Evaluation of the potential use of adipose-derived mesenchymal stromal cells in the treatment of canine atopic dermatitis: a pilot study.

Stem cells and their potential therapeutic uses in human and veterinary medicine have generated considerable interest. These cells have a number of potentially unique immunologic properties; most notable are their reported regenerative and antiinflammatory capabilities. The aim of this prospective pilot study was to evaluate the efficacy of intravenously administered autogenous adipose-derived mesenchymal stem cells (AD-MSCs) in the treatment of canine atopic dermatitis. AD-MSCs administered intravenously at a dose of 1.3 million cells/kg did not significantly reduce the clinical signs of canine atopic dermatitis or the owner-assessed pruritus level.

Expression and characterization of a murine enzyme able to cleave beta-carotene. The formation of retinoids.

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta-carotene 15,15'-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave beta-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzyme with tissue 12,000 x g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.

IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy.

Abnormalities of chromosome 1q21 are common in B cell malignancies, but their target genes are largely unknown. By cloning the breakpoints of a (1;14) (q21;q32) chromosomal translocation in a myeloma cell line, we have identified two novel genes, IRTA1 and IRTA2, encoding cell surface receptors homologous to the Fc and inhibitory receptor families. Both genes are selectively expressed in mature B cells: IRTA1 in marginal zone B cells and IRTA2 in centrocytes, marginal zone B cells, and immunoblasts. As a result of the t(1;14), IRTA1 is fused to the immunoglobulin Calpha domain to produce a chimeric IRTA1/Calpha fusion protein. In tumor cell lines with 1q21 abnormalities, IRTA2 expression is deregulated. Thus, IRTA1 and IRTA2 are novel immunoreceptors implicated in B cell development and lymphomagenesis.

Vitamin A controls epithelial/mesenchymal interactions through Ret expression.

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.

Characteristics of patients with primary acute lateral patellar dislocation and their recovery within the first 6 months of injury.

We prospectively studied the characteristics and early recovery of an unselected population of patients who had acute first-time lateral patellar dislocation. The recovery program used standardized rehabilitation, emphasizing range of motion, muscle strength, and return of function. Patients returned to stressful activities including sports as tolerated when they regained full passive range of motion, had no effusion, and when quadriceps muscle strength was at least 80% compared with the noninjured limb. Seventy-four patients met the enrollment criteria; 37 men and 37 women. The average age was 19.9 years, and preinjury sports participation was similar to that of ligament-injury patients. Four percent of patients (N = 3) had a history of birth complications, 3% (N = 2) had a history of lower extremity problems as an infant or child, and 9% (N = 7) had a family history of patellar dislocation. Radiographs revealed a 50% incidence (N = 37) of patella alta; all patients demonstrated lateral patellar overhang. Patients regained range of motion (mean, 0 degrees to 132 degrees) by 6 weeks. Sports participation remained significantly reduced throughout the first 6 months after injury, with the greatest limitations in kneeling and squatting. At 6 months, 58% of patients (N = 43) noted limitation in strenuous activities. The patients who had acute primary patellar dislocation were young and active. Most injuries occurred during sports, and few patients had abnormal physical features, contradicting any stereotype of an overweight, sedentary, adolescent girl whose patella dislocates with little or no trauma.

Regulation of retinoic acid signaling during lung morphogenesis.

Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development.

Cost-effectiveness analysis of a family physician delivered smoking cessation program.

OBJECTIVES: The objectives were to present a cost-effectiveness analysis of a smoking cessation program delivered by physicians and compare results to other smoking cessation interventions. METHODS: Retrospective effectiveness figures from a previous evaluation of the smoking cessation program were supplemented with estimates based on researched assumptions. Net abstinence rates were determined for smokers, depending on their stage of readiness to quit, that is, "prepared," "contemplative," and "precontemplative," leading to an assessment of the number of smokers achieving abstinence as a result of the Smokescreen intervention. Costs were calculated from the perspectives of smokers, family physicians, organizers, trainers, and all parties combined. Assumptions were varied with a sensitivity analysis. RESULTS: Baseline costs per additional abstainer were $183 based on physicians' intervention costs at 1995 prices. This is the figure most comparable to previously conducted economic evaluations of smoking cessation interventions. Sensitivity analysis varying the perspective and under optimistic and pessimistic assumptions about effectiveness produced a wide variety of estimates. The decision to include or exclude training costs had a particularly important bearing on the estimates. However, under reasonable assumptions the cost per additional quitter compares favorably to smoking and other medical and health care interventions worldwide. CONCLUSIONS: The program appears cost-effective when compared to other smoking cessation and health promotion interventions and illustrates the potential for retrospective cost-effectiveness analysis of interventions.

