Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Expression and regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis

Factors involved in regulating tissue specific gene expression play a major role in cell differentiation. In order to further understand the differentiation events occurring during hematopoiesis, a myeloid specific gene was characterized, the expression pattern during hematopoiesis was analyzed, and the mechanisms governing its regulation were assessed. Previously, our laboratory isolated an anonymous cDNA clone, pD-D1, which displayed preferential expression in myeloid cells. From nucleotide sequencing of overlapping cDNA clones I determined that the D-D1 message encodes a hematopoietic proteoglycan core protein (HpPG). The expression pattern of the gene was assessed by in situ hybridization of bone marrow and peripheral blood samples. The gene was shown to be expressed, at variable levels, in all leukocytes analyzed, including cells from every stage of neutrophil development. In an attempt to ascertain the differentiation time point in which the HpPG gene is initially expressed, more immature populations of leukemic myeloblasts were assessed by northern blot analysis. Though the initial point of expression was not obtained, an up-regulatory event was discovered corresponding to a time point in which granule genesis occurs. This finding is consistent with prior observations of extensive packaging of proteoglycans into the secretory granules of granule producing hematopoietic cells. The HpPG gene was also found to be expressed at low levels in all stages of lymphocyte development analyzed, suggesting that the HpPG gene is initially expressed before the decision for myeloid-lymphoid differentiation. To assess the mechanism for the up-regulatory event, a K562 in vitro megakaryocytic differentiation system was used. Nuclear run-off analyses in this system demonstrated the up-regulation to be under transcriptional control. In addition, the HpPG gene was found to be down regulated during macrophage differentiation of HL60 cells and was also shown to be transcriptionally controlled. These results indicate that there are multiple points of transcriptional regulation of the HpPG gene during differentiation. Furthermore, the factors regulating the gene at these time points are likely to play an important role in the differentiation of granule producing cells and macrophages. ^

Association of biomarkers of lipid modification with functional and morphological indices of coronary stenosis severity in stable coronary artery disease

Biomarkers of blood lipid modification and oxidative stress have been associated with increased cardiovascular morbidity. We sought to determine whether these biomarkers were related to functional indices of stenosis severity among patients with stable coronary artery disease. We studied 197 consecutive patients with stable coronary artery disease due to single vessel disease. Fractional flow reserve (FFR) ≤ 0.80 was assessed as index of a functionally significant lesion. Serum levels of secretory phospholipase A2 (sPLA2) activity, secretory phospholipase A2 type IIA (sPLA2-IIA), myeloperoxydase (MPO), lipoprotein-associated phospholipase A2 (Lp-PLA2), and oxidized low-density lipoprotein (OxLDL) were assessed using commercially available assays. Patients with FFR > 0.8 had higher sPLA2 activity, sPLA2 IIA, and OxLDL levels than patients with FFR ≤ 0.8 (21.25 [16.03-27.28] vs 25.85 [20.58-34.63] U/mL, p < 0.001, 2.0 [1.5-3.4] vs 2.6 [2.0-3.4] ng/mL, p < 0.01; and 53.0 [36.0-71.0] vs 64.5 [50-89.25], p < 0.001 respectively). Patients with FFR > 0.80 had similar Lp-PLA2 and MPO levels versus those with FFR ≤ 0.8. sPLA2 activity, sPLA2 IIA significantly increased area under the curve over baseline characteristics to predict FFR ≤ 0.8 (0.67 to 0.77 (95 % confidence interval [CI]: 0.69-0.85) p < 0.01 and 0.67 to 0.77 (95 % CI: 0.69-0.84) p < 0.01, respectively). Serum sPLA2 activity as well as sPLA2-IIA level is related to functional characteristics of coronary stenoses in patients with stable coronary artery disease.

Suppression by {\it Trypanosoma brucei\/} of anaphylaxis-mediated ion transport in the small intestine of rats

Most tissue-invasive parasitic helminths prime for type 1 hypersensitivity or anaphylaxis during some phase of their life cycles. A prototype in this regard is the nematode Trichinella spiralis. Blood protozoa capable of tissue invasion, such as Trypanosoma brucei, might also be expected to prime for the expression of anaphylaxis. However, this response is usually absent in protozoal infections. The hypothesis tested was that failure of hosts infected with T.brucei to express anaphylaxis is related to this parasite's ability to selectively down-regulate immunoglobulin E (IgE) production, and not to an innate lack of allergenicity on the part of T.brucei-derived antigens. This hypothesis was tested by studying in the intestine of rats, antigen-induced Cl$\sp-$ secretion, which results from a local anaphylactic response mediated by IgE and mucosal mast cells. The Cl$\sp-$ secretory response can be primed either by infection with T.spiralis or by the parenteral administration of antigen. Anaphylaxis-induced Cl$\sp-$ secretion is expressed in vitro, and can be quantified electrophysiologically, as a change in transmural short-circuit current when sensitized intestine is mounted in Ussing chambers and challenged with the sensitizing antigen.^ Rats injected parenterally with trypanosome antigen elicited intestinal anaphylaxis in response to antigenic challenge. In contrast, the intestine of rats infected with T.brucei failed to respond to challenge with trypanosome antigen. Infection with T.brucei also suppressed antigen-induced Cl$\sp-$ secretion in rats sensitized and challenged with various antigens, including T.spiralis antigen. However, T.brucei infection did not inhibit the anaphylactic response in rats concomitantly infected with T.spiralis. Relative to the anaphylactic mediators, T.brucei infection blocked production of IgE in rats parenterally injected with antigen but not in T.spiralis-infected hosts. Also, the mucosal mastocytosis normally associated with trichinosis was unaffected by the trypanosome infection. These results support the conclusion that the failure to express anaphylaxis-mediated Cl$\sp-$ secretion in T.brucei infected rats, is due to this protozoan's ability to inhibit IgE production and not to the lack of allergenicity of trypanosome antigens. ^

Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

Cell-type specific expression and regulation of apolipoprotein D and E in human endometrium.

