Novel bone adhesives: a comparison of bond strengths in vitro.

Fracture fixation using adhesive is a promising alternative in craniofacial surgeries, replacing the plates and screws system. The advantages include the ease of application and avoidance of drilling holes that may weaken the bone and cause fractures. In this study the bond strengths of selected adhesives were evaluated and compared with resorbable plates and screws. Four adhesives, octyl-cyanoacrylate, N-butyl-cyanoacrylate, a novel methyl-methacrylate, and a novel cyanoacrylate derivative, were tested for their microtensile and shear bond strengths. The bone samples were cut into rectangular bars and bonded with selected adhesives for microtensile testing. For the shear bond test, paired bars were bonded at the overlap, while two other sets of bars were attached by a Lactosorb plate using either adhesive or screws. Data were analysed by analysis of variance (ANOVA). The microtensile bond strengths of N-butyl-cyanoacrylate, novel cyanoacrylate derivative, and novel methyl-methacrylate derivative were significantly greater than octyl-cyanoacrylate. When bone sections were fixed with resorbable plates and adhesives, shear bond strength was significantly greater for N-butyl-cyanoacrylate than plate and screws, while the bond strengths of other adhesives were comparable with the plate and screws. N-Butyl cyanoacrylate was shown to have the greatest potential for fixation of fractured bone in craniofacial surgical applications.

Effects of growth hormone-releasing hormone-related peptide on stem cell factor expression in cultured rat Sertoli cells.

The growth hormone-releasing hormone (GHRH) gene produces a precursor molecule that contains GHRH and a carboxyl-terminal peptide that we have named GHRH-related peptide (GHRH-RP). This peptide, like GHRH, stimulates the expression of stem cell factor (SCF), an important reproductive and hematopoietic cytokine, in vitro and in vivo. In the present study, using primary cultures of rat Sertoli cells, we compared the time course of action and the level of SCF stimulation seen following treatment with GHRH-RP and GHRH. Additionally, we investigated the activity of a truncated peptide, p75-92NH2, whose sequence is contained within GHRH-RP. All three of these peptides were shown to stimulate the steady-state levels of SCF mRNA to a comparable degree. However, the time course of action for GHRH-RP differed markedly from that of GHRH. GHRH-RP and p75-92NH2, similar to GHRH, induce SCF expression, at least in part, via the activation of the protein kinase A/cyclic adenosine monophosphate intracellular signaling pathway.

What makes quality assurance effective? Results from a randomized, controlled trial in 16 primary care group practices.

OBJECTIVES: The authors estimate separately contributions of each component intervention to overall effectiveness of quality assurance cycles used to improve practice performance. METHODS: In a randomized, controlled trial, experimental cycles of quality assurance were conducted for eight patient-care guidelines, with two experimental cycles assigned to each of 16 group practices. For three separate interventions per cycle, practitioners: (1) were notified of the name of the experimental guideline, (2) discussed criteria of conformance to the guideline, and (3) received feedback on performance. Actions taken in response to interventions were documented. Using medical records data for a baseline year and for 3 months after each intervention and an additional 9 months, the authors scored each practice for conformance to two experimental guidelines and to control guidelines. RESULTS: For all patient-care guidelines combined, and for four of five guidelines showing improvement, knowledge of guidelines and review criteria alone produced no change. After feedback, performance improved and improvement persisted for at least 9 months. The number of corrective actions implemented contributed significantly to effectiveness of quality assurance. CONCLUSIONS: Feedback to providers of data on their performance is a more powerful stimulus for quality improvement than is knowledge of guidelines or discussion of review criteria.

A novel peptide from the growth hormone releasing hormone gene stimulates Sertoli cell activity.

The growth hormone releasing hormone (GHRH) gene is believed to produce a single functional peptide, GHRH (1). Although GHRH mRNA and peptide have been identified in various tissues, the only accepted function of GHRH is the stimulation of pituitary growth hormone. We have discovered that the GHRH mRNA encodes a second peptide that is abundant in testicular germ cells and specifically activates Sertoli cell expression of stem cell factor (SCF), a factor crucial for the normal progression of spermatogenesis (2). These observations suggest that the GHRH gene produces two distinct peptides, each with tissue-specific activities.

Peptides of the growth hormone-releasing hormone family : Differential expression in rat testis.