A novel pleckstrin homology-related gene family defined by Ipl/Tssc3, TDAG51, and Tih1: tissue-specific expression, chromosomal location, and parental imprinting.

We previously described a gene, Ipl (Tssc3), that is expressed selectively from the maternal allele in placenta, yolk sac, and fetal liver and that maps within the imprinted domain of mouse distal Chromosome (Chr) 7/human Chr 11p15.5 (Hum Mol Genet 6, 2021, 1997). Ipl is similar to TDAG51, a gene that is involved in FAS/CD95 expression. Here we describe another gene, Tih1 (TDAG/Ipl homologue 1), with equivalent sequence similarity to Ipl. Structural prediction indicates that the products of these three genes share a central motif resembling a pleckstrin-homology (PH) domain, and TIH1 protein has weak sequence similarity to the PH-domain protein SEC7/CYTOHESIN. Like Ipl, Tih1 is a small gene with a single small intron. Tih1 maps to distal mouse Chr 1 and human Chr 1q31, chromosomal regions that have not shown evidence for imprinting and, in contrast to Ipl, Tih1 is expressed equally from both parental alleles. Ipl, Tih1, and TDAG51 have overlapping but distinct patterns of expression. Tih1 and TDAG51 are expressed in multiple fetal and adult tissues. In contrast, during early mouse development Ipl mRNA and protein are highly specific for two tissues involved in maternal/fetal exchange: visceral endoderm of the yolk sac and labyrinthine trophoblast of the placenta. These findings highlight the dominance of chromosomal context over gene structure in some examples of parental imprinting and extend previous evidence for placenta-specific expression of imprinted genes. The data also define a new subfamily of PH domain genes.

The targeted disruption of both alleles of RARbeta(2) in F9 cells results in the loss of retinoic acid-associated growth arrest.

F9 teratocarcinoma cell lines, carrying one or two disrupted alleles of the RARbeta(2) gene, were generated by homologous recombination to study the role of RARbeta(2) in mediating the effects of retinoids on cell growth and differentiation. Retinoic acid (RA) does not induce growth arrest of the RARbeta(2)-/- cells, whereas the F9 WT and RARbeta(2)+/- heterozygote lines undergo RA-induced growth arrest. The RARbeta(2)+/- lines also exhibit a faster cell cycle transit time in the absence of RA. The RARbeta(2)-/- stem cells exhibit an altered morphology when compared with the F9 WT parent line, and after RA treatment, the RARbeta(2)-/- cells do not exhibit a fully differentiated cell morphology. As compared with F9 WT cells, the RARbeta-/- cells exhibited a markedly lower induction of several early RA-responsive genes and no induction of laminin B1, a late response gene. The induction of RA metabolism in the F9 RARbeta(2)-/- cells following differentiation was not impaired. The research presented here, and prior research suggest that RARbeta is required for RA-induced growth arrest in a variety of cell types and that RARbeta also functions in mediating late responses to RA. These findings are significant in view of the reduced expression of RARbeta transcripts in a number of different types of human carcinomas.

Stromal cells mediate retinoid-dependent functions essential for renal development.

