, we propose to use the potential of mouse targeting experiments as a model in order to understand human obstetrical pathologies. In this paper, we first review genes that have been demonstrated to be required in mice for implantation, choriovitelline and chorioallantoic placentation. Using examples (integrins, homeoboxs, hepatocyte growth factor or epidermal growth factor receptor...) we demonstrate the reality and efficiency of such an approach. Other candidate genes (receptor of leukemia inhibitory factor, Wnt2 or retinoic acid receptor alpha...) in order to understand, prevent and treat human obstetrical pathologies.

Genomic organization of the mouse plunc gene and expression in the developing airways and thymus.

Few genes have been isolated which display specific expression in the proximal airways. A recently identified mouse cDNA, plunc, appears to be confined to the upper airways and nasopharyngeal epithelium, and may prove a useful marker for these regions. We now report the genomic cloning and characterization of the mouse plunc gene as well as its developmental expression in the nasal and airway epithelium. We also report the novel finding that plunc is also expressed in the medullary compartment of the murine thymus. The mouse gene contains nine exons and the intron-exon boundaries are conserved with those in the human homologue. At e14.5 plunc is expressed in the nasal epithelium and several days later is seen in the thymic lobes, but not in the lining of the tracheobronchial tree. Expression in the trachea and main-stem bronchi first appears at 1--2 days after birth. Tracheobronchial expression persists at high levels throughout adulthood, as do regional areas of nasal and thymic expression. Finally, we show that the human homologue is expressed in bronchial epithelium, suggesting a transcript that is evolutionarily conserved in the mammalian airway.

Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo.

The three murine retinoic acid receptor (RAR) genes each contain two distinct promoters which give rise to protein isoforms differing in their N-terminal regions. This study used in situ hybridization to describe the expression patterns of RARalpha1, RARalpha2, RARbeta1/3, RARbeta2/4, RARgamma1 and RARgamma2 isoform transcripts during mouse embryogenesis. RARalpha1 transcripts are widely distributed, with the exception of the central nervous system. Highest expression is found in developing muscle, pituitary gland and various epithelia. On the other hand, RARalpha2 is essentially expressed along the spinal cord up to the hindbrain 7th rhombomere and in the 4th rhombomere, pons and developing basal ganglia (corpus striatum and pallidum). RARbeta2/4 transcripts account for most of the previously described RARbeta expression features being expressed specifically, or more prominently than RARbeta1/3, in foregut endoderm and its derivatives, olfactory and periocular mesenchyme, urogenital region, proximal limb bud mesenchyme and later within interdigital regions. RARbeta1/3 is more prominently expressed in the developing heart outflow tract mesenchyme, intervertebral disks, midgut loop mesenchyme and umbilical vessel walls. RARbeta1/3 and RARbeta2/4 are coexpressed in the developing corpus striatum. They exhibit, however, distinct dorsoventral distributions along the spinal cord and caudal hindbrain. RARgamma2 is the RARgamma isoform expressed at high levels in the caudal neural groove at embryonic day 8.5. At later stages, both RARgamma isoforms are essentially coexpressed, although the progressive restriction of RARgamma1 transcripts to craniofacial or limb precartilaginous condensations appears to precede that of RARgamma2.

Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa.

Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.