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Background: The homozygous deletion of Pkd1 in the mouse results in embryonic lethality with renal cysts and hydrops fetalis, but there is no precise data on the segmental origin of cysts and potential changes associated with polyhydramnios., Methods: We used Pkd1-null mice to investigate cystogenesis and analyze the amniotic fluid composition from embryonic day 12.5 (E12.5) to birth (n = 257 embryos)., Results: Polyhydramnios was consistently observed from E13.5 in Pkd1(-/-) embryos, in absence of placental abnormalities but with a significantly higher excretion of sodium and glucose from E13.5 through E16.5, and increased cyclic adenosine 3'5-monophosphate (cAMP) levels at E14.5 and E15.5. The Pkd1(-/-) embryos started to die at E13.5, with lethality peaking at E15.5, corresponding to the onset of cystogenesis. The first cysts in Pkd1(-/-) kidneys emerged at E15.5 in mesenchyme-derived segments at the cortico-medullary junction, with a majority of glomerular cysts and fewer proximal tubule cysts (positive for megalin). The cysts extended to ureteric bud-derived collecting ducts (positive for Dolichos biflorus agglutinin lectin) from E16.5., Conclusions: These studies indicate that Pkd1 deletion is associated with a massive loss of solutes (from E13.5) and increased cAMP levels (E14.5) associated with polyhydramnios. These abnormalities precede renal cysts (E15.5), first derived from glomeruli and proximal tubules and later from the collecting ducts, reflecting the expression pattern of Pkd1 in maturing epithelial cells.

Coupling of focal degradation and renewal of the functional layer of menstrual endometrium is a key event of the female reproductive biology. The precise mechanisms by which the various endometrial cell populations control extracellular matrix (ECM) degradation in the functionalis while preserving the basalis and the respective contribution of basalis and functionalis in endometrium regeneration are still unclear. We therefore compared the transcriptome of stromal and glandular cells isolated by laser capture microdissection from the basalis as well as degraded and preserved areas of the functionalis in menstrual endometria. Data were validated by in situ hybridization. Expression profile of selected genes was further analyzed throughout the menstrual cycle, and their response to ovarian steroids withdrawal was studied in a mouse xenograft model. Immunohistochemistry confirmed the results at the protein level. Algorithms for sample clustering segregated biological samples according to cell type and tissue depth, indicating distinct gene expression profiles. Pairwise comparisons identified the greatest numbers of differentially expressed genes in the lysed functionalis when compared with the basalis. Strikingly, in addition to genes products associated with tissue degradation (matrix metalloproteinase and plasmin systems) and apoptosis, superficial lysed stroma was enriched in gene products associated with ECM biosynthesis (collagens and their processing enzymes). These results support the hypothesis that fragments of the functionalis participate in endometrial regeneration during late menstruation. Moreover, menstrual reflux of lysed fragments overexpressing ECM components and adhesion molecules could easily facilitate implantation of endometriotic lesions.

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

Background: Beneficial effects of azithromycin in cystic fibrosis (CF) have been reported, however, its mechanism of action remains unclear. The present study aimed at investigating the effect of azithromycin on CF airway epithelial cells., Methods: Primary cultures of purified tracheal epithelial cells from F508del and normal homozygous mice were established. Responses to lipopolysaccharide from Pseudomonas aeruginosa (LPS, 0.1 microg/ml) on mRNA expression of neutrophil-related chemokines, pro- and anti-inflammatory cytokines were investigated in the presence or the absence of azithromycin (1 microg/ml)., Results: CF airway epithelial cells showed upregulation of MIP-2 and KC responses to LPS, and azithromycin failed to downregulate these responses. In contrast, in CF cells, azithromycin increased KC and TNF-alpha expression under non-stimulated and LPS-stimulated conditions, respectively. In non-CF cells, the macrolide potentiated the LPS response on MIP-2 and on IL-10., Conclusions: Airway epithelial cells contribute to the dysregulated immune processes in CF. Azithromycin rather stimulates cytokine expression in CF airway epithelial cells.

Background: The levonorgestrel-releasing intrauterine system (LNG-IUS) is an effective contraceptive and has many non-contraceptive health benefits. However, it is commonly associated with irregular endometrial bleeding. Metalloproteinases contribute to extracellular matrix (ECM) remodelling and regulate bleeding during the menstrual cycle. Enhanced metalloproteinase expression participates in the pathogenesis of breakthrough bleeding. Thus the objective of this study was to compare matrix metalloproteinase (MMP) expression in endometrium during luteal phase and in short-term (1 month) and long-term (> or =6 months) LNG-IUS users., Methods: MMP expression was analysed by semi-quantitative RT-PCR and immunohistochemistry. Gelatinase activity was determined by gelatin zymography., Results: MMP-1, -2, -3, -7, -9 and -12 mRNAs levels were increased, whereas that of MMP-26 was decreased in the endometrium of LNG-IUS users. MMP-1, -2, -3, -7 and -9 were localized by immunohistochemistry in all biopsies in the short-term group but in only 0-27% in the control group. The incidence of positive immunostaining for MMP-2 and -3 decreased significantly in the long-term compared with short-term LNG-IUS users. MMP-26 was localized in all biopsies from the control group but in only 14 and 25% from the short- and long-term LNG-IUS groups, respectively. In both LNG groups, the numbers of macrophages (the major source of MMP-12) was increased., Conclusions: MMP-1, active MMP-2, MMP-3, MMP-7, MMP-9 and MMP-12 are more prevalent in the short-term LNG-IUS group, suggesting their important contribution to ECM breakdown and transient bleeding. The decrease in the percentage of women expressing MMP-2 and -3 might contribute to the decreased occurrence of unwanted spotting and bleeding in long-term LNG-IUS users.

