One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing.

The role of intravenously administered recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) on the healing of incisional wounds in rats with impaired healing due to age or glucocorticoid administration was investigated. The administration of methylprednisolone to young adult rats decreased wound breaking strength to 50% of normal control. Breaking strength of incisional wounds from 19-mo-old rats was decreased approximately 27% compared with wounds from normal healing young adult rats. A single intravenous administration of rhTGF-beta 1 (100 or 500 micrograms/kg) increased wound breaking strength from old rats or young adult rats with glucocorticoid-induced impaired healing to levels similar to normal healing control animals when determined 7 d after injury. Even though the circulating half-life of systemically administered rhTGF-beta 1 is < 5 min, a sustained stimulatory effect on extracellular matrix secretion was evident in glucocorticoid-impaired rats when rhTGF-beta 1 was administered at the time of wounding, 4 h after wounding, or even 24 h before wounding. These observations indicate a previously unrecognized potential for the active form of TGF-beta 1 to profoundly influence the wound healing cascade after brief systemic exposure.

Rapid publication. TGF-beta 1 induces bone closure of skull defects.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by increasing collagen synthesis and deposition as well as remodeling at sites of soft tissue repair. Large bony defects in the skull heal by fibrous connective tissue and never form bone unless osteoinductive bony fragments or powders are placed in the defect. We have found, however, that the single application of human recombinant TGF-beta 1 in a simple 3% methylcellulose gel to skull defects induced a dose-dependent increase in intramembranous bone formation. Complete bony bridging of defects occurred within 28 days after treatment with 2 micrograms TGF-beta 1. Sites treated with vehicle alone did not heal with bone formation but rather contained dense fibrous connective tissue between the defect margins.

In vivo induction of bone by recombinant human transforming growth factor beta 1.

A single application of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) adjacent to cartilage was found to induce bone formation in rabbit ear full-thickness skin wounds. At doses that optimally promote soft tissue healing, 25-100 ng rhTGF-beta 1 per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF-beta 1 was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF-beta 1 in hard tissue repair.

Characterization of radioiodinated recombinant human TGF-beta 1 binding to bone matrix within rabbit skull defects.

Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-beta 1 induces bone closure of skull defects: temporal dynamics of bone formation in defects exposed to rhTGF-beta 1.

The temporal dynamics of bone repair in a skull defect in rabbits was examined to characterize the in vivo cellular events occurring following a single local application of recombinant human TGF-beta 1 (rhTGF-beta 1). Rabbits received vehicle or 0.4, 1, 2, or 5 micrograms rhTGF-beta 1 applied to 12 mm defects at the time of surgery. The defect sites were subsequently evaluated by radiography and qualitative and quantitative histology at time points ranging from 1 to 180 days. Based on radiographic assessment, the defect area decreased rapidly in a dose-dependent manner through 35 days after surgery in the rhTGF-beta 1-treated groups. Minimal closure occurred in sites administered vehicle control at all time points examined. Sites treated with rhTGF-beta 1 were characterized histologically by an increase in parameters of active bone formation through 49 days, including percentage osteoid surface, percentage osteoblast/total surface, and an increase in the trabecular bone volume. Bone resorption parameters were increased at 16 and 49 days with histologic evidence of remodeling from woven to lamellar bone. By 70 days, no differences were observed among the groups for parameters of either bone formation or resorption. Bone formation rate was not altered with rhTGF-beta 1 treatment at any time point. These results indicate that exogenously applied rhTGF-beta 1 stimulated the recruitment and proliferation of osteoblasts at the defect site, resulting in a rapid deposition of bony matrix, with normal remodeling processes occurring thereafter. This study supports the hypothesis that TGF-beta 1 is a potent osteoinductive growth factor in vivo and may have potential application as a therapeutic aid to nonhealing bony defects.

Transforming growth factor-beta. Effect on soft tissue repair.

Previous studies have demonstrated that TGF-beta possesses many of the biologic properties necessary for acceleration of the normal wound healing process. We report that recombinant human TGF-beta 2 (rhuTGF-beta 1) increases wound strength and accelerates wound closure when applied topically to experimental wounds. Doses of 5 to 1,000 ng/wound increased wound strength in a dose-response manner and wound strength increase as high as 161% above control in the rat incisional wound model. Increased wound strength was observed as early as 3 days following rhuTGF-beta 1 application and continued to Day 28. In the rabbit ear ulcer model, acceleration of wound closure was observed following doses of 5 to 100 ng/wound applied a single topical application. No adverse effects of rhuTGF-beta 1 were observed. The amount of fibrous tissue, scar formation, and mitotic figures were not significantly greater than control. Epithelialization of rhuTGF-beta 1-treated wounds was not impeded. rhuTGF-beta 1 induced bone formation in the rabbit ear ulcer model but not in the rat incisional model, suggesting that precursor cells, such as perichondrial cells, are required for the bone forming activities of TGF-beta 1.