The essential role of vitamin A and its metabolites, retinoids, in kidney development has been demonstrated in vitamin A deficiency and gene targeting studies. Retinoids signal via nuclear transcription factors belonging to the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families. Inactivation of RARaplpha and RARbeta2 receptors together, but not singly, resulted in renal malformations, suggesting that within a given renal cell type, their concerted function is required for renal morphogenesis. At birth, RARalpha beta2(-) mutants displayed small kidneys, containing few ureteric bud branches, reduced numbers of nephrons and lacking the nephrogenic zone where new nephrons are continuously added. These observations have prompted us to investigate the role of RARalpha and RARbeta2 in renal development in detail. We have found that within the embryonic kidney, RARalpha and RARbeta2 are colocalized in stromal cells, but not in other renal cell types, suggesting that stromal cells mediate retinoid-dependent functions essential for renal development. Analysis of RARalpha beta2(-) mutant kidneys at embryonic stages revealed that nephrons were formed and revealed no changes in the intensity or distribution of molecular markers specific for different metanephric mesenchymal cell types. In contrast the development of the collecting duct system was greatly impaired in RARalpha beta2(-) mutant kidneys. Fewer ureteric bud branches were present, and ureteric bud ends were positioned abnormally, at a distance from the renal capsule. Analysis of genes important for ureteric bud morphogenesis revealed that the proto-oncogene c-ret was downregulated. Our results suggest that RARalpha and RARbeta2 are required for generating stromal cell signals that maintain c-ret expression in the embryonic kidney. Since c-ret signaling is required for ureteric bud morphogenesis, loss of c-ret expression is a likely cause of impaired ureteric bud branching in RARalpha beta2(-) mutants.

Effect of training on general practitioners' use of a brief intervention for excessive drinkers.

OBJECTIVE: To determine among general practitioners (GPs) the effect of three different types of training on utilisation of a brief, controlled drinking intervention. DESIGN: A non-randomised intervention study. Setting, participants: 96 GPs (64%) within the South Eastern Sydney Division of General Practice participated; 35 chose workshop training, 39 one-to-one training and 22 received a special kit by mail. MAIN OUTCOME MEASURES: Identification by GPs of excessive drinkers by practice audits; use of the program determined by the number of patients recruited in 3 months and by GPs' use of the intervention 6 months after training. RESULTS: 41 (43%) GPs conducted practice audits, identifying 15.1% of males and 6.6% of females as excessive drinkers (regular excessive weekly consumption and/or binge). 179 patients were recruited by 36 GPs over 3 months, and 32% of these patients reported a reduction of alcohol consumption. 63% who attended workshop training, 57% who received one-to-one training, and 36% who received the kit by mail reported they were current users of the program at 6 months. Significantly fewer GPs who received the kit by mail reported ever using the program (59%) compared to the other groups (p < 0.01). CONCLUSION: This naturalistic study found that workshops and one-to-one training sessions in doctors' surgeries achieved greater uptake of a brief intervention for problem drinkers than distribution of a special kit by mail.

Family physicians' utilization of a brief smoking cessation program following reinforcement contact after training: a randomized trial.

BACKGROUND: Previous studies have examined methods of delivery of brief interventions and reinforcement contact and their effects on physicians' utilization of smoking cessation interventions. In this study the objectives were: (1) to determine the ongoing utilization by family physicians of a brief smoking cessation intervention 6 months after a training workshop and (2) to examine the effect of reinforcement contact on physician utilization. A supplementary aim was to assess point prevalence abstinence among patients identified as ready to quit smoking. METHODS: This was a randomized controlled trial of family physicians (98 in the Contact and 100 in the Noncontact group). Training was conducted in a 2-hr workshop. Doctors in the Contact group received three brief telephone calls at 2 weeks, 2 months, and 4 months after training. Main outcome measures were: (1) utilization, determined by responses to a mailed questionnaire about use of the program, and (2) the number of booklets distributed by full-time doctors, collected by practice secretaries or research assistant. RESULTS: At 6 months 88% of physicians (93% of the Contact group and 84% of the Noncontact group, P = 0.06) were current users of the smoking cessation intervention. Full-time physicians in the Contact group distributed significantly more booklets (40.1) over 6 months than those in the Noncontact group (32.8) (P < 0.05). Twenty-one percent of patients reported not smoking at follow-up at an average of 9.9 months after intervention. CONCLUSIONS: Most doctors continued to use the program 6 months after training and reinforcement contact encouraged greater recruitment of patients.

The risk of venous thromboembolism in the orthopedic patient: epidemiological and physiological data.

