A review of the available clinical therapies for vulvodynia management and new data implicating proinflammatory mediators in pain elicitation.

Localised provoked vulvodynia (LPV) is a common, chronic, and disabling condition: patients experience profound pain and a diminished quality of life. The aetiologic origins of vulvodynia are poorly understood, yet recent evidence suggests a link to site-specific inflammatory responses. Fibroblasts isolated from the vestibule of LPV patients are sensitive to proinflammatory stimuli and copiously produce pain-associated proinflammatory mediators (IL-6 and PGE2 ). Although LPV is a multifactorial disorder, understanding vulvar inflammation and targeting the inflammatory response should lead to treatment advances, especially for patients exhibiting signs of inflammation. NFκB (already targeted clinically) or other inflammatory components may be suitable therapeutic targets. TWEETABLE ABSTRACT: Vulvodynia is a poorly understood, prevalent, and serious women's health issue requiring better understanding to improve therapy.

Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae.

Potocytosis is an endocytic pathway that utilizes glycosylphosphatidylinositol-anchored membrane proteins and caveolae to concentrate and internalize small molecules. We now report that activators of protein kinase C are potent inhibitors of potocytosis. Activators such as phorbol-12-myristate-13-acetate (PMA) inhibit the internalization of receptors for 5-methyltetrahydrofolate but allow the internal receptor pool to return to the cell surface. PMA does not affect the clustering of the folate receptor but instead markedly reduces the number of caveolae. Exposure to PMA totally blocks the intracellular accumulation of 5-methyltetrahydrofolate without affecting receptor-independent uptake or the formation of polyglutamylated species of 5-methyltetrahydrofolate in the cytoplasm. These data suggest that PMA inhibits uptake by inactivating caveolae internalization.

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity.

It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O(2). p38 activity in A549 cells was also increased by exposure to 95% O(2). To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9.

Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.

Genomic structure and promoter activity of the human tissue factor pathway inhibitor-2 gene.

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32 kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type inhibitor domains homologous to tissue factor pathway inhibitor. TFPI-2 strongly inhibits a wide variety of serine proteinases including trypsin, chymotrypsin, plasmin, kallikrein and blood coagulation factor XIa. In this study, we have isolated and characterized a genomic clone from an artificial chromosome genomic library that encodes the entire human TFPI-2 gene. The human TFPI-2 gene spans approximately 7 kb and consists of five exons and four introns. Each Kunitz-type domain is encoded by a single exon, similar to that observed for murine TFPI-2 and other Kunitz-type proteinase inhibitors. A total of 535 bp of the 3'-flanking region contain two probable polyadenylation sites (AATAAA) at +4297 and +4314. A single transcription initiation site was identified by oligo-capping and reverse transcription-PCR analysis. Transient transfection of reporter plasmids containing segments of the 5'-flanking region into human transformed bone marrow endothelial cells and glioblastoma cells identified an 85 bp region (-224 to -139) sufficient for transcription of the human TFPI-2 gene.

The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.

Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.

Expression, purification and characterization of a Kunitz-type protease inhibitor domain from human amyloid precursor protein homolog.

The Kunitz-type protease inhibitor domain from a recently identified homolog of the Alzheimer amyloid precursor protein (APPH KPI) was expressed in yeast, purified and characterized. Its inhibition profile towards several serine proteases was studied and compared to that of APP KPI, the Kunitz domain from the Alzheimer amyloid precursor protein. APPH KPI was shown to inhibit proteases with trypsin-like specificity with an inhibitor profile resembling that of the APP KPI domain. The KPI domains from APP and APPH inhibited trypsin (Ki = 0.02 nM), and plasma kallikrein (Ki = 86 nM) with approximal equal affinity. In comparison to APP KPI (Ki = 82 nM) the KPI domain of the homolog, APPH KPI, (Ki = 8.8 nM) was a more potent inhibitor of glandular kallikrein. APPH KPI was a less potent inhibitor of chymotrypsin than APP KPI (Ki = 78 nM as compared to Ki = 6 nM), plasmin (Ki = 81 nM as compared to 42 nM), and factor XIa (Ki = 14 nM as compared to Ki = 0.7 nM). The affinity of factor XIa for APPH KPI is sufficiently high to allow for an interaction in the blood. It is, however, well possible that the physiological protease ligand for the receptor-like APPH protein has yet to be identified.

