The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis. HMLoviruses . Eighty-oviruses ,20. Sincoviruses ,22.pol and env genes [env mRNA from type 2 proviruses is subspliced to create a rec mRNA that encodes the Rec (formerly cORF) protein, a functional homologue to Rev and Rex, the RNA-binding nuclear export proteins of HIV and HTLV, respectively [rec splice donor site that is located in the 292 bp stretch [np9 mRNA. The corresponding Np9 protein shares only 14 aa with Rec and Env [Two major types of HML-2 proviruses exist in the genome. Type 1 proviruses differ from full-length type 2 proviruses by a 292 bp deletion within the boundary of nv genes ,24env mRectively -29. Rec ectively -32. Type stretch . An alte and Env ,34.env gene [Betaretroviruses that include retroviruses formerly classified as type B and D [Rem. The latter was shown to translocate into nucleoli of murine T cell lymphoma cells [Rem remain to be elucidated.HERV-K(HML-2) displays significant sequence similarities with Mouse Mammary Tumor virus (MMTV), particularly for the env gene . Both HM B and D . MMTV al B and D ,38. Rem ma cells ,40. Specin vitro. Some SPs are further processed by an ER intramembrane cleaving protease, the signal peptide peptidase and released into the cytosol where they can accumulate [Characterization of presecretory eukaryotic and prokaryotic signal peptides (SPs) defined the features essential for their function, such as hydrophobicity and a common sequence for the site of cleavage from its mature protein by signal peptidase -43. For cumulate -46. Impocumulate .Rem, several exceptions exist: HIV-1 gp120 Env SP remains bound to calnexin in the ER membrane and is inefficiently cleaved very late in the maturation process [The orientation of SPs across the ER membrane defines two types of signal peptides. Type I SPs anchor the proteins by transferring it across the ER membrane, leaving the C-terminus of the protein in the cytoplasmic side of the ER. Conversely, type II SPs retain the N-terminus of the protein in the cytosol ,48. Retr process ,52. For  process .Rem targets Rem to the ER, is then cleaved off and accumulates in the nucleoli. Interestingly, this process is independent of cleavage by signal peptide peptidase [More recently, biochemical studies showed that p14/SPeptidase .We describe here for the first time the HERV-K(HML2) Env precursor SP as a 13 kDa signal peptide. By examining features of HML-2 SP, such as subcellular localization, nucleocytoplasmic shuttling, protein stability and RNA export activity, we established functional dissimilarities to Rec. Our data suggest that HML-2 SP exerts a Rec-independent function. Furthermore, the finding of a long-lived SP for HML-2 reveals another similarity between the closely related HML-2 and MMTV retroviruses, thus further establishes their close relationship on the functional level.Retroviridae class and related endogenous retroviral members, using PHOBIUS, SignalP and TMD [Betaretrovirus prototypes, SP N-extensions consist of an unusually long sequence varying from 61 to 78 residues.To gain better insight into the organization of retroviral SPs, we first compared the SP regions of prototype members of each  and TMD -57. As d and TMD . The cen and TMD , encompa and TMD . For HERenv mRNA subsplicing. The first exon of Rec largely overlaps with the env SP coding sequence in that it comprises amino acids 1 to 87 of Env. The second exon of Rec is translated from a different reading frame. The resulting 18aa C-terminus is different in sequence from either the C-terminus of SP or Env. With regard to the resulting protein, Rec mRNA splicing occurs just upstream of the SPase cleavage site  Env is synthesized as a classical retroviral envelope protein. In the ER, the Env precursor undergoes a first cleavage by the signal peptidase releasing the 90 kDa Env precursor which then follows the maturation pathway to the Golgi where it is further cleaved by a furin-like endoprotease into two N-glycosylated domains, a 55 kDa surface subunit (SU) and a 39 kDa transmembrane subunit (TM) . In addition to SU and TM, an accessory protein Rec is encoded by a smaller mRNA resulting from e Figure . ContrarCON/Phoenix [CON [In order to determine conservation of SP among HML-2 proviruses and its sequence relationship to Rec, we compared relevant sequence portions of six HML-2 loci that could potentially encode full-length Env , the seq/Phoenix ,13, repr/Phoenix    that bears the NLS and NES and that is common with Rec. SP96-RFP (aa 1–96) corresponded to the full-length HML-2 SP sequence. To prevent or diminish signal peptidase cleavage during synthesis of SP96-RFP, we deleted the C-terminal two aa residues (GA) of the HML-2 SP, giving rise to construct SP94-RFP. Deletion of those two aa was based on a consensus sequence for signal peptidase cleavage, in which small uncharged residues in position -3 and -1, including a glycine residue, are thought to be important for cleavage [96/94/75-RFP precursor proteins was verified by Western Blot  Figure . The fircleavage . Proper  Figures  and 4B. s Figure , mRFP fu75-RFP was strongly enriched in the nucleoli of transfected cells and could also be detected in the cytoplasm, however, at significantly lower amounts than GFPcORF that was visible only after over-exposure  . HeLa cei Figure . As the 94-RFP, and in some cases that of SP96-RFP, was