Pulsatile Versus Nonpulsatile Flow During Cardiopulmonary Bypass: Extent of Hemolysis and Clinical Significance.

Pulsatile flow has been used during cardiopulmonary bypass (CPB) for decades and its use is increasing with advancing extracorporeal technology. Pulsatile flow generates higher circuit pressures and shear forces than nonpulsatile flow at comparable pump flow and patient mean arterial pressure. Very little is known about the effect this has on erythrocytes. We included 62 adult patients (32 in the pulsatile group and 30 in the nonpulsatile group) undergoing elective coronary artery bypass grafting in this prospective observational study. Blood samples were collected at routine sampling times throughout surgery and were analyzed for the presence of free heme and globin using mass spectroscopy. Patient characteristics, CPB, and aortic cross-clamp times, pump flow as well as patient mean arterial pressure were similar in both groups. Maximum circuit pressure in the pulsatile flow group was statistically significantly higher than that in the nonpulsatile flow group (257.12 vs. 190.64 mmHg, p < 0.0001). Both heme and globin levels were higher in the pulsatile flow group. This reached statistical significance with globin at 30 minutes of CPB and with heme after aortic unclamping. We conclude that pulsatile CPB using roller pumps results in a greater extent of hemolysis. The clinical significance, however, is not yet known.

Regulation of human chorionic gonadotropin beta subunit expression in ovarian cancer.

PURPOSE: Expression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer. METHODS: Expression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR. RESULTS: mRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3-9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue. CONCLUSIONS: In ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3-9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers.

Shotgun metabolomic profiles in maternal urine identify potential mass spectral markers of abnormal fetal biochemistry - dihydrouracil and progesterone in the metabolism of Down syndrome.

In Down syndrome (DS) in particular, the precise cellular mechanisms linking genotype to phenotype is not straightforward despite a clear mapping of the genetic cause. Metabolomic profiling might be more revealing in understanding molecular-cellular mechanisms of inborn errors of metabolism/syndromes than genomics alone and also result in new prenatal screening approaches. The urinary metabolome of 122 maternal urine from women with and without an aneuploid pregnancy (predominantly Down syndrome) were compared by both zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and reversed-phase liquid chromatography (RPLC) coupled to hybrid ion trap time of flight mass spectral analysis. ZIC-HILIC mass spectrometry resolved 10-fold more unique molecular ions than RPLC mass spectrometry, of which molecules corresponding to ions of m/z 114.07 and m/z 314.20 showed maternal urinary level changes that significantly coincided with the presence of a DS fetus. The ion of m/z 314.20 was identified as progesterone and m/z 114.07 as dihydrouracil. A metabolomics profiling-based maternal urinary screening test modelled from this separation data would detect approximately 87 and 60.87% (using HILIC-MS and RPLC-MS, respectively) of all DS pregnancies between 9 and 23 weeks of gestation with no false positives.

Direct analysis of hCGßcf glycosylation in normal and aberrant pregnancy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The analysis of human chorionic gonadotropin (hCG) in clinical chemistry laboratories by specific immunoassay is well established. However, changes in glycosylation are not as easily assayed and yet alterations in hCG glycosylation is associated with abnormal pregnancy. hCGß-core fragment (hCGßcf) was isolated from the urine of women, pregnant with normal, molar and hyperemesis gravidarum pregnancies. Each sample was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis following dithiothreitol (DTT) reduction and fingerprint spectra of peptide hCGß 6-40 were analyzed. Samples were variably glycosylated, where most structures were small, core and largely mono-antennary. Larger single bi-antennary and mixtures of larger mono-antennary and bi-antennary moieties were also observed in some samples. Larger glycoforms were more abundant in the abnormal pregnancies and tri-antennary carbohydrate moieties were only observed in the samples from molar and hyperemesis gravidarum pregnancies. Given that such spectral profiling differences may be characteristic, development of small sample preparation for mass spectral analysis of hCG may lead to a simpler and faster approach to glycostructural analysis and potentially a novel clinical diagnostic test.

Novel insights into the expression of CGB1 & 2 genes by epithelial cancer cell lines secreting ectopic free hCGß.