Growth hormone-releasing hormone (GHRH) belongs to a family of peptides expressed at high levels in the brain and digestive system of mammals. We have identified GHRH mRNA and peptide in rat and human germ cells, and detected a GHRH receptor mRNA in Sertoli cells. GHRH treatment of cultured Sertoli cells results in accumulation of cAMP and increased expression ofc-fos and stem cell factor (SCF), two factors critical for normal germ cell development. The current study was designed to localize within testis the transcription of other members of the GHRH family, and their receptors, and to determine if they also stimulate SCF. RNAs from separated testicular cells were amplified by comparative reverse transcription-polymerase chain reaction (RT-PCR). Southern blot analysis of the PCR products verified the presence of five GHRH family peptide and receptor transcripts in distinct testicular cell types. Transcripts encoding VIP and glucagon, and the receptors for pituitary adenylate cyclase activating peptide (PACAP) and glucagon, were detected predominantly in Leydig cells. In contrast, expression of GHRH, PACAP, secretin, and secretin receptor predominated in germ cells. Receptors for GHRH and VIP were expressed equally in all testicular cell types. To determine if, like GHRH, any of these other peptides activate Sertoli cell expression of SCF, primary Sertoli cell cultures were treated for 4-6 h with 10 or 100 nM of each individual factor. There was no consistent stimulation of SCF mRNA by VIP, PACAP, glucagon, or secretin. Differential expression of these peptides and their receptors suggests that they may each have unique paracrine functions within the testis.

Presence of a spermatogenic-specific promoter in the rat growth hormone-releasing hormone gene.

A GH-releasing hormone (GHRH) messenger RNA (mRNA) has been identified in hypothalamus, placenta, and testicular germ cells. The GHRH mRNA produced by spermatogenic cells is approximately 1700 nucleotides in length, whereas GHRH transcripts in hypothalamus and placenta are 750 nucleotides. To correlate the structure of testicular GHRH mRNA with cell type-specific expression, we determined its sequence. A GHRH clone isolated from a rat testicular complementary DNA library was found to be identical in the coding sequence to hypothalamic GHRH. Rapid amplification of complementary DNA ends analysis of the 5'-end of germ cell GHRH mRNA and comparison with the genomic sequence revealed that GHRH transcription in testis initiates approximately 700 basepairs 5' to transcription initiation in placenta and 10.7 kilobasepairs 5' to that in hypothalamus. Reverse transcription-polymerase chain reaction analysis of germ cell RNA using primers from testicular exons 1 and 4 demonstrated that part of the placental exon 1 sequence is contained in some testicular GHRH transcripts, as an extra exon, between testicular exon 1 and the common exon 2. This was confirmed by a Northern blot of testicular mRNA using a testicular exon 1 probe. The 5'-flanking region of the testicular GHRH gene was analyzed and found to contain a TATA-like motif and sequences homologous to spermatogenic-specific cis-acting elements. Southern blot analysis of rat liver DNA suggested that just one GHRH gene is present in rat. These results indicate that both alternative transcription initiation and splicing of the GHRH gene exist in rat testicular germ cells.

A new target for growth hormone releasing-hormone action in rat: the Sertoli cell.

A GHRH-like mRNA and peptide (t-GHRH) have been detected in rat and human testis. In rat, t-GHRH mRNA is localized to developing spermatogenic cells. We predicted that the most likely target cell of t-GHRH action would be the Sertoli cell. To test this prediction, we evaluated GHRH action on Sertoli cell function. Rat GHRH at a concentration of 10 nM or 100 nM stimulated cAMP production 2-fold over control levels after a 30 min incubation. This stimulation was obliterated by preincubation with a 10-fold excess of the GHRH antagonist (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2. The effect of treatment with [His1,Nle27]GHRH(1-32)-NH2, a GHRH analog, on Sertoli cell mRNAs was also assessed. Treatment with the analog significantly increased levels of c-fos and steel factor (the product of the Steel gene, also termed SCF) mRNAs above controls, but had no effect on sulfated glycoprotein-2 mRNA. We conclude that GHRH acts via adenylate cyclase to modulate specific Sertoli cell products, possibly as part of a network of local interacting factors controlling Sertoli and germ cell function.

Germ cell localization of a testicular growth hormone-releasing hormone-like factor.

