A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage.

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.

beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

Piloting a patient safety and quality improvement co-curriculum.

Background: Despite the push for resident and faculty involvement in patient safety (PS) and quality improvement (QI), there is limited literature describing programs that train them to conduct PS/QI projects. Objective: To determine the effectiveness of a co-learning PS/QI curriculum. Method: The authors implemented a co-learning (residents and faculty together) PS/QI curriculum within our general Internal Medicine program over 1 year. The curriculum consisted of two workshops, between-session guidance, and final presentation. The authors evaluated effectiveness by self-assessment of attitude, knowledge, and behavior change and PS/QI project completion. Results: Thirty-eight of 32 (95%) resident and 8 faculty member participants attended the workshops and 27 of 40 (67%) completed the evaluation. Participants (87-96%) responded favorably regarding workshop effectiveness. The authors found significant improvement in 78% of items pertaining to PS/QI knowledge/skills, but no difference for attitudinal items. The final project evaluation participants rated project content as relevant to learning needs (75%); training as well-organized (75%); faculty mentorship for the project as supportive (75%); and the overall project as excellent or very good (71%). Conclusion: The authors successfully demonstrated a framework for co-teaching faculty and residents to conduct PS/QI projects. Participants acquired necessary tools to practice in an ever-evolving clinical setting emphasizing a patient-centered and quality-focused environment.

In vitro models for studying trophoblast transcellular transport.

In vitro models have proven to be effective in studying the placental transporters that play a role in the exchange of nutrients, waste products, and drugs between the maternal and fetal circulations. Although primary cultures of trophoblast cells can be used to perform uptake, efflux, and metabolism studies, only the rodent HRP-1 and the human BeWo cell lines have been shown to form confluent monolayers when grown on semi-permeable membranes. Protocols for the revival, maintenance, passage, and growth of BeWo cells for transporter expression and transcellular transport studies are provided.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Potential treatment of cerebral global ischemia with Oct-4+ umbilical cord matrix cells.

Potential therapeutic effects of Oct-4-positive rat umbilical cord matrix (RUCM) cells in treating cerebral global ischemia were evaluated using a reproducible model of cardiac arrest (CA) and resuscitation in rats. Animals were randomly assigned to four groups: A, sham-operated; B, 8-minute CA without pretreatment; C, 8-minute CA pretreated with defined media; and D, 8-minute CA pretreated with Oct-4(+) RUCM cells. Pretreatment was done 3 days before CA by 2.5-microl microinjection of defined media or approximately 10(4) Oct-4(+) RUCM cells in left thalamic nucleus, hippocampus, corpus callosum, and cortex. Damage was assessed histologically 7 days after CA and was quantified by the percentage of injured neurons in hippocampal CA1 regions. Little damage (approximately 3%-4%) was found in the sham group, whereas 50%-68% CA1 pyramidal neurons were injured in groups B and C. Pretreatment with Oct-4(+) RUCM cells significantly (p < .001) reduced neuronal loss to 25%-32%. Although the transplanted cells were found to have survived in the brain with significant migration, few were found directly in CA1. Therefore, transdifferentiation and fusion with host cells cannot be the predominant mechanisms for the observed protection. The Oct-4(+) RUCM cells might repair nonfocal tissue damage by an extracellular signaling mechanism. Treating cerebral global ischemia with umbilical cord matrix cells seems promising and worthy of further investigation.

Understanding tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin.

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro.

The yeast Saccharomyces cerevisiae has two genes for alpha-tubulin, TUB1 and TUB3, and one beta-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents approximately 10% of alpha-tubulin in the cell. We determined the effects of the two alpha-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only alpha-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only alpha-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules.

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.