BACKGROUND: Ectopic secretion of human chorionic gonadotrophin free beta (hCGß) by epithelial cancer is associated with aggressive tumors which more readily metastasize, possibly by acting as an autocrine anti-apoptotic agent. hCGß is encoded by six homologous CGB genes, with poorly-understood variable transcriptionally active expression profiles; CGB1 and CGB2 have always been considered pseudogenes. However, transcripts from CGB1 and -2 can be detected in placental, testicular and pituitary tissues. The expression and function of these genes in cancer is less well-known. MATERIALS AND METHODS: Expression profiles of CGB genes in epithelial cancer cells by quantitative polymerase chain reaction (qPCR) were explored, along with the consequence of specific siRNA silencing of CGB1 and 2. Immunohistochemical and immunoassay techniques were used to detect the translation and secretion of hCGß in these cells. RESULTS: CGB1 and -2 gene transcripts were only detected in cells which secreted hCGß. siRNA-mediated silencing of CGB1 and -2 transcripts significantly reduced secreted protein in concordance with a reduction in cell survival to a greater degree than that of other CGB genes. CONCLUSION: CGB genes 1 and 2, previously considered as pseudogenes, are notably expressed by epithelial cancer cell lines. The transcription of these genes, but not other CGB genes, correlates with a functionally expressed protein and propensity for cancer growth.

Stable knockdown of hCGß mRNA expression in bladder cancer cells results in significant growth inhibition.

BACKGROUND: Expression of human chorionic gonadotropin beta subunit (hCGß) by epithelial carcinomas is associated with a poor prognosis and has a proposed autocrine growth effect on cancer cells by inhibition of apoptosis. MATERIAL AND METHODS: We transduced the hCGß-expressing bladder cancer cell line SCaBER with short hairpin (sh) RNA lentiviral gene-specific (CGB) constructs and determined its impact on the synthesis of hCGß and the resultant effect on cancer cell growth. RESULTS: Stable CGB gene-silenced clones exhibited a 60%-80% reduction in the level of hCGß expressed and a reduced growth rate of more than 40% compared to wild-type SCaBER cells. CONCLUSIONS: shRNA Lentiviral particles achieve stable knockdown of hCGß translation in the bladder cancer cell line SCaBER. This transforms the phenotype by reducing hCGß expression and cell growth rate. This is consistent with the proposed autocrine/paracrine function of ectopic hCGß expression during oncogenesis.

Single point biochemical measurement algorithm for early diagnosis of ectopic pregnancy.

OBJECTIVES: Tubal rupture as a result of an ectopic pregnancy is the leading cause of first trimester maternal mortality. Currently, the diagnosis of ectopic pregnancy depends on transvaginal ultrasound and serial serum measurements of human chorionic gonadotrophin (hCG), which requires follow up. The objective of this study was to examine whether single point measurements at presentation could distinguish between women with ectopic pregnancy, viable pregnancy, and spontaneous miscarriage. DESIGN AND METHODS: Serum total hCG (hCGt), hyperglycosylated hCG (hCGh), free beta subunit of hCG (hCGß), progesterone (P), and CA-125 were measured by chemiluminescence immunoassay over a 3 month period in 441 women presenting at the emergency room with abdominal pain and a positive pregnancy test. Patient outcomes were followed and confirmed by histology. 65 samples were excluded due to poor sample storage, or lost to follow up. RESULTS: The pregnancy outcomes were 175 viable pregnancies, 175 spontaneous miscarriages, and 26 ectopic pregnancies. A serum hCGt <3736 mIU/mL cut off was 100% sensitive, with 76% specificity, for distinguishing ectopic pregnancy from viable pregnancy; but did not differentiate spontaneous miscarriage. Serum CA125 <41.98 U/mL produced 100% sensitivity and 43% specificity in distinguishing ectopic pregnancy from spontaneous miscarriage. Sequential application of hCGt and CA-125 cut off followed by ultrasound could detect 100% of ectopic pregnancies with 87% specificity for all intrauterine pregnancies. CONCLUSION: The combination of serum hCGt <3736 mIU/mL, followed by CA125 <41.98 U/mL is a promising algorithm for detecting all ectopic pregnancy at initial presentation.

Human chorionic gonadotropin (hCG) in the secretome of cultured embryos: hyperglycosylated hCG and hCG-free beta subunit are potential markers for infertility management and treatment.