Nucleotide sequence of the gene encoding murine tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a lambda FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3'-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5'-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.

Elevated tissue levels of interleukin-1 beta and tumor necrosis factor-alpha in vulvar vestibulitis.

OBJECTIVE: To compare levels of two inflammatory cytokines, interleukin-1 beta(IL-1 beta) and tumor necrosis factor-alpha(TNF-alpha), in selected regions of the vulva, vestibule, and vagina in women with vulvar vestibulitis and in asymptomatic controls. METHODS: Selective samplings of surgical specimens from 12 women undergoing perineoplasty for vulvar vestibulitis and ten pain-free subjects undergoing posterior vaginal repair were prepared into tissue homogenates and analyzed for concentrations of IL-1 beta and TNF-alpha. Interleukin-1 beta and TNF-alpha concentrations were measured by sandwich enzyme-linked immunosorbent assay. The results were reported after adjustment for total tissue protein concentration. RESULTS: Median tissue levels of IL-1 beta and TNF-alpha were elevated 2.3-fold and 1.8-fold, respectively, in women with vulvar vestibulitis relative to pain-free women. Median IL-1 beta tissue levels were increased significantly from 1.3 pg/mg to 3.0 pg/mg total protein in women with vulvar vestibulitis compared to pain-free women. Median TNF-alpha tissue levels were increased from 83 pg/mg to 148 pg/mg total protein in women with vulvar vestibulitis compared to pain-free women. Analysis by selected anatomic site of women with vulvar vestibulitis revealed a significant 2.2-fold higher median level of TNF alpha at the vulvar site compared to the vestibule. CONCLUSION: Concentrations of IL-1 beta and TNF-alpha were elevated in women with vulvar vestibulitis relative to those in asymptomatic controls. This elevation in inflammatory cytokines with vulvar vestibulitis varied according to anatomic site and was, paradoxically, lowest in the area of highest hyperalgesia, the vulvar vestibule. Inflammatory cytokine elevation may contribute to the pathophysiology of mucocutaneous hyperalgesia.

Effect of vulvovaginal estrogen on sensorimotor response of the lower genital tract: a randomized controlled trial.

OBJECTIVE: To assess the effect of vulvovaginal estrogen on mucocutaneous sensory threshold and circumvaginal motor strength. METHODS: Thirty-nine postmenopausal, hypoestrogenic women with mixed lower-genitourinary-tract complaints were placed in four masked treatment arms by permuted-block randomization for 6 weeks. One group received topical estradiol (E2) cream and pelvic muscle biofeedback training, the second received topical E2 cream and sham biofeedback, the third received placebo cream and pelvic muscle biofeedback training, and the fourth received placebo cream and sham biofeedback. Circumvaginal muscle strength was measured by averaging maximum intravaginal pressure (mmHg) generated over a set of four pelvic muscle contractions. Absolute changes in von Frey threshold (mN) and maximum intravaginal pressure (mmHg) over 4 and 6 weeks were reported as summary measures. Of 39 subjects, 30 completed the study. RESULTS: Topical estradiol cream significantly improved mechanical sensitivity of the vulvar vestibule to von Frey hairs, a -1.2-mN threshold decrease at 4 weeks (F = 10.29; P = .004), and a -1.6-mN threshold decrease at 6 weeks (F = 8.24; P = .009) compared with placebo cream. Stratification by age showed significantly greater improvement in mechanical sensitivity in the older (70-79 years) age group randomized to estrogen cream and a -5.49-mN threshold reduction (F = 17.65; P = .002). Maximum intravaginal pressures during circumvaginal muscle contraction did not differ between estrogen and placebo cream users (F = 0.00; P = .99). CONCLUSION: Improved sensation to mechanical stimuli can result from a rapidly acting, direct effect of topical E2 cream on the vulvar vestibule.

The impact of prenatal care on fetal and neonatal death rates for uninsured patients: a "natural experiment" in West Virginia.