The bone marrow constitutes an unique microenvironment for cancer cells in three specific aspects. First, the bone marrow actively recruits circulating tumor cells where they find a sanctuary rich in growth factors and cytokines that promote their proliferation and survival. When in the bone marrow, tumor cells profoundly affect the homeostasis of the bone and the balance between osteogenesis and osteolysis. As a consequence, growth and survival factors normally sequestered into the bone matrix are released, further fueling cancer progression. Second, tumor cells actively recruit bone marrow-derived precursor cells into their own microenvironment. When in the tumors, these bone marrow-derived cells contribute to an inflammatory reaction and to the formation of the tumor vasculature. Third, bone marrow-derived cells can home in distant organs, where they form niches that attract circulating tumor cells. Our understanding of the contribution of the bone marrow microenvironment to cancer progression has therefore dramatically improved over the last few years. The importance of this new knowledge cannot be underestimated considering that the vast majority of cancer treatments such as cytotoxic and myeloablative chemotherapy, bone marrow transplantation and radiation therapy inflict a trauma to the bone marrow microenvironment. How such trauma affects the influence that the bone marrow microenvironment exerts on cancer is still poorly understood. In this article, the reciprocal relationship between the bone marrow microenvironment and tumor cells is reviewed, and its potential impact on cancer therapy is discussed.

Context: Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown., Objective and Design: Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist., Results: IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal., Conclusions: Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

Objective: To evaluate the risk of finding an infiltrating endometrial carcinoma when atypical hyperplasia (AH) or FIGO stage IA carcinoma were diagnosed on endometrial biopsy by Novak curette during ambulatory hysteroscopy or on pathological analysis of endometrial ablation product during operatoring hysteroscopy., Method: Retrospective unicentric study from 2000 to 2006 including 107 patients. Total hysterectomy with bilateral oophorectomy was performed on 95 of them. All patients had initial diagnosis of AH or FIGO stage IA carcinoma realised either by biopsy with Novak curette performed according to hysteroscopic data (52 cases), or on product of endometrial ablation (43 cases). AH and stage IA carcinoma were voluntarily studied together because of the same surgical treatment and because it is acknowledged that no complementary treatment is necessary if lesion remains intramucous. However, in presence of myometrial infiltration, the risk of lymph node infiltration exists and lymphadenectomy must be discussed., Results: Out of the 95 hysterectomy specimens, 20 infiltrating endometrial carcinoma were diagnosed (21%). The risk of discovering an infiltrating endometrial carcinoma when diagnosis of AH or stage IA carcinoma is done by biopsy with Novak curette is 32.7% (17 out of 52 patients). Let's note that 30% of these ambulatory hysteroscopies (5 out of 17) were non suspect. When diagnosis is done on the products of endometrial ablation, infiltrating carcinoma is observed on the hysterectomy piece in 6.9% of cases (3 out of 43 patients)., Discussion and Conclusion: Presence of AH or stage IA endometrial carcinoma on endometrial biopsies or on products of endometrial ablation can be associated with infiltrating endometrial carcinoma. In presence of AH or stage IA carcinoma diagnosed on biopsy with Novak curette, the incidence of infiltrating carcinoma on hysterectomy product is sizeable (32.7%) and check-up of infiltrating endometrial carcinoma should be realised with the view to state the prognostic factors and the possible indication of lymphadenectomy. Besides, in the case of a patient presenting with post menopausal bleeding with non suspect hysteroscopy, biopsic examination is necessary.

TGF-betas have been reported to mediate the repression by progesterone of several matrix metalloproteinases in the human endometrium, thereby preventing menstrual breakdown. Because of conflicting reports on the expression profiles, source, and regulation of the TGF-beta system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-betas (total and mature forms) and their two receptors throughout the menstrual cycle, and their regulation by ovarian steroids in cultured explants including their microdissected epithelial and stromal compartments. Regulation by cAMP and MAPK was further investigated. This comprehensive study on a large collection of endometrial samples evidenced a differential regulation of TGF-beta isoforms expression, both in vivo and in explant culture. In vivo, TGF-beta2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-beta3 increased at menstruation and remained high during the proliferative phase; TGF-beta1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-beta2 and -beta3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-beta2 and -beta3 in stroma but only TGF-beta2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-beta2 but not -beta3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-beta3 but inhibited TGF-beta2 expression. In conclusion, TGF-beta2 and -beta3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-beta2 and -beta3 by cAMP and MAPK could account for their distinct expression in vivo.

Ovarian remnant syndrome is a rare but known complication of bilateral salpingo-oophorectomy associated or not with hysterectomy. This complication is frequently related to a history of multiple surgery, pelvic inflammatory disease, or endometriosis. Here we report the eighth documented case of a primary ovarian adenocarcinoma developing in an ovarian remnant but, to our knowledge, the first case to occur after laparoscopic hysterectomy and bilateral salpingo-oophorectomy. We discuss the management of pelvic masses suspected of malignancy after bilateral salpingo-oophorectomy and the possible role of endometriosis in the development of malignancy in ovarian remnant syndrome.

We report the occurrence of a small cell carcinoma of pulmonary type developed within a large borderline mucinous cystic tumor of the ovary, with another focus of microinvasive mucinous adenocarcinoma, in a 32-year-old woman. The small cell carcinoma expresses several neuroendocrine markers, thyroid transcription factor 1 and parathyroid hormone. Carbohydrate antigen 15.3 and carcinoembryonic antigen were focally detected but not cancer antigen 125. Cytokeratin 20 was immunolabeled in several mucinous carcinomatous glands but not in the neuroendocrine cells. The p53 protein was abnormally abundant, including in the borderline tumor. Progesterone receptor was heterogeneously immunodetected, whereas estrogen receptor labeling was weak and limited. This unusual immunohistochemical pattern further enhances the singularity of this quite exceptional tumor association.