GH-releasing hormone (GHRH)-like mRNA and immunoreactivity (t-GHRH-li) are present in the testes of rats and humans. To learn more about the physiology of t-GHRH-li mRNA, we performed a series of experiments that disrupted various testicular functions. We employed ethylene dimethanesulfonate, a Leydig cell toxin, to assess the effects of Leydig cell ablation on t-GHRH-li mRNA and protein levels in prepubertal and postpubertal male rats. The ethylene dimethanesulfonate treatment resulted in decreases in serum testosterone, but had no effect on t-GHRH-li mRNA or peptide levels. To assess the effect of GHRH on Leydig cell steroidogenesis, Leydig cells were isolated by Percoll gradient centrifugation and cultured in the presence or absence of hCG, GHRH, or hCG plus GHRH. GHRH had no effect on steroidogenesis by Leydig cells, either alone or in combination with hCG. To localize t-GHRH-li mRNA within rat testis, in situ hybridization analysis was performed on testicular sections from normal rats, using a [35S]GHRH riboprobe. Grains were detected in spermatogenic cells with the antisense probe, whereas none was detected with the sense strand (control) probe. To verify these results, Northern blot analysis of RNA from separated testicular cells was performed. t-GHRH-li mRNA was detected in spermatocytes and round spermatids and to a lesser extent in Sertoli cells, but not in elongating spermatids, Leydig cells, or peritubular myoid cells. t-GHRH-li mRNA was also not found in epididymis. Since our experiments localized t-GHRH-li mRNA to spermatogenic cells, methoxyacetic acid (MAA), a pachytene spermatocyte toxin, was administered to postpubertal rats to determine whether t-GHRH-li is expressed primarily in pachytene spermatocytes. MAA treatment caused a decrease in testicular weight, which gradually returned to control levels by 42 days. Serum FSH levels in the treated animals fluctuated over the course of the experiment, while those in control animals remained steady. However, there was no difference in testicular GHRH-li mRNA levels between control and treated animals at any treatment time. Insulin-like growth factor-I and -II mRNA levels were also unaltered by the MAA treatment. We conclude from these results that t-GHRH-li is synthesized in early spermatogenic cells, but not in mature sperm, and that testicular GHRH-li does not play a major role in steroidogenesis by the Leydig cell.

Inhibition of aldosterone production by pinacidil in vitro.

Pinacidil, an antihypertensive agent that opens potassium channels, lowers plasma aldosterone levels in hypertensive patients by an unknown mechanism. In the present study, pinacidil's direct effects on production of aldosterone were assessed using isolated cells from bovine adrenal glomerulosa. Pinacidil was found to inhibit aldosterone production, both basally and during stimulation with either potassium, angiotensin II (Ang II), or adrenocorticotropic hormone (p less than 0.001), with half maximal inhibition occurring at 10(-5) M. As assessed by the exclusion of trypan blue from cells, pinacidil did not inhibit secretion through injurious effects on glomerulosa cells. Also, washing of cells previously exposed to pinacidil restored secretory responsiveness. Pinacidil did not alter cytosolic calcium (Ca2+) concentrations when aequorin was used as a photoluminescent indicator of Ca2+ levels, suggesting that pinacidil acted by a non-Ca(2+)-mediated mechanism. Consistent with direct inhibition of the late pathway in steroidogenesis was that pinacidil decreased conversion of pregnenolone and corticosterone to aldosterone. Pinacidil did not block binding of Ang II to its receptor, nor did it appear to affect adrenocorticotropic hormone-receptor binding, since stimulation by cyclic AMP, the post-receptor second messenger of adrenocorticotropic hormone, was also inhibited. In summary, pinacidil inhibited directly the adrenal's production of aldosterone. The mechanism whereby the inhibition occurred was unclear.

Fluoride mobilizes intracellular calcium and promotes Ca2+ influx in rat proximal tubules.

In the renal proximal tubule, external Ca2+ ([Ca2+]o) is required for parathyroid hormone to elevate cytosolic Ca2+ ([Ca2+]i). However, other hormones increase [Ca2+]i in the absence of [Ca2+]o. These differences may arise from a diversity of signal transduction pathways acting on external and internal Ca2+ pools. However, Ca2+ influx may be necessary to expedite and maintain the rise of [Ca2+]i for a period after the initial surge. In this study, F- was used to probe the roles of intracellular Ca2+ mobilization, Ca2+ influx, and phosphoinositide (PI) hydrolysis on the surge of [Ca2+]i in rat proximal tubules. In the presence of external Ca2+; 1-20 mM F- evoked incremental rises of [Ca2+]i in tubules loaded with aequorin. Whereas 10 mM F- increased [Ca2+]i in the absence of [Ca2+]o, the time constant for the [Ca2+]i surge was increased. These findings are consistent with a role of Ca2+ influx on the effect of F- on [Ca2+]i. Indeed, 10 mM F- also enhanced the uptake of 45Ca2+, and promoted Ca2+ influx in aequorin- and fura-2-loaded, Ca(2+)-deprived tubules. In tubules, F- also activated PI hydrolysis with a time course that paralleled Ca2+ mobilization. The effect of F- on [Ca2+]i was not altered when the 39-kDa pertussis toxin substrate was inactivated with the toxin. This G protein was most likely Gi, because prostaglandin E2, an activator of Gi in tubules, dissociated the pertussis toxin-sensitive protein. The results support the notion that activation of a signal-transduction complex, the F- substrate, causes Ca2+ influx, mobilizes internal Ca2+, and activates PI hydrolysis in rat proximal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Na(+)-Ca2+ exchange and Ca2+ depletion in rat proximal tubules.