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGß) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGß was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGß was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGß expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer.

Arsenic trioxide induces cervical cancer apoptosis, but specifically targets human papillomavirus-infected cell populations.

OBJECTIVES: Human papillomavirus (HPV) is directly associated with the occurrence and development of cervical cancer. Targeting HPV infection has become the priority in treatment and prevention. Some treatment strategies have shown a limited therapeutic potential in suppressing and reversing the oncogenic effects of HPVs, but are compromised by the toxicity, immune suppression and the expense. Arsenic trioxide (As2O3) has shown therapeutic efficacy in the treatment of haematological and solid cancer and has been demonstrated to effectively induce apoptosis of HPV-infected cervical cancer cells in vitro. Here, we examined the effects and possible molecular pathway of As2O3-induced apoptosis in HPV-infected and noninfected cervical cancer cells. METHODS: As2O3 was added to HPV-infected cell lines HeLa and CaSki and the HPV-negative cell line C33A at concentrations from 1 to 10 µmol/l. Cell proliferation rates were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after exposure. Expressions of tumour suppressor gene p53, activated caspase-3 and cell cycle distribution were evaluated in relation to HPV-E6 protein expression by confocal microscopy immunofluorescent staining and flow cytometry. RESULTS: As2O3 reduced cell populations by 16% in C33A but by 48-60% in HPV-infected cell lines CaSki and HeLa. The expression of HPV-E6 proteins was drastically down-regulated in a dose-dependent manner, whereas p53 and activated caspase-3 expressions increased in the HPV-infected cell lines. Flow cytometry demonstrated cell cycle arrest in S-G2/M phases, and increasing apoptotic bodies were seen in HPV-infected lines only. CONCLUSION: As2O3, at low concentrations, inhibited HPV-E6 protein expression, leading to up-regulated p53 levels, induced S to G2/M arrest and apoptosis.

Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles.

BACKGROUND: The production of sex steroids by follicular cells is proposed to be influenced by the maturity of the incumbent oocyte. Thus steroid levels may reflect suitability of an oocyte for IVF. We examined follicular fluids and granulosa cell production of steroid from IVF patients in order to test the relationship between steroid levels and fertilization. METHODS: Follicular fluid and granulosa cells were extracted from 206 follicles of 35 women undergoing controlled ovarian stimulation. Follicular fluid was assayed for estradiol, progesterone and testosterone. Granulosa cells were cultured from individual follicles and their culture media assayed for production of these hormones after 24 hrs in vitro. Levels of steroids were correlated with follicular diameter, oocyte recovery and subsequent fertilization. RESULTS: Follicular fluid levels of progesterone were 6100 times higher than that of estradiol, and 16,900 times higher that of testosterone. Despite the size of follicle triggered after controlled luteinization, the levels of progesterone and testosterone were maintained at relatively constant levels (median 98.1 micromoles/L for progesterone, and 5.8 nanomoles/L for testosterone). However, estradiol levels were slightly lower in the larger follicles (follicular diameter 10-15 mm, median 25.3 nanomoles/L; follicles > = 15 mm, median 15.1 nanomoles/L; linear correlation r = -0.47, p < 0.0001). With respect to oocyte recovery, no steroid showed a significant association in follicular fluid levels. Similarly no difference in follicular fluid steroid levels was found for those oocytes that did or did not fertilize. Significant quantities of progesterone were produced by the granulosa cells but production was constant regardless of the size of follicle from which the cells originated. Estradiol levels were only detectable in 10 of 121 cultures examined, and testosterone in none. Interestingly, when an oocyte was present follicular estradiol levels correlated with progesterone levels. However, when absent, follicular estradiol levels correlated with testosterone levels but not with progesterone. CONCLUSIONS: The principle steroid product of luteinized pre-ovulatory granulosa is progesterone, a differentiation triggered by the gonadotropin surge. However, absolute steroid levels are associated with follicular size, not oocyte maturation/ability to fertilize.

Bony metastases from breast cancer - a study of foetal antigen 2 as a blood tumour marker.

