Joseph D. Campeau

Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome

Serum measurements of bioactive (bio) luteinizing hormone (LH), immunoreactive (i) LH, iLH/follicle-stimulating hormone (FSH) ratios, serum androgens and estradiol (E2) were determined in 20 women with the clinical diagnosis of the polycystic ovary syndrome (PCO), and compared with the levels of 10 women with chronic anovulation (CA) and 10 control subjects in the early follicular phase. Women with CA and control subjects had similar levels of E2, androgens, bioLH, iLH, and iLH/FSH ratios. Fourteen of 20 women with PCO had levels of iLH exceeding 3 standard deviations (SD) of the levels of control women (21 mIU/ml), and 13 of 20 had iLH/FSH ratios above 3.2 (3 SD of control levels). Nineteen of 20 women, however, had bioLH levels above 70 mIU/ml (3 SD of control levels). Mean levels for bioLH were 131 +/- 18 in PCO, 39 +/- 3 in control subjects, and 40 +/- 3 in women with CA. The ratio of bioLH/iLH was 3.5 +/- 0.4 in control subjects and 3.2 +/- 0.3 in women with CA but significantly elevated in PCO (4.6 +/- 0.4, P less than 0.05). There was, however, a significant positive correlation between bioLH and iLH values in PCO (r = 0.64, P less than 0.01). A significant correlation was found between bioLH and serum testosterone as well as between bioLH and serum dehydroepiandrosterone sulfate (DHEA-S) (P less than 0.05), although no correlation was found between iLH and serum DHEA-S. Weight and obesity also did not correlate with either iLH or bioLH in women with PCO and CA. These data suggest that bioLH may be an important hormonal marker in the clinical diagnosis of PCO.

Decreased endopelvic fascia elastin content in uterine prolapse.

Background. Genital prolapse is a debilitating manifestation of pelvic floor dysfunction. The cause of this condition has not been elucidated. The purpose of this study was to determine elastin content and RNA expression of related enzymes of elastin synthesis in uterosacral ligament biopsies from women with severe prolapse, and controls with normal pelvic support. Methods. Biopsies were taken from the uterosacral ligament tissue of 31 women with Grade III or greater prolapse and 29 women with normal pelvic support. Elastin content was assessed by measuring desmosine using radioimmunoassay, and quantitative real time PCR was performed to quantify mRNA levels of lysyl oxidase (LOX), lysyl oxidase like‐1 (LOXL1), LOXL2 and fibulin‐5 (FIB‐5). Results. The mean desmosine concentration found in uterosacral ligaments of women with prolapse (n =26) was 103.3±59.3 pmolD/mgP compared to controls (n =29) 120.5±47.4 pmolD/mgP (p =0.1943). In the subgroup of subjects with complete procidentia (n =8), mean desmosine concentration was 50.6±25.8 and 127.1±42.2 pmolD/mgP in age‐matched controls (n =12) (p <0.05). In tissue from subjects with more than 2 vaginal deliveries (n =18), the mean desmosine concentration was 99.9±60.7 and 133.0±44.0 pmolD/mgP in controls (n =17) (p <0.05). Expression of LOX, LOXL1 and LOXL2 decreased 8.2‐fold±3.4, 5.0‐fold±1.7 and 15.2‐fold±5.2, respectively (mean±SD) in cases versus controls (p<0.05). Expression of FIB‐5 was increased 3.1‐fold±0.7 compared to controls (p<0.05). Conclusions. Significantly decreased desmosine content was measured in the uterosacral ligament tissue from women with prolapse versus controls in women with parity >2 and in women with complete procidentia. Suppression of mRNA for LOX and two LOX isoenzymes was correspondingly present. These results suggest that altered elastin metabolism is present in women with uterine prolapse.

Comparative electrophoretic analysis of human and porcine plasminogen activators in sds-polyacrylamide gels containing plasminogen and casein

Electrophoretic analysis of plasminogen activators from pig heart, human uterus, human plasma and human melanoma cells was performed in SDS-polyacrylamide gradient slab gels containing plasminogen and casein. Direct visualization of activator activity bands in polyacrylamide gels was achieved after removal of SDS, incubation in buffer, and staining with Coomassie brilliant blue. Tissue activator extracted from pig hearts displayed a molecular weight of 72000 and migrated similarly to activator secreted by human melanoma cells and to one activator component present in extracts of human uterus. Immunoadsorption experiments with melanoma cell activator antiserum indicated that these 72-kDa activators are all related immunologically. Human uterus also contained a second activator component with a molecular weight 55000, which migrated similarly to a higher molecular weight component of urokinase and cross-reacted with urokinase antiserum. We conclude that the 72-kDa uterine activator component represents a tissue activator and the 55-kDa component represents a urokinase-like activator. A euglobulin solution from venous occlusion plasma displayed multiple bands of plasmin activity in the Mr range 85000-96000. Two activator components were also present, one of Mr 72000 and another of Mr 62000. The 72-kDa euglobulin activator was adsorbed by MCA antiserum, and we conclude that this component represents vascular activator. The 62000 activator also had weak plasminogen-independent caseinolytic activity and was not affected by either melanoma cell activator or urokinase antisera. Conclusions concerning its identity cannot be made at this time.

Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits

OBJECTIVE To examine the effect of hyaluronic acid, a high-molecular-weight glucosaminoglycan found in the extracellular matrix, on the formation of adhesions, a major source of postoperative complications. DESIGN The ability of hyaluronic acid to reduce adhesion formation was evaluated using a standardized rabbit model. The material was administered i.p. at the end of surgery. SETTING University laboratory. ANIMAL(S) New Zealand White female rabbits. INTERVENTION(S) Intraperitoneal administration of various formulations of hyaluronic acid at the end of surgery. MAIN OUTCOME MEASURE(S) One week after surgery, a second laparotomy was performed and the extent of adhesion formation was determined. RESULT(S) Five separate molecular weight ranges of hyaluronic acid representing eight viscosities between 1,000 and 12,000 centipoise (CPS) were shown to reduce adhesion formation in this model. All volumes, 1 to 30 mL, of hyaluronic acid tested reduced adhesion formation. In addition, the low-viscosity, low-molecular-weight hyaluronic acid significantly reduced adhesion formation when added to the trauma site or when injected at a site remote from the trauma area. CONCLUSION(S) This study showed that hyaluronic acid administered at the end of surgery reduced adhesion formation.

Acceleration of dermal tissue repair by angiotensin II

Angiotensin II is a naturally occurring peptide which has been shown to possess angiogenic properties. In the studies reported here, angiotensin II was shown to increase the proliferation of cultured bovine aortic arch endothelial cells in a concentration‐dependent manner. Acute administration of angiotensin II in Hydron accelerated the repair of dermal injuries in a full‐thickness excisional rat model. Additional studies were done to determine the best vehicle for delivery of angiotensin II to a dermal injury. Several vehicles, including 10% low‐viscosity carboxymethyl cellulose, 4% medium‐viscosity carboxymethyl cellulose, and 3% high‐viscosity carboxymethyl cellulose, were found to be effective in this regard. Daily administration of angiotensin II for days 0 to 4 after injury (day 0 being the time of surgery) was determined to provide the optimal dosage for acceleration of wound repair by angiotensin II. In addition, dose‐response studies indicated that angiotensin II accelerated wound repair in a dose‐dependent fashion with 0.03 and 0.01 µg/rat/day of angiotensin II administered on days 0 to 4 being the minimally effective and no‐effect doses, respectively. Administration of 100 µg/day of angiotensin II in 10% carboxymethyl cellulose for 5 days after injury to animals with impaired healing (steroid‐ and adriamycin‐treated rats and diabetic mice) was also found to accelerate the rate of repair. In conclusion, angiotensin II accelerated the closure of full‐thickness skin injuries in a dose‐dependent manner in normal and impaired animal models.

The control of bioactive luteinizing hormone secretion in women with polycystic ovary syndrome.

Serum bioactive luteinizing hormone (LH) is elevated in virtually all patients with polycystic ovary syndrome, whereas serum immunoreactive LH may not be increased. The resultant increase in the bioactive: immunoreactive LH ratio in polycystic ovary syndrome leads to the suggestion that a more biologically active form of LH may be secreted in patients with polycystic ovary syndrome. This study was designed to investigate the control of bioactive LH in polycystic ovary syndrome. Compared to matched control subjects, seven patients with polycystic ovary syndrome had higher levels of serum immunoreactive LH (24 +/- 3 mlU/ml), immunoreactive LH: follicle-stimulating hormone (FSH) ratios (4.6 +/- 0.6), bioactive LH (98 +/- 27 mlU/ml), and bioactive: immunoreactive LH ratios (4.6 +/- 0.5). Serum testosterone (64 +/- 10 ng/ml), unbound testosterone (16 +/- 3 mg/dl), and unbound estradiol (49 +/- 5 pg/ml) were also higher. In response to 150 micrograms of intravenous gonadotropin-releasing hormone, increments of both bioactive LH and immunoreactive LH were higher than those in control subjects, but the bioactive: immunoreactive LH ratio was unaltered. Although urinary homovanillic acid was lower in polycystic ovary syndrome, it did not correlate with the bioactive: immunoreactive LH ratio. Similarly, the bioactive: immunoreactive LH ratio was not altered by 1 week of L-dopa (500 mg) or after another week of L-dopa (400 mg) with carbidopa (100 mg) 1 month later. Although baseline unbound estradiol correlated with the delta maximum response of bioactive LH after gonadotropin-releasing hormone (r = 0.65, p less than 0.05), unbound estradiol did not correlate with the bioactive: immunoreactive LH ratio. However, there was a significant positive correlation between the baseline bioactive: immunoreactive LH and the increased delta maximum responses of both immunoreactive LH (r = 0.55) and bioactive LH (r = 0.58), p less than 0.05. These data suggest that, although gonadotropin-releasing hormone stimulation, dopamine, and estrogen may not selectively increase the pituitary secretion of bioactive LH, the sensitivity of the pituitary gland itself and the hyperdynamic state of gonadotropin secretion in polycystic ovary syndrome may result in the increased secretion of bioactive LH.