Background: Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice., Methods: We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the DeltaF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated., Results: In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-alpha and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation., Conclusion: Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

Pericytes occur in tumour blood vessels and are critical for the development of a functional vascular network. Targeting tumour pericytes is a promising anti-angiogenic therapy but requires identifying the mechanisms of their recruitment in tumour and addressing whether these mechanisms can be selectively harnessed. Among the pathways involved in pericyte recruitment during embryonic development, the contribution of platelet-derived growth factor B and sphingosine 1-phosphate is confirmed in tumour angiogenesis. The effect of angiopoietin 1 depends on the tumour model. Transforming growth factor-beta1 enhances tumour vascularization and microvessel maturation. Recent reports suggest a participation of matrix metalloproteinases (MMP) in tumour pericyte recruitment that is consistent with the effect of certain MMPs in the development of microvasculature in embryonic development and in in vitro models of vascular remodelling. Here, we discuss the possibility for MMPs to contribute to pericyte recruitment at six levels: (1) direct promotion of pericyte invasion by extracellular matrix degradation; (2) stimulation of pericyte proliferation and protection against apoptosis by modification of the ECM; (3) activation of pericytes through the release of growth factor bound to the ECM; (4) transactivation of angiogenic cell surface receptor; (5) propagation of angiogenic signalling as cofactor; and (6) recruitment of bone marrow-derived stem cells.

The vacuolar H(+)-ATPase (V-ATPase) is a ubiquitous multisubunit pump that is responsible for acidification of intracellular organelles. In the kidney, a particular form of V-ATPase, made of specific subunits isoforms, has been located at the plasma membrane of intercalated cells (IC). Mutations in genes encoding IC-specific subunits cause infant distal renal tubular acidosis (dRTA), suggesting that the segmental distribution of these subunits is acquired at birth or during early infancy. However, the comparative ontogeny of the IC-specific versus the ubiquitous subunits of V-ATPase and the mechanisms involved in their segmental expression remain unknown. Real-time reverse transcription-PCR, in situ hybridization, immunoblotting, immunostaining, and subcellular fractionation analyses characterized the expression and distribution of V-ATPase subunits, transcription factors, and differentiation markers during mouse nephrogenesis. Ubiquitous A, E1, B2, G1, and C1 subunits showed an early (embryonic day 13.5 [E13.5]) and stable expression throughout nephrogenesis, followed by a slight increase around birth. The developmental pattern of a1 was bimodal, with early induction, gradual decrease during organogenesis, and neonatal increase. These patterns contrasted with the later (from E15.5) and progressive expression of IC-specific a4, B1, G3, and C2 subunits, after the induction of the forkhead transcription factor Foxi1. From E15.5, Foxi1 mRNA was detected in IC, where it co-distributed with B1 in late nephrogenesis. Immunostaining showed that the distribution of ubiquitous E1 and B2 was acquired from E15.5, whereas a4 was located in IC during late nephrogenesis. Subcellular fractionation showed that in both fetal and mature (cortex and medulla) kidneys, E1 and a4 were located in endosomes. These data demonstrate a differential expression and a coordinate regulation of IC-specific versus ubiquitous V-ATPase subunits during nephrogenesis. They provide new insights into the complex regulation of V-ATPase subunits, the maturation of IC along the nephron, and the pathophysiology of hereditary dRTA.

Context: Cyclic remodeling and breakdown of the extracellular matrix, a unique feature of the human endometrium, depends on matrix metalloproteinases (MMPs). These enzymes are globally controlled by estradiol and progesterone or their withdrawal, but various MMPs and their tissue inhibitors (TIMPs) show distinct responses., Objective and Design: To clarify the role of ovarian steroids in the differential regulation of MMP-1, MMP-3, MMP-7, MMP-8, MMP-10, TIMP-1, TIMP-2, and TIMP-3 mRNAs, we compared their variations in the cycling endometrium in vivo with their response to hormone addition or withdrawal in corresponding explants., Results: Different patterns were identified in vivo according to the time frame (secretory vs. perimenstrual increase), sharpness (peak vs. progressive increase or decrease), and magnitude of the changes. In vivo ratios between early/midsecretory and perimenstrual phases ranged from more than 1000 (MMP-1, MMP-3, and MMP-10) to less than 10 (TIMPs). Differential response to ovarian steroids of the various MMPs and TIMPs mRNAs tested in cultured explants matched the same ranking and varied according to the phase at sampling. Remarkably, ovarian steroids repressed MMPs and TIMP-1 and TIMP-2 but, in secretory explants, increased TIMP-3 mRNA. Finally, in situ hybridization evidenced the major contribution of fibroblasts to the increase in MMP-8 mRNA at menstruation or in explants cultured without hormones., Conclusions: Both phase- and gene-specific modulators finely tune in space, time, and amplitude the global control of MMPs and TIMPs mRNAs by estradiol and progesterone in the cycling human endometrium.

