R. Stan Williams

Effect of transforming growth factor β on postoperative adhesion formation and intact peritoneum

Transforming growth factor beta (TGF beta) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and fibroblasts. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell growth. TGF beta plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGF beta could affect postoperative wound healing in a rat model. In the first experiment, 20 rats were divided into two groups and received either 2 micrograms TGF beta or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0-3. The TGF beta group showed a higher adhesion score at 2 weeks compared to control, 2.9 +/- 0.34 and 1.6 +/- 0.61, respectively (P less than 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and fibroblasts in the TGF beta-treated animals. A comparison trial of bone-derived TGF beta (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGF beta (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGF beta were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGF beta groups, 3.0 +/- 0 for both TGF beta groups, and 2.2 +/- 0.91 for control (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Laparoscopic oophoropexy and ovarian function in the treatment of Hodgkin disease.

Hodgkin disease commonly affects women of reproductive age. Total lymph node irradiation (TNI) typically delivers a dose of 2000–4000 centigray (cGy) to the ovaries, which invariably results in premature ovarian failure (POF) and infertility unless the ovaries are shielded. Transposition of the ovaries at staging laparotomy has had mixed success and may be remote in time from pelvic radiation.

Gene Expression Profile of Leiomyoma and Myometrium and the Effect of Gonadotropin Releasing Hormone Analogue Therapy

Objective: To determine the profile of differentially expressed genes in leiomyoma and matched unaffected myometrium and to identify the genes whose expression are altered after gonadotropin releasing hormone analogue (GnRHa) therapy. Methods: Using total RNA isolated from untreated and GnRHa-treated leiomyoma and myometrium subjected to Clontech Atlas 1.2 K cancer microarray containing 1176 known genes, we found transcripts for many cytokines, growth factors and their receptors, adhesion molecules, extracellular matrix, proteases, signaling intermediates, and transcription factors in both tissues. Based on the overall normalization and reproducibility of the results, 328 differentially expressed and regulated genes with at least threefold change in their expression in untreated and GnRHa-treated tissues were chosen for further analysis. Results: The expression value of the 328 genes subjected to significance analysis of microarray showed a total of 100 genes with relative significant change in expression. Of these genes, the expression of 18 was up-regulated and 82 down-regulated in untreated leiomyoma compared with myometrium. In GnRHa-treated tissues, the expression of 34 genes showed a significant decrease in leiomyoma, whereas 27 genes increased and 15 decreased in myometrium compared with their respective untreated tissues. There was no difference in the expressive profile of these genes when comparing leiomyoma and myometrium from the GnRHa-treated group. Hierarchical duster analysis revealed that these differentially expressed or regulated genes, classified based on their biologic functions, are involved in regulation of cell growth/cycle, signal transduction, transcription factors, and cell and tissue structure. Conclusion: Microarray analysis allowed us to identify the profile expression of several specific genes in leiomyoma and myometrium whose expression appears to be differentially regulated after GnRHa therapy.

Differential expression of interleukins (IL) IL-13 and IL-15 throughout the menstrual cycle in endometrium of normal fertile women and women with recurrent spontaneous abortion

Interleukins (IL)-13 and IL-15 are novel cytokines and key regulators of immune/inflammatory related responses that are critical in the outcome of various normal biological and associated abnormalities of the endometrium. The present study determined the temporal and spatial expression of IL-13 and IL-15 mRNA and protein in endometrium of normal fertile women throughout the menstrual cycle, and examined whether profiles of their expression differ from endometrium of women with unexplained recurrent spontaneous abortion (RSA). Using quantitative RT-PCR, ELISA and immunohistochemistry we found that IL-13 and IL-15 mRNA and protein are expressed in control endometrium throughout the menstrual cycle and in RSA (cycle days 21-23) with peak expression detected immediately after and prior to onset of menses, and two distinct periods corresponding to late proliferative and the early-mid secretory phases, respectively. The ratio of IL-13:IL-15 expression revealed a predominance in IL-13 expression during late proliferative/early secretory phase and IL-15 during the mid secretory phase. Compared to control endometrium, endometrium of women with RSA expresses elevated levels of IL-13 and IL-15, with IL-13:IL-15 ratio favoring IL-13. The immunoreactive IL-13 and IL-15 were localized primarily in endometrial luminal epithelial cells with an increased intensity in glandular epithelial and stromal cells in RSA. In conclusion, the results indicate that endometrium of normal fertile women expresses IL-13 and IL-15, with a distinct profile during the menstrual cycle and elevated expression in women with RSA. Although the biological significance of IL-13 and IL-15 in human endometrium and their elevated expression in RSA await investigation, these cytokines with distinct biological functions may regulate endometrial inflammatory/immune responses, tissue repair and receptivity for embryo implantation.