Combination of bone marrow and TGF-beta1 augment the healing of critical-sized bone defects.

A 1.5 cm segmental defect in the radius of rabbits was used to compare healing at sites administered TGF-beta, with or without autologous bone marrow, to autogenous cortical bone graft. The carrier for TGF-beta consisted of tricalcium phosphate (TCP) granules and hetastarch. The efficacy of TGF-beta formulations and bone marrow (BM) was compared to autogenous bone, carrier control, and untreated defect sites. Bone measurements taken at necropsy included the anterior-posterior (AP) diameter and medial to lateral (LAT) diameter of the defect; the AP and LAT diameters of both radii measured 1 cm proximal to the distal epiphysis, and the AP and LAT diameters of the mid-shaft of the femora. The bones from each group were subdivided for either histological evaluation or for mechanical testing. Strength (maximum torque), energy, angle of rotation and stiffness were determined for both the treated and contralateral radii. Results of the radiographic, necropsy, and mechanical data for defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone were similar and indicated superior healing compared to defects left blank or administered the carrier control with or without bone marrow. Defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone had high mechanical strength relative to the control groups and were characterized histologically as healed primarily with lamellar bone. The results from the defects left blank or administered carrier control were similar and generally characterized by poor healing or nonunion. This study demonstrated substantial equality of healing between 1.0 microgram of TGF-beta1 + BM and autograft indicating that this formulation could function as a substitute for autologous grafts.

Coronary blood flow during cardiopulmonary resuscitation in swine.

Recent papers have raised doubt as to the magnitude of coronary blood flow during closed-chest cardiopulmonary resuscitation. We will describe experiments that concern the methods of coronary flow measurement during cardiopulmonary resuscitation. Nine anesthetized swine were instrumented to allow simultaneous measurements of coronary blood flow by both electromagnetic cuff flow probes and by the radiomicrosphere technique. Cardiac arrest was caused by electrical fibrillation and closed-chest massage was performed by a Thumper (Dixie Medical Inc., Houston). The chest was compressed transversely at a rate of 66 strokes/min. Compression occupied one-half of the massage cycle. Three different Thumper piston strokes were studied: 1.5, 2, and 2.5 inches. Mean aortic pressure and total systemic blood flow measured by the radiomicrosphere technique increased as Thumper piston stroke was lengthened (mean +/- SD): 1.5 inch stroke, 23 +/- 4 mm Hg, 525 +/- 195 ml/min; 2 inch stroke, 33 +/- 5 mm Hg, 692 +/- 202 ml/min; 2.5 inch stroke, 40 +/- 6 mm Hg, 817 +/- 321 ml/min. Both methods of coronary flow measurement (electromagnetic [EMF] and radiomicrosphere [RMS]) gave similar results in technically successful preparations (data expressed as percent prearrest flow mean +/- 1 SD): 1.5 inch stroke, EMF 12 +/- 5%, RMS 16 +/- 5%; 2 inch stroke, EMF 30 +/- 6%, RMS 26 +/- 11%; 2.5 inch stroke, EMF 50 +/- 12%, RMS 40 +/- 20%. The phasic coronary flow signal during closed-chest compression indicated that all perfusion occurred during the relaxation phase of the massage cycle. We concluded that coronary blood flow is demonstrable during closed-chest massage, but that the magnitude is unlikely to be more than a fraction of normal.

Organ blood flow and the cause of death following massive hemorrhage.