BACKGROUND: Foetal antigen 2 (FA-2), first isolated in the amniotic fluid, was shown to be the circulating form of the aminopropeptide of the alpha 1 chain of procollagen type I. Serum concentrations of FA-2 appeared to be elevated in a number of disorders of bone metabolism. This paper is the first report of its role as a marker of bone metabolism in metastatic breast cancer. METHODS: Serum FA-2 concentrations were measured by radioimmunoassay in 153 women with different stages of breast cancer and in 34 normal controls. RESULTS: Serum FA-2 was significantly elevated in women with bony metastases (p < 0.015). Its levels were not significantly different among women with non-bony metastases, with non-metastatic disease, as well as among normal controls. CONCLUSIONS: FA-2 is a promising blood marker of bone metabolism. Further studies to delineate its role in the diagnosis and management of bony metastases from breast cancer are required.

High follicular fluid adenosine levels may be pivotal in the metabolism and recycling of adenosine nucleotides in the human follicle.

This study investigated the biochemical relationship between human follicular/oocyte maturity and the levels of follicular fluid purines. Intrafollicular levels of purine metabolites and creatinine are associated with oocyte presence, and the presence of such high levels of adenosine indicates a privileged site with no adenosine deaminase activity. Subgrouping according to oocyte recovery and fertilization revealed differences in correlation between the purine metabolites: Only where an oocyte was recovered and subsequently fertilized did follicular fluid adenosine, adenine, and hypoxanthine levels correlate with each other. Significantly, purines' correlation with levels of the terminal degradation product, uric acid, could only be seen in failed fertilization samples. Given the established metabolic pathways for adenosine triphosphate/adenosine diphosphate/adenosine monophosphate degradation, the results indicate maximization of 2 purine salvage pathways (from adenine and hypoxanthine) that pivot on the presence of high adenosine levels. Such optimized recovery may be necessary to build a store of salvaged adenosine phosphate for oocyte survival.

Towards the development of an electrochemical biosensor for hCGbeta detection.

The free beta subunit of human-chorionic-gonadotropin (hCGbeta) is critical for various aspects of human health. Detection and quantification of this protein are essential during pregnancy as it provides clinicians valuable information regarding the progress of a pregnancy and the health of a foetus. Furthermore, it can be used as a biomarker for gestational trophoblastic disease (GTD), germ cell tumours and some non-trophoblastic gynaecological cancers and common epithelial tumours. Monitoring hCGbeta levels is particularly important for patient treatment monitoring and relapse detection especially in GTD. This paper presents an investigation of the characteristics of the first two stages necessary for the development of a bio-impedance hCGbeta sensor, using impedance spectroscopy and commercially available microelectrodes. Additionally, electrical equivalent circuit models based on the experimental results of these stages are presented. The biosensor is based on the formation of stable antibody-antigen complexes on golden microband electrodes covered with a layer of a self-assembled monolayer (SAM) or with both SAM and protein G. The preliminary results and analysis relate the interfacial processes and physical structure of the sensor to its electrical behaviour. Finally, preliminary results obtained from the sensor without protein G, which strongly indicate hCGbeta detection, are also presented.

Human granulosa-lutein cell in vitro production of progesterone, inhibin A, inhibin B, and activin A are dependent on follicular size and not the presence of the oocyte.

OBJECTIVE: To investigate inhibin A, inhibin B, activin A, and P production by cultured granulosa cells (GCs) and what relationship this hormone production has to fertility. DESIGN: Luteinized GCs from individual follicles were cultured, and inhibin A, inhibin B, activin A, and P production were measured by ELISA at 24 and 72 hours. SETTING: Research laboratory and university hospital. PATIENT(S): Fifteen women who undertook an IVF-ICSI program, yielding 58 follicles. INTERVENTION(S): Individual follicular aspiration and preparation of GCs for culture. MAIN OUTCOME MEASURE(S): Inhibin A, inhibin B, activin A, and P production; oocyte retrieval; and fertility outcome. RESULT(S): Inhibin A, inhibin B, and P continued to be secreted by GCs in vitro, and activin A levels were detected only marginally in 56% of cultures. The rate of production also was dependent on the size of follicle from which the GCs originated but not on oocyte presence or ability to fertilize. Granulosa cell stimulation with hCG had no effect on inhibin A but increased P and decreased inhibin B production. CONCLUSION(S): A marked effect of luteal differentiation appears to be the inhibition of inhibin B production in response to hCG stimulation. Luteinized GC function, with respect to inhibins, activin A, and P production, was not influenced by the presence or absence of an oocyte and did not correlate with fertility outcome. However, follicle size did influence rates of local hormone production.