The removal of external Ca2+ ([Ca2+]o) reduces cytosolic Ca2+ ([Ca2+]i) in rat proximal tubules. In this report the role of external Na+ ([Na+]o) on the changes of [Ca2+]i and Ca2+ efflux caused by withdrawal of [Ca2+]o is described in rat renal proximal tubules. In aequorin-loaded tubules [Ca2+]i decreased from 235 +/- 25 to 48 +/- 16 (n = 4, P = 0.017), and 45Ca2+ fractional efflux ratio (45Ca2+ FER) increased from 0.94 +/- 0.03 to 1.64 +/- 0.19 (n = 6, P = 0.021) when Ca2+ was withdrawn from the bathing media of Krebs buffer (KB). The fall of [Ca2+]i, as well as the activation of 45Ca2+ FER, was reversed when [Na+]o in Ca(2+)-free KB was lowered isosmotically from 150 to 15 mM. However, when tubules were superfused with only 5 mM [Na+]o before [Ca2+]o was removed, [Ca2+]i also declined, but 45Ca2+ FER did not increase. The Na(+)-Ca2+ exchange inhibitor dichlorobenzamil (DCB) added after [Ca2+]o was removed evoked responses similar to [Na+]o removal, although DCB also inhibited internal Ca2+ release. These results are congruous with stimulation of Na+ influx in exchange for [Ca2+]i in Ca(2+)-free KB. However, even though total tubular Na+ was higher in Ca(2+)-free KB after 10 min, the initial rate of 22Na+ influx was not different without or with [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that angiotensin-II and potassium collaborate to increase cytosolic calcium and stimulate the secretion of aldosterone.

Angiotensin-II (AII) and potassium (K+) as stimuli of aldosterone secretion enhance each other's stimulatory potential. In the present study we looked for evidence that AII and K+ act through a common mechanism of signal transduction to affect secretion. Bovine adrenal glomerulosa cells were loaded with the calcium (Ca2+) probe aequorin to permit detection over prolonged time periods of the changes in cytosolic Ca2+ that occur in response to AII and K+. Perfusion fractions were collected for simultaneous measurement of aldosterone production rates. AII (10(-7) M) produced an immediate and transient increase in Ca2+, followed by a Ca2+ plateau that remained above baseline for as long as AII was present. An increase in K+ concentration (from 5 to 12 mM) produced a slow and eventually sustained increase in cytosolic Ca2+, which resembled the plateau produced by AII. Nitrendipine (10(-5) M) completely inhibited the secretory response to AII and K+ (during 60-min incubations) and inhibited the typical K+-induced increase in Ca2+. The sustained increase in Ca2+ with AII (the plateau) required extracellular Ca2+ and was proportional to the prevailing extracellular K+ concentration. When glomerulosa cells were incubated with AII, the aldosterone secretory response to K+ was substantially enhanced (P less than 0.001). In summary, stimulation by both AII and K+ resulted in a sustained increase in Ca2+ influx. AII-induced Ca2+ influx was dependent on the ambient K+ concentration. These results indicate that AII and K+ act together to determine the optimal rate of Ca2+ entry, which may then lead to the appropriate secretory rate of aldosterone.

Na+ electrochemical gradient and Na+-Ca2+ exchange in rat proximal tubule.

The basolateral cell membrane of the rat proximal tubule contains a Na+-Ca2+ exchanger that may participate in the regulation of cytosolic calcium (Cai) and Ca2+ transport. In this work, the activity and orientation of the Na+-Ca2+ exchanger was studied in rat proximal tubules. The experiments were based on the thermodynamic notion that the exchanger is driven by the prevalence of either of two electrochemical gradients, that for Na+ (delta mu Na+) or for Ca2+ (delta mu Ca2+). Reductions in delta mu Na+, achieved by lowering extracellular Na+ (Nao) from 150 to 15 mM, increased Cai, decreased 45Ca efflux, and increased 45Ca influx. These changes occurred concurrently. When delta mu Na+ was reduced by increasing intracellular Na+ (Nai) with 10(-3) M oubain, Cai also increased. The effect of ouabain was probably dependent on Nai accumulation because the surge in Cai was prevented by exposure of the tubules to 5 mM Nao before ouabain exposure. On the other hand, when delta mu Na+ was lowered mM Nao and then by reducing Nao to 15 mM, Cai rose in two additive stages. We conclude from these data that in the rat proximal tubule the basal state of the Na+-Ca2+ exchanger is in forward mode, Nao-Cai. Moreover, the function of the Na+-Ca2+ exchanger is in accord with predictions derived from a thermodynamic analysis of its function.