We tested the hypothesis that death occurring shortly after massive hemorrhage can be attributed to the development of positive feedback loops arising from deficient blood flow to one or more vital organs such as the heart, brain, and respiratory muscles. The radiomicrosphere technique was used to measure organ blood flow in unmedicated, chronically instrumented swine subjected to removal of 41 ml/kg of blood in 15 min. Ten swine survived the experiments and five died, all within 2 hr after completion of hemorrhage. Cardiac output but not blood pressure was lower in nonsurvivors (mean +/- 1 SD). No difference was found in regard to blood flows to the heart, brain, and diaphragm (flow in ml/gm/min) between survivors and nonsurvivors. Although blood flow posthemorrhage to kidney, organs of the splanchnic bed, and the carcass tended to be greater in survivors compared to nonsurvivors, the difference reached statistical significance only in the small intestine. We conclude that in this model there is no evidence that death following massive hemorrhage results from positive feedback loops arising from deficient perfusion of the coronary or cerebral circulation. It is more likely that death results from abnormalities arising in the carcass and/or splanchnic bed.

Immediate hemodynamic consequences of MAST inflation in normo- and hypovolemic anesthetized swine.

Use of the military antishock trouser (MAST) remains controversial in part because its mechanism(s) of action are poorly understood. We studied two aspects of the hemodynamic response to MAST inflation in 14 anesthetized swine. First, in six swine the relation that existed between inferior vena cava flow and aortic pressure/cardiac output was determined before and during inflation of the MAST, and then before and after removal of 30% of the calculated blood volume. Inflation of the MAST before hemorrhage had little effect on cardiac output but increased aortic pressure by 25%. Inflation of the MAST after hemorrhage increased cardiac output by 41% and increased aortic pressure by 62%. Three different inflation pressures were studied (40, 80, and 120 mm Hg) and were found to give equivalent results. Inflation of the MAST translocated about 3 ml/kg of blood to the heart. A second group of eight swine were instrumented so that the radiomicrosphere technique could be used to measure organ blood flow. Inflation of the MAST following hemorrhage increased coronary perfusion by 50% and cerebral perfusion by about one third. Flow to kidney, liver, and small intestine was not changed. We conclude that, in addition to tamponade of venous bleeding and the splinting of lower extremity fractures, use of the MAST might cause a clinically important increase in the perfusion of the heart and brain in some trauma patients.

Early administration of methylprednisolone promotes survival in rats with intra-abdominal sepsis.

We determined how the following drugs affected survival of 350-gm Sprague-Dawley rats subjected to intra-abdominal sepsis according to the method of Wichterman et al (J Surg Res 29:189-201, 1980): gentamicin (4.5 mg/kg/day), clindamycin (30 mg/kg/day), naloxone (2 mg/kg/hr), or methylprednisolone given either as a continuous infusion (2 mg/kg/hr) or as a bolus (30 mg/kg). A control group received only saline in a volume equal to the drug vehicle volume. Treatment was started immediately after cecal ligation and puncture. Drugs not given by bolus were infused by Alzet mini-pump (Model 2001) for 7 days. Percent of original population surviving at 10 days was (size of original population): saline--48% (92), antibiotics--86% (43), naloxone--30% (43), continuous methylprednisolone--14% (43), bolus methylprednisolone--93% (45). Survival of animals receiving either antibiotics or bolus methylprednisolone was significantly increased over the control population.

Recent nonsteroidal anti-inflammatory drug use increases the risk of early recurrence of bleeding in patients presenting with bleeding ulcer.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID) use is a well-known risk factor for ulcer formation and ulcer complications. The purpose of this study was to determine whether recent NSAID use increases the risk of early recurrence of bleeding in patients who present with bleeding ulcer. METHODS: Clinical and endoscopic data were collected prospectively. Dose, frequency, and duration of recent NSAID use were quantified. Recent NSAID use was defined as consumption of over-the-counter or prescription NSAIDs or aspirin for at least 5 days of the 2-week period preceding the index episode of bleeding. Endoscopy was performed within 24 hours of admission to confirm the source of bleeding and endoscopic intervention was applied for stigmata of bleeding. Early recurrence of bleeding was defined as melena, hematochezia or blood per nasogastric tube with a 2 gm or greater decrease in hemoglobin during a period of 48 hours, occurring less than 2 weeks from index episode of bleeding. RESULTS: One hundred twenty patients (52 NSAID users and 68 nonusers) were enrolled in the study; mean age was 56 years. NSAID users were older than nonusers (p = 0.003); nonusers were more likely to have a history of ulcer disease (p < 0.0005) and higher prevalence of Helicobacter pylori infection (p = 0.05). Recent NSAID use was associated with a significantly higher frequency of early recurrence of bleeding and in-hospital recurrent bleeding compared with nonusers: 19% vs. 6%, p = 0.02, and 17% vs. 6%, p = 0.04, respectively. In multivariate logistic regression analysis, the significant association between recent NSAID use and early recurrence of bleeding persisted (p = 0.0048) while controlling for age and other covariates. CONCLUSIONS: Recent NSAID use predisposes bleeding ulcer patients to early and in-hospital recurrent bleeding, probably via its effects on platelet function, mucosal prostaglandins, and ulcer healing.