Venous thromboembolism is responsible for 500,000 deaths annually in industrialized countries. It is probably the most common preventable cause of death in elective orthopedic surgery patients. Rates of deep vein thrombosis (DVT) and fatal pulmonary embolism (PE) in unprotected orthopedic patient populations are high. The overall DVT rate is > 40% in patients undergoing hip or knee arthroplasty or suffering from multiple injuries. The proximal DVT rate for these patients is > or = 15%, and the fatal PE rate is > or = 1%. Risk factors associated with venous thromboembolism are related to the vascular injury, activation of blood coagulation, and venous stasis. Lower extremity orthopedic procedures carry a risk greater than that of surgery itself. Thus, orthopedic patients are at high risk for venous thromboembolic conditions. A systematic assessment of this risk should be performed in every patient, and an appropriate management plan should be implemented.

The risk of venous thromboembolism in the orthopedic patient: epidemiological and physiological data.

ABSTRACTVenous thromboembolism is responsible for 500,000 deaths annually in industrialized countries. It is probably the most common preventable cause of death in elective orthopedic surgery patients. Rates of deep vein thrombosis (DVT) and fatal pulmonary embolism (PE) in unprotected orthopedic patient populations are high. The overall DVT rate is >40% in patients undergoing hip or knee arthroplasty or suffering from multiple injuries. The proximal DVT rate for these patients is ≥15%, and the fatal PE rate is ≥1%. Risk factors associated with venous thromboembolism are related to the vascular injury, activation of blood coagulation, and venous stasis. Lower extremity orthopedic procedures carry a risk greater than that of surgery itself. Thus, orthopedic patients are at high risk for venous thromboembolic conditions. A systematic assessment of this risk should be performed in every patient, and an appropriate management plan should be implemented.

Physical examination of the knee.

Knee injuries are common, and many primary providers are uncomfortable with the clinical diagnosis of these conditions. The following article emphasizes the importance of obtaining a good history from the patient and accurately determining the mechanism of injury to narrow the diagnostic possibilities. It briefly reviews some pertinent points of anatomy to facilitate the physical examination and to understand the rationale of some clinical tests. A brief description of the most useful clinical tests in an acute situation also is given.

Developmental roles of the retinoic acid receptors.

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.

Roles of retinoic acid receptors and of Hox genes in the patterning of the teeth and of the jaw skeleton.

Retinoic acid receptors and transcriptional factors encoded by Hox genes play key roles in vertebrate development and belong to an integrated functional network. To investigate the actual functions of these molecules during ontogenesis and in particular in the patterning of the cranial neural crest cells giving rise to the teeth and to the jaw bones, we have generated null mutant mice lacking functional retinoic acid receptors or Hox genes by gene targeting in embryonic stem cells.

Retinoic acid receptor beta 2 (RAR beta 2) null mutant mice appear normal.

Vertebrates are highly sensitive to both retinoic acid (RA) deficiency and excess. The RA signal is thought to be transduced by nuclear receptors (the RAR and RXR families) which activate the expression of target genes via cis-acting transcriptional enhancer elements. Each of the three RAR genes, RAR alpha, RAR beta, and RAR gamma, gives rise to several isoforms by differential usage of two promoters and alternative splicing. RAR beta 2 is the most abundant of the four RAR beta isoforms, and its transcription is spatially and temporally restricted in developing embryos, suggesting that it might perform specific functions. Furthermore, RAR beta 2 expression can be induced via a retinoic acid response element located in its promoter region. This RA effect is particularly interesting since under conditions of RA excess, RAR beta 2 promoter activity and transcript accumulation are induced in regions of developing embryos in which malformations subsequently appear, such as the craniofacial region, the hindbrain, and the limbs. These findings have led to the suggestion that the RAR beta 2 isoform might mediate some of the teratogenic effects of RA. In this study, we have eliminated RAR beta 2 expression by targeted gene disruption. RAR beta 2 null mutants exhibit an apparently normal phenotype, indicating that other RARs must compensate for RAR beta 2 sufficiently well to allow normal prenatal and postnatal development to proceed. By challenging RAR beta 2 null embryos with teratogenic doses of RA, we have also directly addressed the question of whether RAR beta 2 is required for mediating RA-induced malformations.