The hemostatic effect of deacetylated chitin membrane on peritoneal injury in rabbit model

In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion. Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4×4 cm) or an abrasion of liver surface (3×2 cm). The injured surface was then covered with a 0.2 mm thick DAC-80 membrane. On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid. The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9±0.8; control: 24.6±5.9×108 cells/peritoneal cavity, P<0.005). This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model. We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate. The DAC-80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC-80: 2.8±0.7; control: 3.9±0.9 mPU/ml). The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion. No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models.These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.

Comparative Efficacy of Nonsteroidal Anti-Inflammatory Drugs and Anti-Thromboxane Agents in a Rabbit Adhesion-Prevention Model

A variety of nonsteroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit postsurgical peritoneal adhesion formation in a number of animal models. A rabbit uterine horn adhesion model was used to directly compare several commonly used NSAIDs of different chemical classes in a single animal study to evaluate their ability to prevent adhesion formation. The effect of thromboxane inhibitors on adhesion prevention was also evaluated. Each of the NSAIDs tested (tolmetin, ibuprofen, aspirin, and indomethacin) showed significant and comparable efficacy. In this same study, imidazole, a thromboxane synthetase inhibitor, also showed significant efficacy. In a second study, ridogrel, an inhibitor of thromboxane synthetase as well as a thromboxane A2 receptor blocker, also showed significant efficacy in reducing peritoneal adhesion severity. These results further support the view that NSAIDs act to prevent adhesions through a common mechanism. In addition, thromboxane A2 inhibitors were also shown to be efficacious in adhesion prevention, suggesting that platelets may play a substantial role in adhesion formation.

Reduction of Adhesion Formation by Intraperitoneal Administration of Anti-inflammatory Peptide 2

Adhesion formation is a major source of postoperative morbidity and mortality. Therefore, the reduction of postoperative adhesion formation would be of clinical benefit. Various modalities have been shown to reduce adhesion formation, including fibrinolytic enzymes, nonsteroidal anti-inflammatory drugs, and barriers that reduce the apposition of sites of potential adhesion formation. This study examined the ability of a phospholipase A2 inhibitor, anti-inflammatory peptide 2 (antinflammin), to reduce the formation of intraperitoneal adhesions in two rabbit models of adhesion formation. In the sidewall model, antinflammin was administered via Alzet miniosmotic pump for the entire postoperative interval, and there was a dose-dependent reduction in the area of the sidewall injury that was involved in adhesions to the cecum and the bowel. In the double uterine horn model, antinflammin was administered via Alzet miniosmotic pump to the area of injury for either 1, 2, 3, or 7 days. Administration of antinflammin for as little as 24 h after surgery significantly reduced the extent of adhesion formation. Administration of the peptide for longer periods of time did not further increase the reduction in adhesion formation. These studies clearly demonstrate that postoperative administration of antinflammin to the site of injury reduced the formation of postoperative adhesions in two animal models.

Differential secretion of plasminogen activator activity by postsurgical activated macrophages

Macrophage functions during postsurgical tissue repair include secretion of neutral proteases like plasminogen activator. Accordingly, we studied the differential secretion of plasminogen activator by postsurgical macrophages. Rabbits underwent resection and reanastomosis of their small bowel (three rabbits/time group). Postoperatively (up to 28 days), they underwent a second laparotomy for collection of ascites cells by lavage. Cells were incubated with culture medium containing 10% acid-treated fetal calf serum. After 2 days, the medium was removed (1-2 days media, M1) and replaced for 2 more days (3-4 days media, M2). Plasminogen activator activity of the medium (M1 and M2) was determined using a modified indirect solid-phase radioassay. The specific plasminogen activator activity (plasminogen-dependent-plasminogen-independent activator activity) in M1 was 43 mPU/10(6) cells in nonsurgical control rabbits. Exudative cells on Day 1 had less activity than control, and by Day 5, the activity significantly increased to 290% of control levels, reaching peak value on Day 14 (360%). The specific plasminogen activator activity in M2 was 310 mPU/10(6) cells in nonsurgical control media. Exudative cells on Day 1 had less activity than control, while, on Days 3-7, they were the same as control. By day 10, specific activity significantly increased to peak levels 188% of control and then gradually decreased. Since a marked increase in the number of macrophages parallels the increase in these metabolic activities through postsurgical Day 10, postsurgical activated macrophages appear to play an important role in peritoneal healing.