Military antishock trousers prolong survival after otherwise fatal hemorrhage in pigs.

Military antishock trousers (MAST) have not received universal acceptance for use during hypovolemia because cardiac output results have been conflicting, lower extremity compartment syndromes have been reported, and a prospective clinical trial has not been performed. We completed survival studies in a fatal porcine hemorrhage model which simulates human exsanguination. Trousers were specifically made for the weight of the swine in this study and then were calibrated by observing transmitted pressure to the lower abdomen and upper hind limbs. When the trousers were inflated to 60 torr, 77% of the inflation pressure was transmitted to the proximal lower leg (46 +/- 8 torr) and 58% was monitored in the lower abdomen (35 +/- 4 torr). Compared to a noninflation control group, we found the survival time significantly prolonged after an otherwise fatal hemorrhage. The mechanism appears to be through increased tissue perfusion probably not related to a transient translocation of lower extremity blood volume.

Assessment of technical competence during ERCP training.

BACKGROUND: Successful performance of diagnostic and therapeutic ERCP requires skillful manipulation of the duodenoscope and accessories. The evaluation process for assessing competency is still in evolution. Recommendations for the number of examinations has ranged from 35 to 200, made without the benefit of prospective data. METHODS: Pancreatic and common bile duct cannulation rates were prospectively recorded for 21 trainees and 9 proctors over 6 years in a large university-based training program. Trainee success rates were compared to those of the proctor and learning curves were constructed. RESULTS: Trainees performed 641 examinations over 6 years. Each did an average of 31 examinations (range, 10 to 96). For both pancreatic duct and common bile duct cannulation, there was a rapid linear rise of the success curve extending up to the fortieth procedure. Pancreatic duct cannulation rates exceeded those of the common bile duct. CONCLUSIONS: This is the first prospective evaluation of acquisition of skills in ERCP. Although the rapid rise of the learning curve ends at the fortieth examination, the 85% level of selective cannulation is not reached for the pancreas duct until the seventieth procedure and is not reached for the common bile duct even at 100 procedures. These data suggest a threshold of at least 100 procedures.

Clinical and histologic predictors of response to interferon-alpha in patients with chronic hepatitis C viral infection.

To evaluate if any pretreatment characteristics of patients with chronic hepatitis C (HCV) can be used to predict response to the current recommended dose (3 million units three times a week) and higher doses of interferon-alpha (IFN), we retrospectively assessed the response of 37 patients with HCV who were treated with IFN. Sixteen patients (43%) responded to the standard dose of IFN with normalization of ALT. Weight and liver histology were found to be significant factors for response. The responders weighed significantly less than nonresponders (161.8 +/- 35.5 lb versus 200.3 +/- 45.4 lb, P = 0.008). Seventy-five percent of patients with chronic lobular or persistent hepatitis were responders, whereas only 28% of patients with more advanced hepatitis responded (P = 0.01). There was no correlation between the degree of bile duct damage or steatosis and response rate. This study suggests that obesity and severe histologic injury are negative predictive factors of response to the current recommended dose of IFN. The adequacy of the current recommended dose of IFN in overweight patients needs to be investigated.

Fluid resuscitation after an otherwise fatal hemorrhage: II. Colloid solutions.

We developed a fixed-volume porcine hemorrhage model that simulates the rapid exsanguination of combat or civilian trauma victims. In this study we compared the ability of colloid resuscitation solutions to prevent death after an otherwise lethal hemorrhage in 100 swine. The shed blood was replaced in a 1:1 ratio with either autologous whole blood (WB), untyped swine fresh frozen plasma (FFP), typed FFP, 5% human serum albumin (ALB), or normal saline (NS). Survival rate analysis indicated that WB was significantly better than FFP (untyped), ALB, or NS but not better than typed FFP. The 24-hour survival rates were: WB = 90%, typed FFP = 79%, untyped FFP = 56%, ALB = 57%, and NS = 25%. All deaths in the untyped FFP group suddenly occurred during or within 15 minutes after treatment in a recovering animal. Deaths in the ALB group steadily occurred for up to 2 1/2 hours after treatment. Analysis of hemodynamic, arterial blood gas, and acid-base data indicated that WB and FFP provided a better acid-buffering capacity in surviving animals than NS or ALB. We conclude that compatible FFP is a better resuscitation agent than ALB after an otherwise fatal hemorrhage because FFP is a better acid buffer.