OBJECTIVE To assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. STUDY DESIGN Endometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45(+) leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. RESULTS ApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. CONCLUSION We demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.

The mammary gland in small ruminants: major morphological and functional events underlying milk production – a review

The importance of small ruminants to the dairy industry has increased in recent years, especially in developing countries, where it has a high economic and social impact. Interestingly and despite the fact that the mammary gland is the specialised milk production organ, very few authors studied the modifications occurring in the mammary gland through the lactation period in production animals, particularly in the small ruminants, sheep (Ovis aries) and goat (Capra hircus). Nevertheless, understanding the different mammary gland patterns throughout lactation is essential to improve dairy production. In addition, associating these patterns with different milking frequencies, lactation number or different diets is also of high importance, directly affecting the dairy industry. The mammary gland is commonly composed of parenchyma and stroma, which includes the ductal system, with individual proportions of each changing during the different periods and yields in a lactation cycle. Indeed, during late gestation, as well as during early to mid-lactation, mammary gland expansion occurs, with an increase in the number of epithelial cells and lumen area, which leads to increment of the parenchyma tissue, as well as a reduction of stroma, corresponding macroscopically to the increase in mammary gland volume. Throughout late lactation, the mammary gland volume decreases owing to the regression of the secretory structure. In general, common mammary gland patterns have been shown for both goats and sheep throughout the several lactation stages, although the number of studies is limited. The main objective of this manuscript is to review the colostrogenesis and lactogenesis processes as well as to highlight the mammary gland morphological patterns underlying milk production during the lactation cycle for small ruminants, and to describe potential differences between goats and sheep, hence contributing to a better description of mammary gland development during lactation for these two poorly studied species.

Colostrogenesis: IgG1 Transcytosis Mechanisms

Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

Enhancing chemotherapy efficacy in Pten-deficient prostate tumors by activating the senescence-associated antitumor immunity

Prosenescence therapy has recently emerged as a novel therapeutic approach for treating cancer. However, this concept is challenged by conflicting evidence showing that the senescence-associated secretory phenotype (SASP) of senescent tumor cells can have pro- as well as antitumorigenic effects. Herein, we report that, in Pten-null senescent tumors, activation of the Jak2/Stat3 pathway establishes an immunosuppressive tumor microenvironment that contributes to tumor growth and chemoresistance. Activation of the Jak2/Stat3 pathway in Pten-null tumors is sustained by the downregulation of the protein tyrosine phosphatase PTPN11/SHP2, providing evidence for the existence of a novel PTEN/SHP2 axis. Importantly, treatment with docetaxel in combination with a JAK2 inhibitor reprograms the SASP and improves the efficacy of docetaxel-induced senescence by triggering a strong antitumor immune response in Pten-null tumors. Altogether, these data demonstrate that immune surveillance of senescent tumor cells can be suppressed in specific genetic backgrounds but also evoked by pharmacological treatments.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis.

PURPOSE To assess endometrial gene as well as protein expression of neuroendocrine and supposedly endometriosis-associated product PGP9.5 and pain symptoms in women with endometriosis and controls undergoing laparoscopy, using molecular biological and immuno-histochemical approaches in the same patients. METHODS Biopsy of eutopic endometrium from 29 patients by sharp curettage, and preparation of paraffin blocks. Determination of PGP9.5 gene expression and protein abundance using qPCR and immuno-histochemistry. RESULTS qPCR; The PGP9.5 mRNA expression level between women with (N = 16) and without (N = 13) endometriosis was not different, regardless of pain symptoms or menstrual cycle phase. PGP9.5 expression was higher in women who reported pain compared to those who did not; however, this association was not statistically significant. The expression of PGP9.5 mRNA was higher in women with endometriosis and pain during the proliferative than in the secretory phase (P = 0.03). Furthermore, in the first half of the cycle, the abundance of the PGP9.5 transcript was also significantly higher in endometriosis patients compared to those without (P = 0.03). Immuno-histochemistry; Thirteen of the 16 endometriosis patients showed positive PGP9.5 immuno-reactivity in the endometrium, whereas no such signal was observed in women without endometriosis. The absolute number of nerve fibres per mm(2) in women with endometriosis was similar, regardless of the pain symptoms. CONCLUSIONS PGP9.5 mRNA expression is increased in the proliferative phase of endometriotic women with pain. The presence of nerve fibres was demonstrated by a PGP9.5 protein signal in immuno-histochemistry and restricted to patients with endometriosis. Based on these results, however, there did not appear to be a direct association between the gene expression and protein abundance in women with and without endometriosis or those that experienced pain.