Various matrix metalloproteinases (MMPs) participate in the menstrual breakdown of the human endometrium. MMP-9/gelatinase B is proposed as a major factor because it degrades many extracellular matrix constituents, including in the vasculature. Although globally under ovarian steroids control, endometrial MMP-9 seems expressed differently than other MMPs, and conflicting publications prevent a clear understanding of its regulation. We therefore quantified MMP-9 expression in the cycling human endometrium, defined its localization, and analyzed its regulation by estradiol and progesterone and by LEFTY-A/endometrial bleeding-associated factor in explant cultures. In fresh tissues, a major increase in MMP-9 mRNA expression occurred at menstruation, after a larger increase in LEFTY-A mRNA. MMP-9 was immunodetected in all cell types throughout the cycle, especially in foci of stromal cells during menstruation. MMP-9 synthesis by these cells was confirmed in cultured explants. In proliferative explants, ovarian steroids slightly decreased MMP-9 mRNA. They had no consistent effect on MMP-9 release in culture medium but strongly inhibited proMMP-9 activation. Addition of recombinant LEFTY-A to explants induced MMP-9 in most samples, a response prevented by ovarian steroids. We propose that endometrial MMP-9 activity is overall controlled by the ovarian steroids and locally adjusted through a network of modulators, including LEFTY-A.

Menstruation was thought to be the consequence of ischemic necrosis of the endometrium, resulting from vasoconstriction of the spiral arterioles. However, a new paradigm recently emerged when it was shown that human endometrium produces a lot of matrix metalloproteinases (MMPS) at menstruation. These MMPS are active and degrade almost all constituents of the extracellular matrix at neutral pH, allowing for tissue fragmentation and shedding. The tight control of their expression by progesterone and the close relationship in time and space between their expression and the lysis of the mucosa suggest their involvement in triggering menses as well as abnormal bleeding. A proof of their involvement was provided by culturing explants in conditions that mimic ex vivo the menstrual degradation of the endometrium, because specific inhibitors of MMPS prevent lysis of the extracellular matrix. Several cytokines have been shown to focally modulate the inhibition of MMPs expression by progesterone in a paracrine way.

The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.

Irregular dysfunctional bleeding of the endometrium (ie, metrorrhagia without organic lesion) is common in women, whether treated or not with ovarian hormones. Several matrix metalloproteinases (MMPs) become normally expressed and/or activated at menstruation and cause extracellular matrix breakdown. We therefore explored whether episodes of irregular dysfunctional bleeding could be associated with untimely MMP activity. By histology, foci of stromal breakdown were exclusively found in the endometrium of metrorrhagic women at bleeding. In these foci, 1) expression of estrogen receptor-alpha and progesterone receptor was altered; 2) collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) became detected in stromal cells, together with MMP-9 in neutrophils; and 3) gelatinase A (MMP-2) was more expressed and immunolocalized at the membrane of stromal cells. By biochemistry, endometrial lysates from nonbleeding metrorrhagic patients contained more latent and active MMP-2 and -9 than age-matched controls; at bleeding, collagenase activity, MMP-9, and active MMP-2 were strikingly increased whereas tissue inhibitor of metalloproteinases-1 (TIMP-1) was considerably decreased. As a functional assay, in situ gelatin zymography revealed large areas of gelatinolytic activity only in endometrium of bleeding patients. Altogether, these results strongly suggest that inappropriate focal expression and activation of several MMPs, combined with decreased inhibition, trigger irregular dysfunctional endometrial bleeding.

Background: Neck metastasis is a major prognostic factor of head and neck carcinoma, but its preoperative detection is currently unreliable. Molecular markers of the metastatic potential of a carcinoma would help to avoid unnecessary neck dissection in patients with nonmetastatic cancer., Methods: The fractions of cancer cells immunostained for proliferating cell nuclear antigen (PCNA) and Ki67 were determined in 80 preoperative biopsy specimens of head and neck carcinomas. The value of both indexes to detect metastasis in the subsequent neck dissection was compared with that of the clinical and radiologic examinations., Results: PCNA and Ki67 indexes correlated with neck metastasis. Cutoff points were determined for both indexes to discriminate metastatic from nonmetastatic carcinomas. By multivariate logistic regression, these indexes were significant predictors of metastases, together with clinical T stage and neck palpation., Conclusion: The fraction of cancer cells immunolabeled for PCNA or Ki67 in preoperative biopsy specimens is helpful to predict neck metastasis., (Copyright 2003 Wiley Periodicals, Inc.)

In an attempt to identify molecular prognostic markers, a series of laryngeal and hypopharyngeal carcinomas was examined for PCNA, Ki67, p27(Kip1), p53, E-cadherin and CD44 by immunohistochemistry and for DNA content by flow cytometry. No correlation was found between E-cadherin, CD44, p53 or DNA ploidy and the clinicopathological data. The fraction of cancer cells immunolabelled for p27(Kip1) correlated with tumour differentiation, but not with lymph node metastasis. In contrast, the PCNA, Ki67 and S-phase fractions of cancer cells were significantly higher in tumours with lymph node metastasis than in those without lymph node metastasis and were correlated with pathological T-stages and with tumour dedifferentiation. In univariate analysis, advanced pathological T-stage, lymph node metastasis and high fractions of cancer cells immunolabelled for PCNA or Ki67 inversely correlated with overall and disease-free survival. In multivariate analysis, lymph node metastasis was the only factor significantly associated with poor survival. The data suggest that immunohistochemical investigation of PCNA and Ki67 and flow cytometric analysis of S-phase fractions may be useful predictive markers of biological aggressiveness in laryngeal carcinomas.