Expression of matrix metalloproteinase-26 and tissue inhibitor of matrix metalloproteinase-3 and -4 in endometrium throughout the normal menstrual cycle and alteration in users of levonorgestrel implants who experience irregular uterine bleeding

OBJECTIVE To determine the expression of matrix metalloproteinase (MMP-26) and tissue inhibitor of MMP (TIMP) in the endometrium of women with normal menstrual cycles compared with users of levonorgestrel implants. DESIGN Prospective observational study. SETTING Academic research center. PATIENT(S) Fifty patients with normal menstrual cycles who requested permanent surgical sterilization (tubal ligation) and 35 users of levonorgestrel implants. INTERVENTION(S) Endometrial biopsy. MAIN OUTCOME MEASURE(S) Expression of MMP-26, TIMP-3, and TIMP-4 by immunohistochemistry and semiquantitative analysis of staining intensity by using the H score. RESULT(S) Endometrium from women with a normal menstrual cycle and users of levonorgestrel implants expresses MMP-26, TIMP-3, and TIMP-4. These substances are present in various types of endometrial cells; expression is strongest in surface and glandular epithelial cells, followed by vascular endothelial and endometrial stromal cells. Inflammatory and immune-related cells also stained strongly for MMP-26 and TIMPs. Semiquantitative analysis of the staining intensity of endometrial epithelial and stromal cells indicated that expression of MMP-26, TIMP-3, and TIMP-4 peaks during the early to mid-luteal phase. Expression of MMP-26 is elevated in users of levonorgestrel implants who experienced irregular uterine bleeding. CONCLUSION(S) Endometrial expression of MMP-26 and TIMP-4 is present throughout the menstrual cycle and is elevated during the early to mid-luteal phase in normally cycling women. Further elevations in MMP-26 are seen in users of levonorgestrel implants who experience irregular uterine bleeding. These substances thus seem to play a role in hormonal regulation and endometrial tissue remodeling.

Expression of integrin messenger ribonucleic acid in human endometrium: a quantitative reverse transcription polymerase chain reaction study

OBJECTIVE To assess the expression of selected integrin subunits messenger RNA (mRNA) in human endometrium during the menstrual cycle. DESIGN A prospective comparative study. SETTING Academic research environment. PATIENT(S) Premenopausal women with histologically normal endometrium who were undergoing hysterectomy. INTERVENTION(S) Endometrial tissues were collected. RESULT(S) Human endometrium expresses integrins alpha2, alpha3, alpha4, alpha5, alpha6.1, alpha6.2, alpha v, beta1, beta2, beta3, and beta5, as well as fibronectin mRNA. The levels of endometrial integrins mRNA expression varied significantly, with the lowest levels observed for alpha2, beta3, and fibronectin and the highest for alpha5, beta2, and beta5. The levels of integrins alpha2, alpha3, and alpha5 mRNA expression were significantly higher during the proliferative phase, whereas alpha4, alpha6.2, alpha v, beta1, beta2, beta3, beta5, and fibronectin were higher during the secretory phase of the menstrual cycle. The alpha6.1 mRNA was found to be equally expressed in the endometrium during the menstrual cycle, whereas the most dramatic changes occurred in alpha v and beta3 expression, compared with other integrin subunits. CONCLUSION(S) Human endometrium expresses mRNA for several integrins and fibronectin, with up-regulation of alpha4, alpha v, beta1, and beta3 during the secretory phase of the menstrual cycle, suggesting that their differential expression may be regulated in part by ovarian steroids.

Identification of epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor in surgically induced pelvic adhesions in the rat and intraperitoneal adhesions in the human