A three-county program in southern West Virginia was developed by an obstetric practice to deliver prenatal care to a population of uninsured patients. Between January 1984 and December 1986, 1331 (29.4%) of 4534 patients were delivered at a level 2 hospital after prenatal care within the clinic program. The hospital-wide fetal death ratio declined from 11.8 to 7.2 per 1000 live births during the years of clinic operation, a statistically significant reduction (P = .02). Uninsured patients experienced a statistically significant reduction in fetal death ratio during the program, from 35.4 to 7.0 per 1000 live births (P = .02), whereas those covered by medical assistance did not experience a reduction. Privately insured patients also had a significant decrease, from 10.0 to 3.1 per 1000 live births (P less than .001). The increasing operating expense, mainly due to rising malpractice insurance premiums, required suspension of the program in December 1986. The fetal death ratio returned to 10.3 deaths per 1000 live births in 1987. Factors that varied significantly during the "clinic" phase included: higher rates of cesarean, diagnosed maternal hypertension, and diabetes mellitus; and lower rates of premature rupture of membranes and non-white population. Other factors, including age over 35 years, postdatism, incidence of twins, incidence of lethal congenital anomalies, and single marital status, did not vary significantly before, during, or after the clinic program. This study identified a high-risk population of patients who did not qualify for medical assistance coverage and were de facto "uninsured." The results suggest that prenatal care for this high-risk population of uninsured patients can reduce the fetal death rate.

The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line.

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tissue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not involved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gladomain in this reaction. Antibody experiments revealed that this effect was not due to trace levels of a tissue factor pathway inhibitor contaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of heparin resulted in a factor VIIa-tissue factor neutralization rate essentially identical to that observed for antithrombin III alone. Plasma levels of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pronounced as that of factor X. Other vitamin K-dependent plasma proteins including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of mixed phospholipids. Our collective finding suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin molecule resulting in a transient ternary complex of antithrombin III-heparin-factor X that represents the anticoagulant species. Factor X conceivably guides complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue factor complex and facilitates rapid neutralization of factor VIIa. Our findings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than previously recognized.

Inhibitory properties of human recombinant Arg24-->Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2).

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.

Evidence that an Arg79-->Gln substitution in human factor VII is not associated with a reduction in coagulant activity.

A recent report hypothesized that an Arg79-->Gln mutation in the first epidermal growth factor-like domain of human factor VII is the molecular basis for a severe (< 1%) factor VII functional deficiency. In the present study, a site-specific mutant human factor VII cDNA (Arg79-->Gln) was constructed, subcloned and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity and characterized with respect to gamma-carboxyglutamic acid content, ability to activate, tissue factor-dependent amidolytic activity and expression of factor VIIa proteolytic activity on tissue factor-bearing cells. Mutant factor VII was fully carboxylated and exhibited the same molecular weight and coagulant activity as plasma factor VII. Mutant factor VII was activated by factor Xa at the same rate, and to the same extent, as plasma factor VII. In the presence of tissue factor, mutant factor VII was converted to factor VIIa in an autocatalytic manner at a rate indistinguishable from that observed with plasma factor VII. In addition, the amidolytic activities of mutant factor VIIa and plasma factor VIIa towards S-2288 in the presence of relipidated tissue factor were identical. Finally, following complex formation with cell surface tissue factor, mutant factor VIIa activated factor X at essentially the same rate as plasma factor VIIa under comparable conditions. These results are not consistent with the notion that the arginine-79 residue in the first epidermal growth factor-like domain of human factor VII is essential for the expression of tissue factor-dependent factor VIIa proteolytic activity.

Vulvitis and vaginitis.

Although the vulva and vagina are embryologically and histologically different, proximity results in inflammatory conditions which commonly affect both regions. Patients often confuse 'vulvitis' and 'vaginitis' in characterizing symptoms. It is the job of the clinician to recognize inflammatory conditions specific to vulva and vagina, as well as to understand that inflammatory conditions can often involve both areas. This review of recent literature does not pretend to be comprehensive. Rather, it highlights specific conditions and research questions of recent interest.