The human endometrium is a unique tissue that is periodically shed during menstruation. Although overall triggered by ovarian steroids withdrawal, menstrual induction of matrix metalloproteinases (MMPs) and resulting tissue breakdown are focal responses, pointing to additional local modulators. LEFTY-A, a novel member of the transforming growth factor-beta family identified originally as an endometrial bleeding-associated factor (EBAF), is a candidate for this local control. We measured LEFTY-A and beta-ACTIN mRNA concentration during the menstrual cycle in vivo and found that their ratio was dramatically ( approximately 100-fold) increased at the perimenstrual phase. A similar increase was seen when proliferative explants were cultured for 24 h in the absence of ovarian steroids; this was followed by spontaneous production of proMMP-1, -3, and -9. Both responses were inhibited by progesterone. Moreover, addition of recombinant LEFTY-A to proliferative explants was sufficient to stimulate the expression of proMMP-3 and -7; this response was also blocked by ovarian steroids. Collectively, these data indicate that LEFTY-A may provide a crucial signal for endometrial breakdown and bleeding by triggering expression of several MMPs. Progesterone appears to exert a dual block, upstream by inhibiting LEFTY-A expression and downstream by suppressing its stimulatory effect on MMPs.

Recent studies strongly suggest that matrix metalloproteinases (MMPs) play a key role in the initiation of menstrual bleeding in the human endometrium upon the fall of ovarian steroid serum concentrations by inducing the degradation of the extracellular matrix of this mucosa. MMPs are also involved in abnormal endometrial bleeding and have been identified in endometriotic foci. In all cases, they are associated with areas of extracellular matrix breakdown. This paper reviews the literature on the regulation by estradiol and progesterone of the expression and activation of MMPs, and of the expression of their tissue inhibitors (TIMPs), (i) in the endometrium in situ during normal cycle, (ii) during artificial cycles in spayed monkeys, and (iii) in cultures of endometrial explants or purified cells. Whereas progesterone consistently decreases the activity of endometrial MMPs, its effects vary in intensity, duration, and pattern between MMPs as well as among experimental systems. The contribution and limitations of the various investigations are therefore discussed. The focal heterogeneity points to additional local controls of the expression and activation of MMPs in human endometrium, acting beyond the general inhibitory role of progesterone, for example, by cytokines. Focal changes in type or abundance of sex steroid receptors also could be responsible for spatial variation in the expression of MMPs in the endometrium and endometriotic lesions.

Most matrix metalloproteinases (MMPs) are secreted as inactive proenzymes. Their expression is well documented in several human tissues, but their activators in vivo are still unknown. To address this question, the activation of progelatinase B (proMMP-9) in the human endometrium was selected as a model system. ProMMP-9 was detected by gelatin zymography in homogenates of fresh endometrial tissue sampled during all phases of the menstrual cycle, whereas its active form was observed only during the late secretory and menstrual phases. Furthermore, proMMP-9 was expressed and activated in endometrial explants sampled outside the perimenstrual phase and cultured in the absence of both progesterone and oestradiol, mimicking the menstrual condition in vivo. Analysis of such tissue cultures by gelatin zymography and Western blotting showed that activation of proMMP-9 depended on a secreted factor and was selectively inhibited by either a synthetic inhibitor of stromelysin 1 (MMP-3) or a monoclonal antibody that specifically blocks MMP-3, thus providing strong evidence for the activation of proMMP-9 in vivo by MMP-3. The activation of proMMP-3 was itself inhibited by a broad-range MMP inhibitor in most cultures, but seemed to involve multiple pathways, implying both serine proteinases and metalloproteinases, which could operate in parallel or sequentially.

The pathogenesis of irregular endometrial bleeding, the main reason for stopping contraception with progestins only, is unknown. Based on the recent reappraisal of the mechanisms of menstrual bleeding, we hypothesized that matrix metalloproteinases initiate this disorder. Volunteers upon Norplant treatment provided endometrial biopsies at the start of a bleeding episode and during nonbleeding intervals. Focal stromal breakdown, collagen fiber lysis, and collagenase-1 messenger ribonucleic acid were evidenced in most bleeding endometria, but never in the nonbleeding ones. In the breaking down areas, immunolabeling for gelatinase A was strongly increased, and that of progesterone and estrogen receptors was decreased. Explants from bleeding endometria produced high collagenase and gelatinase activities, whereas release from nonbleeding endometria was negligible. Bleeding endometria released more latent and active forms of collagenase-1 and active gelatinases A and B, but less tissue inhibitor of metalloproteinases-1, than nonbleeding endometria. Collagenase-1 release closely correlated with that of interleukin-1alpha. In contrast, N:-acetyl-beta-hexosaminidase and tissue inhibitor of metalloproteinases-2 were similarly released in both groups. Thus, endometrial bleeding occurs together with focal stromal breakdown, collagen lysis, expression and activation of several matrix metalloproteinases, and decreased production of tissue inhibitor of metalloproteinases-1. These results may lead to new pharmacological treatment of this common medical problem.

Objective: To investigate the regulation of perimenstrual MMP-1 expression in human endometrium., Design: In vitro study utilizing epithelial-stromal co-cultures., Setting: Cell Biology Unit, International Institute of Cellular and Molecular Pathology, and Departments of Pathology and Gynecology, Saint-Luc University Clinics, Louvain University Medical School, Brussels, Belgium., Methods: Contact-dependent and contact-independent co-cultures were established and resulting MMP-1 gene and protein expression was analyzed by RNase protection assays and soluble-collagen assays., Results: MMP-1 expression in endometrial fibroblasts is markedly stimulated by epithelial cell-conditioned medium. This stimulation can be prevented by antibodies directed against interleukin 1 alpha (IL-1 alpha). Ovarian steroids inhibit MMP-1 production by IL-1 alpha-stimulated fibroblasts in vitro., Conclusion: Taken together, our results suggest that epithelium-derived IL-1 alpha is the most important paracrine induced of MMP-1 in endometrial fibroblasts. However, IL-1 alpha-stimulated MMP-1 production in the human endometrium is effectively blocked by ovarian steroids. We believe that this mechanism responsible for the MMP-1 repression that occurs when systemic sex steroid concentrations are high and the MMP-1 production and activity during the perimenstrual phase when estrogen and progesterone concentrations are low.

