Acceleration of tissue repair by transforming growth factor beta 1: identification of in vivo mechanism of action with radiotherapy-induced specific healing deficits.

BACKGROUND: Transforming growth factor beta 1 (TGF-beta 1) is an effective accelerator of soft tissue repair in both normal and impaired healing models; however, its in vivo mechanism of action remains unclear. Modern radiation techniques can create unique healing deficits, allowing for a more specific definition of tissue response to growth factor therapy. In the rat linear skin incision model, cobalt 60 photon beam total body irradiation (TBI), 800 rads, causes a marked depression of circulating monocytes and largely spares the skin tissue. Megavoltage electron beam surface irradiation (SI), 2500 rads, markedly impairs surface healing while sparing the bone marrow. With these models of selective healing deficits, the ability of TGF-beta 1 to accelerate tissue repair directly in the absence of circulating macrophage precursors (TBI) or in the presence of damaged dermal fibroblasts (SI) was evaluated. METHODS: Adult male Sprague-Dawley rats were randomly assigned to groups of TBI, SI, or nonirradiated sham controls and received radiation 2 days before wounding. Paired linear full-thickness skin incisions were created and a single dose of TGF-beta 1 (2 micrograms/wound) or vehicle control was applied to each wound. RESULTS: Both radiation techniques produced a marked healing deficit when assessed on postwounding days 7 and 12. TBI treatment was characterized by severe monocytopenia, confirmed by a tissue macrophage-specific immunohistochemical technique. On days 7 and 12 after wounding, TGF-beta 1 significantly accelerated soft tissue repair and wound-breaking strength in the TBI-treated rats, demonstrating an ability to directly promote the induction of collagen synthesis in the absence of monocytes/macrophages. In contrast, TGF-beta 1 was unable to reverse the SI-induced healing deficit characterized by impaired function of dermal fibroblasts. CONCLUSIONS: These in vivo observations provide further evidence for a direct mechanism of action by TGF-beta 1 on collagen synthesis by wound fibroblasts during soft tissue repair.

Pathology and treatment of impalpable breast lesions.

With the increasing use of mammography, more needle-localized breast biopsies (NLBB) are being done. The purpose of this study was to analyze the pathology of impalpable breast lesions and the impact of NLBB on treatment strategies. From 1985 to 1990, 1,605 NLBB were performed, of which 321 (20%) were malignant. Twenty-five percent of malignant biopsies demonstrated in situ disease only. The average size of all lesions detected was 16 mm, and, for invasive cancer, 12 mm. Eighteen percent of invasive cancers had metastasized to the axillary lymph nodes. Surgical management consisted of mastectomy in 74% of patients and breast conservation treatment (BCT) in 26%. No significant difference in surgical management for women 50 years of age or younger compared with those older than 50 years of age was noted. Although the use of BCT for eligible women is recommended by the National Institutes of Health, it is not widely practiced, possibly reflecting less physician acceptance of BCT. These observations suggest that the detection of smaller, impalpable breast cancers has had no impact on treatment strategies.

Pulsatile activation of the hypothalamic-pituitary-adrenal axis during major surgery.

To examine the response of the hypothalamic-pituitary-adrenal (HPA) axis to severe surgical stress, we measured the immunoreactive plasma levels of corticotropin-releasing hormone (CRH), corticotropin, cortisol, arginine-vasopressin (AVP), atrial natriuretic factor (ANF), neuropeptide Y (NPY), interleukin-1 (IL-1), IL-6, interferon gamma (INF), and tumor necrosis factor-alpha (TNF-alpha) in eight patients with Zollinger-Ellison syndrome (ZES) or mediastinal parathyroid carcinoma, all undergoing major surgery with a standardized anesthetic technique. Blood samples were drawn the morning before surgery, every 10 to 30 minutes throughout surgery (average, 308.7 +/- 15 minutes), and every morning for the next 4 postoperative days (POD). During surgery, plasma CRH concentrations were slightly but not significantly elevated compared with those before surgery and with those of the next 4 POD. However, the values were within the normal range (less than 2.2 pmol/L) and showed 8.9 +/- 0.6 pulses (one pulse every 34.7 +/- 1.6 minutes). Plasma corticotropin, on the other hand, was quite elevated, but was also released in a pulsatile fashion during the surgical procedure (one pulse every 36.7 +/- 1.6 minutes). Most of these secretory episodes of corticotropin were temporally related to those of CRH. Corticotropin returned to basal levels on the first POD and remained so for all 4 POD. Plasma cortisol concentrations increased steadily during surgery and remained elevated the first POD. Cortisol showed 6.2 +/- 1.1 pulses during the operative sampling period (one pulse every 71.8 +/- 13 minutes). Plasma AVP concentrations were also markedly elevated during surgery, but individual secretory pulses were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Acceleration of soft tissue repair by a thrombin-derived oligopeptide.