TGF-beta 1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits.

TGF-beta modulates events of normal wound healing through multiple pathways that influence cell infiltration, proliferation, angiogenesis, extracellular matrix synthesis and remodeling. The effects of topically applied TGF-beta 1 on wound healing in two models of healing were evaluated when the healing response was impaired by the administration of methylprednisolone to rats or rabbits. TGF-beta 1 increased the healing of linear incision wounds on rats, as measured by breaking strength, to that of normal rats. Full thickness open wounds were also created on the inner ears of rabbits to simulate a non-contracting wound with limited blood supply. Healing was further impaired by the administration of methylprednisolone. The single application of TGF-beta 1 improved the healing of open wounds. TGF-beta 1 stimulated increased granulation tissue formation, as well as reepithelialization. The amount of granulation tissue and epithelialization were similar to wounds from normal-healing control rabbits. The delayed healing caused by methylprednisolone permitted the evaluation of multiple applications of TGF-beta 1 to wounds. Two applications of TGF-beta 1 spaced 7 days apart further improved the healing response when compared to a single application. Thus, single or multiple topical applications of TGF-beta 1 reversed impaired healing conditions secondary to methylprednisolone when used on incisional or open wounds. These observations support the hypothesis that growth factors, such as TGF-beta 1, may be useful as accelerators of wound repair in patients with impaired healing conditions.

The validation of the Peer Assessment Rating index for malocclusion severity and treatment difficulty.

The Peer Assessment Rating (PAR) index is a British occlusal index that measures the severity of dental malocclusion and has been used in several investigations that have evaluated the effectiveness of orthodontic treatment provision in Europe. As part of its development, the PAR index was validated for malocclusion severity, by using the opinions of a panel of 74 dentists and orthodontists. The present investigation was carried out to validate the PAR index, by using the opinion of an American panel of orthodontists. Eleven orthodontists examined a sample of 200 sets of study casts and rated them for malocclusion severity and perceived treatment difficulty. Multiple regression techniques were used to evaluate the predictive power of the components of malocclusion on the panel's scores. Weightings were calculated from the partial regression coefficients and, when these weightings were applied to the PAR index, the association between the panel's opinion and the PAR index scores was increased.

Diagnosis of acute hepatitis E infection utilizing enzyme immunoassay.

Acute hepatitis E infection was diagnosed in a Pakistani immigrant admitted to the University of Illinois Hospital. Utilizing enzyme immunoassay (EIA) tests, specific IgG and IgM class antibodies to three different epitopes of hepatitis E virus (HEV) were detected 12 weeks after the onset of illness and in the early convalescent stage. Sixteen months after the onset of hepatitis, IgM anti-HEV was no longer detectable. Low levels of IgG class anti-HEV antibodies continued to be detected. We demonstrate the utility of the EIA HEV assay to diagnose prospectively acute HEV infection.

Regional blood flow during hypothermic arrest.

Little is known about the efficacy of CPR in the setting of hypothermia-induced cardiac arrest. We measured organ blood flow produced by conventional closed-chest CPR in eight swine following normothermic KCl-induced cardiac arrest and in seven swine surface-cooled until cardiac arrest occurred. Radiomicrospheres were injected in the unanesthetized basal state, after five minutes of CPR, and after 20 minutes of CPR. After five minutes of CPR, the cardiac output and cerebral and myocardial blood flows (mean +/- SD) of hypothermic animals were 15.3 +/- 7.5 mL/min/kg, 0.16 +/- 0.11 mL/min/g, and 0.20 +/- 0.15 mL/min/g, respectively. Mean percentage flows were 7%, 15%, and 8%, respectively, of those measured in the unanesthetized prearrest state, and 50%, 55%, and 31%, respectively, of the flow produced during CPR in normothermic animals. Blood flow during hypothermic CPR did not change significantly over time; however, during normothermic CPR, cardiac output and cerebral and myocardial flows decreased so that at 20 minutes there were no significant differences from those values measured in hypothermic animals. The reduction in organ flow produced by external chest compression in hypothermic animals may be a result of the changes in the viscoelastic properties of the thorax that occur during profound hypothermia.