Monoclonal antibody (Mab) 57B, which was raised against a recombinant MAGE-A3 protein, was tested for its ability to stain cells expressing various members of the MAGE-A gene family. COS-7 cells transfected with cDNAs encoding MAGE-A1, A2, A3, A4, A6, or A12 were stained, whereas those transfected with MAGE-A8, A9, A10, or A11 cDNAs were not. However, in tissue sections, we observed a different pattern of staining: the antibody effectively stained the tumors that expressed MAGE-A4 and only these tumors, regardless of the expression of the other MAGE-A genes. It seems, therefore, that at the level of MAGE gene expression found in tumors, a level clearly lower than that observed in transfected COS cells, only the MAGE-A4 protein can be reliably detected. We conclude that the 57B Mab should be useful for tumor diagnosis related to therapeutic vaccination involving MAGE-A4., (Copyright 2000 Wiley-Liss, Inc.)

The endometrium is the only human tissue to undergo cyclic breakdown and regeneration. This physiological alternation renders it an advantageous system for studying tissue remodelling. Our previous observations indicate that menstrual endometrial breakdown is initiated by matrix metalloproteinases (MMPs), which are controlled overall by ovarian steroids but are also locally regulated by cytokines. We have therefore compared the effect of several endometrial cytokines on the gene expression of eight MMPs and their tissue inhibitors (TIMP)-1, -2 and -3, in primary cultures of human endometrial fibroblasts. Three categories of gene expression were identified: (a) MMP-13, -15 and -16 mRNAs were not detected despite stimulation by various cytokines; (b) MMP-2 and -14 as well as TIMP-1, -2 and -3 mRNAs were constitutively expressed but not markedly affected by the six cytokines tested; (c) mRNAs for MMP-1, -9 and -11 were selectively induced by specific cytokines: insulin-like growth factor-II, epidermal growth factor (EGF), platelet derived growth factor (PDGF)-BB and interleukin (IL)-6 stimulated MMP-11 expression; MMP-1 was induced by EGF, PDGF-BB, tumour necrosis factor (TNF)alpha and IL-1alpha, which also exerted additive effects. In contrast with MMP-1 and MMP-11 gene expression, which was sustained for 48 h, MMP-9 mRNA was quickly induced by TNFalpha, but disappeared within 12 h despite continuing stimulation. These results show that several cytokines are able to induce the selective expression of MMPs in cultured human endometrial fibroblasts and are thus good candidates for involvement in local triggering of menstrual tissue breakdown.

This review will focus on prognostic factors that can be disclosed by the pathological and molecular analysis of squamous cell carcinomas of the head and neck. The following pathological prognostic factors are considered and discussed: location, histopathology, lymph node metastasis. In addition, a number of molecular genetic markers have been identified such as: DNA content, chromosomal abnormalities, cell proliferation, RAS gene family, epidermal growth factor and its receptor, tumor-suppressor genes, cell adhesion molecules and proteinases.

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1alpha (IL-1alpha) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1alpha and repress MMP-1 production by IL-1alpha-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1alpha and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1alpha is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1alpha, thereby exerting an effective control via a "double-block" mechanism.

Anal and perianal condylomata acuminata are warts caused by infection with the human papillomavirus (HPV). The annual incidence of genital warts seems to have increased during the past few decades. Approximately 1.5 million consultations per year take place in the United States with this condition (1). Papillomavirus is a sexually transmitted disease, and is associated with several other venereal infections as well as with intraepithelial neoplasia and invasive squamous carcinoma. Only certain genotypes of HPV are carcinogenic, and can be precisely identified by in situ hybridisation techniques. There are many therapeutic alternatives, possibly reflecting the wide variability in treatment response.

Recently advanced computerized technology was applied to the investigation of morphometric, immunohistological and three-dimensional changes of the endometrial mucosa in order to evaluate quantitatively the effects of three doses of a new slow-release vaginal progesterone on the endometrium in post-menopausal women. A total of 20 menopausal women, deprived of ovarian function, were given oestrogen for 12 days and a combined therapy of oestrogen (administered orally) and progesterone for another 12 day period. Progesterone was administered vaginally through a new gel (Crinone) utilizing a bioadhesive, biocompatible polymer as a base to achieve a sustained release effect. An endometrial biopsy was taken before treatment, after oestrogen-only treatment and after the oestro-progestogen therapy. Before treatment, all the patients exhibited an atrophic endometrium. After oestrogen-only treatment, typical proliferative changes occurred: an increase in the endometrium thickness, an increase in the mitotic index, numerous cylinder-like glands and no coiled glands, and high concentrations of oestrogen receptors (ER) and progesterone receptors (PR). After the oestro-progestogen therapy, whatever the dose of progesterone given, a secretory transformation of the endometrial mucosa occurred, mitotic activity decreased significantly, more ramified and coiled glands were observed, and a decrease in PR content was noted in epithelial and stromal nuclei, and a decrease in PR content was also observed in epithelial nuclei but not in stromal nuclei. Accurate new techniques of image analysis have shown that crinone therapy could eliminate the proliferative effects of oestrogen treatment in post-menopausal women, despite doses as low as 45 mg of progesterone administered vaginally every other day. The results suggest that the sustained release effects of Crinone are clinically relevant.