Augmentation of thrombin-modulated chemotaxis and mitogenic activity within the early phase of soft tissue repair is now possible. Identification of high-affinity thrombin receptor binding domains within thrombin has enabled the synthesis of a family of peptides which interact with thrombin receptors and enhance in vitro mitogenesis. A single (5.0 micrograms/wound) application of the thrombin receptor-activating peptide (P517-30) significantly increased wound breaking strength from Day 5 (31% over controls) to Day 12. Two models of impaired healing created by radiotherapy (RT) were used to elucidate possible mechanisms of P517-30 action. Although P517-30 did not completely overcome the RT-induced healing impairments, it increased breaking strength under conditions of penetrating whole body RT-induced pancytopenia by 22% and of nonpenetrating surface RT-induced dermal cell damage by 42%. This suggests that P517-30 directly stimulates resident endothelial cells, fibroblasts, or other cells to overcome dermal and circulating monocytic deficits. These results suggest a method to accelerate wound healing with potential clinical applications and emphasize the activity of thrombin as a growth factor.

The local effects of cachectin/tumor necrosis factor on wound healing.

Previous experimental studies have suggested that tumor necrosis factor (TNF) may have either a beneficial or a detrimental role in wound healing. Control and doxorubicin-treated (6 mg/kg, intravenously) rats underwent paired dorsal 5-cm linear wounds and had either vehicle or recombinant (r)TNF (0.5, 5, or 50 micrograms) applied locally to the wound. Paired wounds were harvested at 7 and 14 days after wounding and analyzed for wound-bursting strength (WBS) and activity of the gene for type 1 collagen and TNF. Doxorubicin treatment decreased WBS at 14 days but not at 7 days after wounding. Local application of 50 micrograms of rTNF decreased WBS in saline-treated rats and concentrations of 5 and 50 micrograms decreased WBS in doxorubicin-treated rats when measured 7 days after wounding. These effects dissipated when WBS was measured 14 days after wounding. Doxorubicin decreased wound collagen gene expression and local TNF treatment decreased wound collagen gene expression in saline-treated rats and further decreased it in doxorubicin-treated rats. The decrement in collagen gene expression induced by rTNF increased as the local dose of rTNF increased. The gene for TNF was not detectable in wounds from normal or doxorubicin-treated rats at 3, 7, 10, or 14 days after wounding. These data suggest that the gene for TNF is not expressed in wounds and that the local application of TNF is detrimental to wound healing as it decreases WBS and activity of the gene for collagen.

Current concepts in wound healing: growth factor and macrophage interaction.

Growth factors are potent wound healing promoters which accelerate incisional wound repair by distinct mechanisms. Transforming growth factor-beta (TGF-beta), a chemotactic factor, increases synthesis of extracellular matrix and stimulates granulation tissue. We demonstrated that a single topical dose of TGF-beta increased the wound breaking strength in normal models of tissue repair as well as in models of impaired wound repair, characterized by severe monocytopenia. PDGF, a chemotactic agent for inflammatory cells, with mitogenic activity, activates monocytes and stimulates collagen production, significantly increased the wound breaking strength with effects that lasted for up to 47 days. In contrast to TGF-beta, PDGF was only active in normal models of wound healing and its effects were dependent upon the presence of macrophages. PAF is a glycerophospholipid which chemotaxes and activates macrophages, but differs from growth factors in lacking mitogenic activity. A single topical dose of PAF significantly increased the wound breaking strength and promoted macrophage migration.

Localization and surgical treatment of occult insulinomas.

Management of patients with biochemical evidence of insulinoma and negative preoperative imaging studies (occult) tumors is controversial, varying from primarily medical management to aggressive, blind nearly total pancreatectomy to extirpate the tumor. Since 1982, 12 consecutive patients with occult insulinoma underwent preoperative portal venous sampling (PVS) for insulin followed by surgical exploration with intraoperative ultrasound (IOUS). Eleven of twelve patients (92%) had insulinoma removed and were cured. Portal venous sampling correctly predicted the location of the insulinoma in 9 patients (75%) and that no tumor would be found in another patient. A fourfold insulin gradient in the pancreatic tail of one patient correctly predicted that a distal pancreatectomy would remove the insulinoma despite the fact that neither palpation nor IOUS identified any tumor. Intraoperative ultrasound was the single best method to identify occult tumors because it correctly identified 10 of 11 insulinomas that were found, including five pancreatic head tumors that were not palpable. Palpation identified five insulinomas. Of the 10 tumors that were identified during operation by palpation or ultrasound, IOUS identified significantly more (100% versus 50%, p = 0.03) and guided the successful enucleation of each. The results support the strategy of preoperative PVS and operation with IOUS to localize and remove insulinoma in patients with occult tumors. Most tumors (75%) will be correctly localized to a specific pancreatic region by preoperative PVS and identified by IOUS (83%), allowing simple enucleation and biochemical correction of hypoglycemia. Morbid blind pancreatic resections are no longer indicated and long-term medical management of hypoglycemia should be reserved for the occasional patient (8%) who fails preoperative PVS and operation guided by IOUS.

Differential acceleration of healing of surgical incisions in the rabbit gastrointestinal tract by platelet-derived growth factor and transforming growth factor, type beta.