A randomized controlled trial of quality assurance in sixteen ambulatory care practices.

A crossover randomized controlled trial of cycles of quality assurance in 16 primary care (8 medical, 8 pediatric) group practices was conducted. Of four medical and four pediatric tasks important to patient outcome, two were randomly assigned to experimental intervention (a quality assurance cycle), and two were also measured and used as blinded controls for each medical or pediatric group practice. Task performance was measured in each group for 12 months prior to, 9 months during, and 9 months after the experimental intervention, using as a performance score the percentage of evaluation criteria failed of those applicable to a case. As a result of quality assurance intervention, quality of performance was significantly improved in two of the tasks (P less than 0.0001, with 6.7, and 9.8 percentage points improvement), and marginally improved in one task (P = 0.06, 5.7 percentage points improvement). Surprisingly, tasks with lower perceived effect on patient health (low physician motivation) had greater improvement in quality. Unimproved tasks were associated with the perceived need for delivery system changes beyond the immediate control of the individual practitioner.

Resource requirements for evaluating ambulatory health care.

We implemented the most frequently used form of quality assurance activity: abstracting information on the quality of patient care from medical records and communicating findings to providers in 16 ambulatory care groups. Site providers accepted the evaluation criteria, agreed that deficiencies in care were detected, and, for some medical tasks, effected improvements in care. Direct costs in 1980 dollars for the quality assurance cycle including data system development were $46 per evaluated case. Per-case costs varied considerably among tasks, decreased with larger numbers of cases and as experience grew, and were reduced through computerization. Measured costs were high due to: a demanding research design; our extended accounting of direct, indirect, and induced costs; and the substantial resource requirements of rigorously performed evaluations.

Quality assurance in eight adult medicine group practices.

Four evaluations of ambulatory medical care tasks were developed for use in quality assurance. The evaluations used medical records data and explicit criteria incorporating branching logic. They were implemented in eight general medicine provider groups in two teaching hospitals and six related health centers. Agreement with criteria among 316 provider responses to questionnaires varied from 57% to 100%. The percentage of cases with one or more variation from evaluation criteria, confirmed on peer review to have a deficiency in care, ranged by task from 6% to 42%, with substantial variation between sites. Physician reviewers from each site varied in leniency. Numbers of actions taken to correct deficiencies ranged by site and task from zero to six. Multisite evaluations revealed differences in performance and efforts to improve that are not apparent when each site conducts its own evaluations. More uniformly effective and impartial quality assurance is needed to correct some important deficiencies in care observed in this study.

The cost of quality assurance in medicine.

Cost analysis has been frequently neglected in program evaluations but is currently of high relevance in policy decisions on quality assurance in medicine. The Ambulatory Care Medical Audit Demonstration (ACMAD) Project implemented and evaluated a program of medical record-based quality assurance in eleven sites for nine medical topics. Total direct costs for the project were $1.22 million over five years; indirect costs, $694,000. A computerized data system enabled disaggregation of the cost data by person, timing, type of work, project phase, health topic, health center, and research or operational nature. Of the costs incurred, 79% were for operational reasons, with 21% incurred for research reasons. Costs per audited case were 31% higher in hospitals than in neighborhood health centers. Audit topics of low per-case costs tended to have automated case findings, straightforward and limited abstracting, little need to examine multiple visits, and a low proportion of case-found patients ineligible for audit.

Evaluation of operational failures in clinical decision making.

Medical decision analysts are interested in the changes in health benefits from inappropriate ordering of tests, imperfect information from tests, and imperfect results of treatments. Operational failures in the decision-making sequence, caused by human and system factors, may also alter health benefits. An evaluation of operational failures in follow-up of positive urine cultures in pediatric patients is reported. Branching criteria were developed by physician representatives from each of eight pediatric group practices to evaluate the care of patients aged 6 months to 16 years, inclusive. Of 858 cases evaluated, 52 percent failed at one or more nodes in the criteria sequence. Rates of failure at individual nodes among cases reaching these nodes varied from 2 percent to 48 percent. Like those of other researchers, our findings suggest that decision analyses that do not take operational failures into consideration may inaccurately predict the yield of health benefits achieved in actual practice.