Human endometrial tissue, sampled at different periods of the reproductive cycle, expressed interstitial collagenase mRNA, protein and activity only just before and during the menstrual period. This clear-cut correlation and the inhibition of collagenase expression by progesterone and oestradiol in tissue culture point to a pivotal role of this proteinase in the mechanism of menstrual tissue breakdown and bleeding.

Longterm administration of topical antiglaucoma therapies had been indicated to be a serious risk for failure of trabeculectomy through inducing a subclinical inflammation of conjunctival tissues and stimulating secondary fibroblastic proliferation. Our study had 3 main aims: (1) to check if this pejorative effect is present in our population of glaucomatous patients, (2) in order to improve the rate of surgical success in theoretically high risk patients (duration of topical treatment longer than 36 months), to determine the usefulness of the following actions: suppression of beta blocking agents 8 days before surgery, suppression of miotics and eventual topical administration of fluorometholone one month before surgery. (3) to correlate the observed clinical results to the cellular profile of preoperative conjunctival biopsies. We presented in a first step the results of a retrospective analysis including 88 trabeculectomies performed between January '93 and April '95 in 62 GCAO patients older than 40 years and without any previous ocular surgery. The mean age of patients was 66,8 years (41 to 87 years). Patients were classified into "complete success", "qualified success" and "failures" according to the "target" IOP was or was not reached in the last examination. Mean follow up was 5,8 months (1 to 26 months). The second prospective part of our study included 20 trabeculectomies performed in 16 patients we chose according to the nature, the duration of the preoperative topical therapy and the preoperative eventual preparation of the conjunctiva. This study also included a semiquantitative count of the different conjunctival cellular colonies in peroperative biopsies. Mean follow up of this study was 2,3 months (1 to 5 months). 31 "complete success", 52 "qualified success" and 5 "failure" were observed in the retrospective study. The reduction of IOP following trabeculectomy was statistically significant at all examinations (mean IOP in the last examination: 14,9 +/- 4,3 mmHg) but the level of success was not significantly different according to the nature, (mono against polytherapy), the duration of preoperative medical topical treatment, as well as the preoperative actions in order to improve the operative success in high risk patients. Preliminary results of our second prospective study did not yet show any failure and did not exhibit any clear correlation between the counts of the different cellular colonies and the level of operative success on one hand and the different topical therapies in the other hand. The results and limitations of this study were compared to those of the literature.

Granulocytic sarcoma of the larynx. Case report and review of the literature. Granulocytic sarcoma is a rare extramedullary tumor. It occurs either as an isolated tumor or associated with acute or chronic myelogenous leukemia. It has also been reported in patients with myelodysplastic syndromes when the granulocytic sarcoma is a harbinger of imminent disease progression. Granulocytic sarcoma can occur anywhere in the body. The larynx is a rare site of this lesion. A first case of glottic localisation is presented. The treatment of granulocytic sarcoma must include surgery, radiation and chemotherapy.

Odontogenic fibroma is an infrequent benign tumor, originating in the mesenchyma and including an inactive odontogenic epithelial component. The central form, of which one case is reported here, is expressed by a relatively well-delineated radiolucent area, whose interest and difficulty lie in the differential diagnosis. On the basis of a review of the literature, details are given on the clinical, clinicopathologic, diagnostic and therapeutic data.

The solitary plasmocytoma of the mandible is a rare affection. This article describes the case of a man, 42 years old. A short review of the pathology is done. The treatment (surgery and radiotherapy) with their failures and complications is exposed.

Explants of human endometrium were cultured to study the release of matrix metalloproteinases (MMPs). Analysis of conditioned media by zymography revealed latent and active forms of collagenase (MMP-1, EC 3.4.24.7), 72-kDa gelatinase A (MMP-2, EC 3.4.24.24), and 92-kDa gelatinase B (MMP-9, EC 3.4.24.35). These proteinases were identified by their M(r), their inhibition by tissue inhibitor of metalloproteinases, and the activation of their zymogens by trypsin or aminophenylmercuric acetate. In the absence of sex hormone, explants released large amounts of enzyme activities, as measured by densitometry of zymograms or in soluble assays. Physiological concentrations of progesterone (10-200 nM) almost totally abolished the release of collagenase, of total gelatinase activity, and of the active form of gelatinase B and largely inhibited the release of the active form of gelatinase A. These effects, which were antagonized by mifepristone (RU 38486), suggest that progesterone restrains endometrial tissue breakdown by blocking the secretion and activation of MMPs.

We studied the healing process in surgically created cleft lips in fetal mice and compared it with that in newborn mice with cleft lips. Our purpose was to determine the time for optimal healing, defined as minimal scarring, for a repaired cleft lip. Full-thickness paramedian lip incisions were made in NMRI mice in utero, in 2- and 4-day-old neonates, and in adults (n = 10 in each experimental and control group). The healing process was studied by biochemical analysis of hyaluronic acid and hydroxyproline content in the repaired cleft tissue. We found that the production of hyaluronic acid remained stable during the healing period and was similar in all experimental groups. However, there was an unexplained but consistent depression in the hyaluronic acid content of fetal tissue 2 days after repair. Hydroxyproline was present in the fetal healing tissue, but in a low concentration, starting 4 days after surgical incision of the lip. The production of hydroxyproline in 2-day-old neonates was similar to that in the fetuses throughout the healing period (p less than 0.0005). However, the production of hydroxyproline increased in 4-day-old neonatal and adult tissues. In conclusion, we found an optimal healing period for mice with minimal collagen production in the late fetal stage, and this lasted 2 days after birth.