Anastomotic dehiscence is a major cause of morbidity and mortality in gastrointestinal surgery. A unique model system of a gastric incision was developed to test the potential of polypeptide growth factors to enhance wound healing. Paired, deep partial-thickness incisions to but not including the gastric mucosa were made. A single topical application of transforming growth factor, type beta 1 (TGF-beta), platelet-derived growth factor, or control vehicle at the time of wounding was given. Wound breaking strength and detailed histologic analyses of wounds were evaluated as a function of time after wounding. TGF-beta (0.1 to 2.0 micrograms/wound) demonstrated a bimodal, dose-dependent acceleration of wound breaking strength 7 days after gastric wounding. An approximate 4-day acceleration of gastric wound breaking strength by TGF-beta (2 micrograms/wound) was seen at 7 and 11 days. Wounds treated with platelet-derived growth factor (10 micrograms/wound) displayed an increased cellular response but no enhancement of breaking strength at 7 and 11 days. These results demonstrate the ability of TGF-beta to accelerate gastrointestinal tissue repair by topical application and suggest significant potential for the use of growth factors in enhancing repair of surgical wounds of the gastrointestinal tract.

Are complications in intraoperative radiation therapy more frequent than in conventional treatment?

To evaluate whether intraoperative radiation therapy (IORT) results in higher complication rates than conventional radiotherapy, 119 patients were studied who entered four prospectively randomized clinical trials that compared IORT with conventional therapy. Malignant neoplasms included 33 gastric carcinomas, 35 retroperitoneal sarcomas, 22 resectable pancreatic cancers, and 29 unresectable pancreatic cancers. One hundred thirty-six complications developed among 66 patients who received conventional therapy, and 108 complications developed among 53 patients who received IORT. There was no statistical significance between treatment groups with respect to the overall incidence of complications. Analysis of types of complications by tumor type using Fisher's exact test revealed only one significant complication: an increased rate of sepsis among the patients with retroperitoneal sarcoma who received conventional therapy compared with their IORT cohorts. The overall complication rate associated with IORT was equivalent to conventional radiotherapy in the treatment of these malignant neoplasms and supported the use of IORT where clinically indicated.

Intraoperative ultrasonographic localization of islet cell tumors. A prospective comparison to palpation.

The purpose of the present study was to evaluate prospectively the value of intraoperative ultrasound scanning (IOUS) in localizing islet cell tumors by comparing results of IOUS to those of palpation during 44 consecutive laparotomies for gastrinoma (36) or insulinoma (8). All patients had preoperative radiographic imaging studies and selective venous sampling for hormones, which guided the subsequent laparotomy. Any suspicious finding by palpation and/or IOUS was resected. Pathologic evidence of islet cell neoplasm served as the reference standard. Five patients were excluded from analysis because neither palpation nor IOUS had suspicious findings and no islet cell tumor was found. Seven pancreatic insulinomas were found in seven patients. IOUS was as sensitive as palpation at localizing insulinomas. Twenty-three pancreatic gastrinomas were found in 19 patients. IOUS was equal to palpation in the ability to localize gastrinomas. Gastrinomas that were successfully imaged by IOUS were significantly larger than gastrinomas that were not imaged. Twelve extrapancreatic gastrinomas were found in nine patients, and palpation was more sensitive than IOUS at localizing these small duodenal wall tumors. Five patients (11%) had their surgical management changed by IOUS. Two patients had pancreatic tumors (one gastrinoma and insulinoma) enucleated that would not have been found without IOUS, and three patients had resections of pathologically proven malignant islet cell tumors based on sonographic findings. All five patients were cured with short follow-up. The present results demonstrate that palpation and IOUS are complementary because IOUS can image tumors that are not palpable and IOUS can provide additional information concerning malignant potential not detected by palpation.

Transforming growth factor beta levels in rat wound chambers.

Exogenous TGF-beta accelerates healing in both normal and doxorubicin-treated rats, but whether it plays an intrinsic role in the natural healing process is unknown. Subcutaneous wound chambers in 16 F344 rats were aspirated from postwounding Day 3 through Day 16 for TGF-beta levels and cytology. A soft agar assay and a competitive radioreceptor binding assay were used to determine TGF-beta levels. Papanicolau staining and differential cell counts were used to determine cytology. Results were similar using either method for the determination of TGF-beta levels. With the sensitive radioreceptor assay, low TGF-beta levels on postwounding Day 4, mean 2.6 ng/ml, rose to a peak mean level of 20.4 ng/ml on Day 7 and fell significantly from peak level to a level of 5.4 ng/ml of Day 16. All TGF-beta levels for postwounding Days 6 through 14 were significantly increased over the baseline TGF-beta levels of Days 4 and 5 (P less than 0.05). Day 16 TGF-beta levels were not different from baseline. Cytologic changes were characterized by a liner decrease in total neutrophil count over the exam period and a concurrent linear increase in total lymphocyte and macrophage counts. TGF-beta levels changed in a bell-shaped temporal sequence during healing, apparently unrelated to percentage lymphocyte, macrophage, or neutrophil count. Peak TGF-beta levels occurred during the fibroblast proliferation and collagen synthesis phase of healing. This study presents the first evidence that TGF-beta is present in a healing wound and suggests that it may be an intrinsic mediator of the healing process.