Gonadotropins and prostate cancer: revisited.

Luteinizing hormone and follicle-stimulating hormone are called gonadotropins, because they stimulate the gonads - in males the testes and in females the ovaries. They are not necessary for life, but are essential for reproduction. In addition, the association of these hormones with prostate cancer has been the interest of many researchers. Their detection in the human prostate has been investigated using different methods, including immunologic and RT-PCR techniques. In addition, the increasing evidence of paracrine/autocrine functions of the gonadotropic glycoprotein hormones, their allocation to the superfamily of cystine knot growth factors, and luteinizing hormone/chorionic gonadotropin receptor gene expression in non-gonadal tissues led many researchers to investigate intraprostatic glycoprotein hormones and their receptor gene expression. We aim in this review to shed light on the physiology of the gonadotropins and their association with prostate cancer and highlight the future possibilities of their use as targets in treating this disease.

Follicular fluid levels of inhibin A, inhibin B, and activin A levels reflect changes in follicle size but are not independent markers of the oocyte's ability to fertilize.

OBJECTIVE: To investigate the biochemical relationship between follicular/oocyte maturity and follicular inhibins and activin levels. DESIGN: Prospective study. SETTING: Research laboratory in university hospital. PATIENT(S): Thirty-five women undertook IVF/ICSI program. INTERVENTION(S): Individual follicular fluid aspirations, oocyte isolation, follicular fluid storage. MAIN OUTCOME MEASURE(S): Inhibin A, inhibin B, and activin A concentrations, oocyte retrieval, and fertility outcome. RESULT(S): Inhibin A, inhibin B, and activin A concentrations varied from 7.9 to 436 ng/mL, 9.7 to 786 ng/mL, and 1.7 to 267.9 ng/mL, respectively. There was no change of inhibin A concentrations, whereas inhibin B and activin A concentrations dropped dramatically as the follicles enlarged. Total follicular content of inhibin A and activin A increased, and inhibin B remained constant. Both inhibin A and inhibin B levels were significantly higher in those follicles from which an oocyte could be recovered, but they did not differ with respect to subsequent oocyte fertilization. CONCLUSION(S): Inhibin A is actively produced throughout follicular growth to retain a set concentration. In contrast, inhibin B appears not to be actively produced, and the concentration drops as follicles enlarge. Activin A concentrations also decrease, but there is some extra synthesis. Higher levels of inhibin A and B are associated with oocyte presence but not with fertilization rates.

Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques.

A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of this complex showed three polypeptides at approximately 6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-beta (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-beta in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-beta, which may have transforming growth factor-beta agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonadotrophin preparations.

The free monomeric beta subunit of human chorionic gonadotrophin (hCG beta) and the recently identified homodimeric beta-beta subunit (hCG beta beta) both have autocrine growth effects.

The ectopic production of free hCG beta is a common phenomenon in epithelial tumours, a phenomenon originally believed to have no biological significance. However, it is now apparent that hCG beta may significantly effect tumour development by increasing cell populations through inhibition of apoptosis. The recently identified hCG beta beta homodimer, with topological similarities to cystine knot growth factors, has been suggested to be the responsible mediator of these novel tumourigenic responses. In this study we isolated hCG beta monomer from hCG beta beta homodimer using size exclusion chromatography and confirmed the separation by Western blotting. Using a tetrazolium bromide incorporation cell number quantification assay (MTT), we measured the growth effects of separated hCG beta fractions corresponding to monomeric (hCG beta) and dimeric (hCG beta beta) forms on the hCG beta responding cell line T24. Maximal increases in cell number corresponded to the elution peak of dimeric and monomeric hCG beta. In conclusion, it would appear that the recently observed hCG beta beta homodimer is no more bioactive than its monomeric counterpart, in stimulating bladder cancer cell growth. This strengthens the proposition that hCG beta may exert its antiapoptotic effects by antagonistic inhibition of other cystine knot growth factor receptors and not by a specific receptor-mediated homodimeric interaction as seen for its topological counterparts TGF, PDGF-B and NGF.