Exudative laryngopathies are represented by the nodule, the polyp and the Reinke's oedema. Their origin is found in exudative processes located in the Reinke's space. Evolution towards one or another lesion depends on the etiological factor (vocal abuse, tobacco, alcohol, acute vocal fold trauma) and can happen according three modalities: oedema, fibrosis and angiectasies development. Apparition of fibrosis inside the lesion or too important haemorrhage require microsurgical treatment in addition to the medical treatment and the speech therapy.

A report is presented of the first histological study of GAX-collagen injected into the human vocal cord. The study concerns a specimen from an arytenoidectomy performed 28 months after injection. As it had already been the case in the animal, the excellent integration of GAX-collagen with the host-tissues has been noted, as well as the stability of the result more than 2 years later. It also appeared that colonization of GAX-collagen by the host cells is less rapid than for the ZCI. While inflammatory reaction is weak in the region of the GAX-collagen, there is to be seen some atrophy and fibrosis of the muscular fibres around the deposit, probably due to compression during injection.

We compared the morphologic fate of three types of soluble bovine collagen injected into vocal folds of dogs: 1) Zyderm collagen implant containing 35 mg/mL of collagen (ZCI I), 2) Zyderm collagen implant containing 65 mg/mL of collagen (ZCI II), and 3) GAX collagen containing 35 mg/mL of collagen minimally cross-linked with 0.0075% glutaraldehyde. Light and electron microscopy showed good host tolerance and a long-term persistence of the three types of collagen at the site of injection. The collagen implants were progressively colonized by fibroblasts and capillaries accompanied by host collagen deposition. No inflammatory process or foreign body reaction occurred, in contrast to the typical host reaction to injected Teflon. The colonization of GAX collagen was slower than for ZCI I and ZCI II. No other significant difference was evident among the three types of injectable collagen. All three appear to be very suitable materials for laryngeal implants; moreover, they seem to best meet accepted requirements for a good implant: they are easy to inject, they are well tolerated, and they persist for a long time. We prefer GAX collagen for clinical use because of its lower reactivity and higher stability, and because overinjection is not necessary (GAX shrinks less than ZCI I and ZCI II).

The activities of four lysosomal enzymes, i.e. N-acetyl-beta-hexosaminidase, acid phosphatase, alpha-D-mannosidase and alpha-L-fucosidase have been measured in extracts of endometrial biopsies from untreated and levonorgestrel-treated women of fertile age. Values were compared with protein and DNA content, as well as with lactate dehydrogenase activity, used as reference constituents. In parallel, organ cultures were established from the same endometrial specimens and the release of lysosomal enzymes into the medium was followed. The human endometrium possesses a rich lysosomal equipment, comparable to that found in the human liver. In the untreated cycles, the activities of lysosomal enzymes show a coordinate response to the hormonal changes, decreasing by about 40% from the proliferative to the mid-late secretory phase. Long-term levonorgestrel treatment causes a marked cytoplasmic atrophy, as shown by decreased protein content and lactate dehydrogenase activity, whereas DNA content remains unchanged. In contrast, N-acetyl-beta-hexosaminidase, one of the most active lysosomal enzymes studied, shows a higher specific activity upon levonorgestrel. In both untreated and treated endometria, the organ cultures provide biochemical evidence for a higher release of N-acetyl-beta-hexosaminidase than of lactate dehydrogenase, indicating active secretion of the lysosomal enzyme. During levonorgestrel treatment, there was no correlation between clinically recognized spotting-bleeding patterns and lysosomal enzyme content in, or release from, the endometrium.

The clinical observation of an oral pulse granuloma allows to detail the clinical and pathological characteristics of this lesion. The etiology of this particular granuloma is explained by the introduction of alimentary particles into maxillary bone. A review of the literature about this subject is also made.

Using the dog as an animal model, we have tested an implant material composed of purified fibrillar collagen (PFC) and particulate hydroxylapatite (HA) in the mandible and in surrounding tissues. Bone and tissue samples were taken at 2, 4 and 6 months for histological study. After 2 months, the PFC was replaced by fibro-connective host tissues. After 4 months, some small areas of ossification were observed around the HA particles. After 6 months, the fibro-connective tissue was replaced by neo-formed bone in the mandible. PFC was found to increase the interfaces between the HA particles and the host tissues, permitting HA integration into the bone. The PFC/HA implant was also molded when moistened by blood or saline solution and then became mis-sharpen by local pressures exerted. These findings show that the implant should preferably be reserved for the restoration of bones not subjected to significant forces or local stresses.

Thirty-nine patients were treated with GAX collagen for vocal fold augmentation. The objective results were evaluated by measurement of the maximum phonation time, the phonatory quotient, vocal frequency analysis, and laryngostroboscopy. These results are as good as those with Zyderm Collagen II (ZCI). Avoidance of useless overcorrection, better resistance to collagenase, and less induced hypersensitivity differentiate GAX from ZCI.

The use of Gax collagen is proposed for correcting velopharyngeal insufficiency with posterior pharyngeal wall augmentation. The indications for this procedure are velopharyngeal insufficiency that remains after surgery and treatment by a speech pathologist. The procedure is also valuable after velum paralysis. Collagen (2.5-5cc) is injected through a device designed for vocal-fold and laryngeal rehabilitation. Several injection points are necessary to raise a good Passavant's ridge. The muscular activity of the posterior pharyngeal wall models the collagen pad for the first 3 weeks; afterwards, the collagen shape remains unchanged due to its integration in the host tissues. At this time, five patients have been injected with Gax collagen and have improved. The oldest case dates from 28 months ago; the most recent dates from 8 months ago. There were no adverse reactions, and the results remain stable at the present time.