OBJECTIVE Our purpose was to determine the presence and cellular distribution of epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor in surgically induced pelvic fibrous adhesions in rat uterine horns subjected to burn, crush, and debridement injury and intraperitoneal fibrous adhesions formed to various organs in the human. STUDY DESIGN A total of 15 injured and five uninjured rats were used in this study, and fibrous adhesions and intact peritoneum were removed for processing 2 weeks after surgery. Fibrous adhesions formed to uterine, ovarian, and oviductal tissues and the peritoneal wall from eight patients who had gynecologic surgery were also collected. The tissues were processed for immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor with specific antibodies to human and rat epidermal growth factor and transforming growth factor-alpha and the extracellular binding domain of the epidermal growth factor receptor. RESULTS All the cell types in the rat fibrous adhesion immunostained for epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor. The highest immunostaining intensity for epidermal growth factor was associated with inflammatory cells infiltrated into the fibrous adhesion, followed by arteriole endothelial and smooth muscle cells, fascial striated muscle, and fibroblasts of the fibrous adhesion. In the uterine tissue at the site of injuries myometrial smooth muscle cells, in addition to inflammatory cells that migrated among stromal cells, also immunostained for epidermal growth factor. Fibrous adhesions also immunostained for transforming growth factor-alpha with three separate polyclonal antibodies to the amino and carboxy termini of transforming growth factor-alpha precursor and the mature transforming growth factor-alpha, with no substantial differences in their intensity and pattern compared with epidermal growth factor. The pattern and cellular distribution of epidermal growth factor receptor was similar to that seen for epidermal growth factor and transforming growth factor-alpha. Fibrous adhesions from patients with intraperitoneal adhesions immunostained for epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor with a pattern and intensity similar to that observed in fibrous adhesions in the rats. CONCLUSIONS The data suggest that epidermal growth factor and transforming growth factor-alpha may play a key role both in normal mechanism of peritoneal repair after injury and formation and maintenance of fibrous adhesions.

Angelman syndrome imprinting center encodes a transcriptional promoter

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11–q13 is responsible for both Angelman syndrome (AS) and Prader–Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader–Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS–PWS locus. The PWS–smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS–SRO element generates maternal allele identity by epigenetically inactivating the PWS–SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS–SRO, the element necessary for maternal allele identity. We find that, in humans, the AS–SRO is an oocyte-specific promoter that generates transcripts that transit the PWS–SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription.

Endometriosis associated with massive ascites and absence of pelvic peritoneum

Although massive ascites associated with endometriosis has been reported in rare cases, this patient was also noted to have massive destruction of the pelvic peritoneum. Failure of medical suppression necessitated total abdominal hysterectomy and bilateral salpingo-oophorectomy. Several months after surgery ascites resolved, possibly with reestablishment of the pelvic peritoneum.

Postsurgical intraperitoneal exposure to glove powders modulates inflammatory and immune‐related cytokine production

The objective of the present study was to determine whether intraperitoneal exposure to glove powders modulates the inflammatory and immune responses by altering the influx of inflammatory and immune cells and peritoneal fluid cytokines and thus the outcome of surgically induced peritoneal wound healing. Peritoneal wall injuries were made by scraping the tissue until bleeding occurred in 360 mice. One of the following fluids was then introduced into the peritoneal cavity: phosphate‐buffered saline solution, phosphate‐buffered saline solution containing glove powders (Biosorb and Keoflo, 100 µg/ml), Hydrocote (Hydrogel film, Biogel 100 µg/ml), latex proteins (1 mg/ml), or lipopolysaccharides (12.5 µg/ml). At intervals of 1 to 28 days after injury, 10 mice per treatment per day and 10 uninjured mice were killed, peritoneal fluids were collected to determine the cytokine levels, the rate of fibrous adhesions formed at the site of injuries was graded, and peritoneal walls with attached fibrous adhesions were removed to determine the degree of inflammatory and immune cell infiltration into the wound. The results indicated that, with the exception of interferon‐γ, the peritoneal fluid levels of transforming growth factor‐β1, tumor necrosis factor‐α, interleukin‐1β, and granulocyte‐macrophage‐colony stimulating factor in the phosphate‐buffered saline solution‐treated injured group significantly increased, reaching maximum between days 4 and 7 (p < 0.05) compared with the uninjured group and returned to uninjured values by day 14 after injury. The level of transforming growth factor‐β1 was higher in glove powders and Hydrocote‐treated groups than in latex, liposaccharides, or phosphate‐buffered saline solution‐treated groups until day 14 after surgery (p < 0.05). The levels of tumor necrosis factor‐α and interleukin‐1β increased in all treatment groups during the first week after injury compared with uninjured controls, with the exception of Hydrocote. The number of T helper/inducers (CD4), total leukocytes (CD11a), B lymphocytes (CD45R), granulocytes (Gr‐1), and mononuclear phagocytes (Mac‐3) in the wound increased during the first week after peritoneal wounding with no significant difference between treated and untreated groups. The rate of adhesion formation was not significantly altered in treated compared with untreated groups. These data suggest that a mechanism which mediates glove powder‐induced peritoneal inflammatory and immune reactions in the postsurgical setting involves augmentation of cytokine production without influencing the influx of inflammatory and immune cells or adhesion formation.