due to an aberrant conformation generated by the C-terminal RFP fusion, we determined HML-2 SP's localization after cleavage from its natural Env precursor. To facilitate analysis of SP in the Env context and to eliminate Rec production, we introduced silent mutations in rec splice donor and acceptor sites at nt positions 6708–6716 and 8404–8414, respectively (numbers refer to the HERV-K(HML-2.HOM) sequence [env and rec transcripts was analyzed by RT-PCR using appropriate primers. While env transcripts were readily observed, no transcript corresponding to rec mRNA could be detected (data not shown). Hence, the EnvΔRec construct predominantly produces SP and Env but no Rec. Furthermore, Env maturation and trafficking were not affected by the introduced point mutations, and as predicted, as more env mRNA was available for translation, EnvΔRec-expressing cells showed an increased amount of Env (data not shown).After cleavage from the native polypeptidic chain, SPs are usually released from the ER membrane and subsequently degraded. In some cases and in particular when the N-extension is long, SPs can be released into the cytosol and exert biological activities . As abovsequence  . That anti-SP antibody detected a protein of approx. 15 kDa, as predicted for Rec, in Env-expressing cells Figure . In EnvΔWe used EnvΔRec-expressing cells to determine the subcellular localization of HML-2 SP and compared it to that of Rec. Considering that the epitope designed for anti-SP production is also present in Rec, the antibody thus detecting both SP and Rec in Env-expressing cells, we preferred to employ a Rec expression plasmid . Subcell75-RFP, SP94-RFP and GFPcORF-expressing cells , an RNA polymerase II inhibitor, causes redistribution of nucleolar proteins, such as B23 and C23, into the nucleoplasm ,65. ActDt Figure . Similart Figure . Interesl Figure . These rAlthough HML-2 SP and Rec proteins share large parts of their amino acid sequence and both localize to nucleoli, the steady state protein levels in the context of HML-2 Env expression were significantly different Figure . To addrRem [To better understand the molecular mechanism responsible for reduced HML-2 SP stability, when compared to Rec, proteasome inhibition was achieved by addition of carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) to a final concentration of 10 μM. MG132 was added to Env- and EnvΔRec-expressing cells 16 hours post-transfection. Cells were collected every hour and analyzed by Western blot using the anti-SP antibody. As shown in Figure Rem . Altoget75-RFP, SP94-RFP or GFPcORF expression plasmids and co-cultured with mouse NIH3T3 cells 16 hours post-transfection. 24 hours later, protein synthesis was blocked by CHX treatment and fusion was induced. Syncytia formation was verified by May-Grünwald-Giemsa staining (not shown). Rec (GFPcORF) was readily detected in human and mouse nuclei, confirming its nucleo-cytoplasmic shuttling activity. SP75-RFP also shuttled into mouse nucleoli, even more efficiently than Rec/GFPcORF, demonstrating that the NES motif in SP75-RFP is functional. However, SP94-RFP was never found in mouse nucleoli, suggesting that in the context of the full-length sequence, the NES motif has lost its functionality. Thus, heterokaryon assays demonstrated that full-length HML-2 SP carries a non functional NES. HML-2 SP therefore does not appear to be a nucleo-cytoplasmic shuttle protein.The observed differences in protein stability and responsiveness to ActD suggested that HML-2 SP and Rec may have distinct biological properties. Rec's activity in export of genomic RNA strictly depends on functional NLS and NES motifs. While our above analysis demonstrated efficient nuclear import of SP, functionality of the putative NES sequence in SP , namely a 13 kDa SP that is produced by cleavage of the HML-2 Env precursor. Sequence analysis revealed that HML-2 SP is common to HERV-K(HML-2) type 2 proviruses and that the first 87 residues, out of 96, are shared with Rec, the protein exporting HERV-K(HML-2) mRNA out of the nucleus -29. LikeConsidering that HML-2 SP shares 87 of its 96 residues with Rec, the functional differences between both proteins are remarkable. These differences do not stem from altered subcellular localization. Surprisingly, while the nuclear import motif is functional in HML-2 SP, mediating its transport into the nucleoli, the NES motif, functional and well characterized for Rec, is no longer functional in HML-2 SP. Shuttle activity, as well as distinct protein stability, and RNA export inactivity are therefore probably determined by the HML-2 C-terminal 9 aa that distinguish SP from Rec. One may argue that reduced half-life of HML-2 SP, when compared to Rec, hampers its ability to export HML-2 RNA. Addition of ubiquitin on protein chains is known to target them to the proteasome ,72. SUMORem, also trafficking to nucleoli after its cleavage, showed that its release was independent of Signal Peptide Peptidase activity. The release was a time- and temperature-dependent process, and SPRem/p14 was found in the nucleolar fraction for at least 90 minutes [Rem and HML-2 SP are processed in the same manner.Signal peptide fragments resulting from processing of signal peptides by signal peptide peptidases are known to be released in the cytosol. Such processing could also occur in the hydrophobic region of HML-2 SP. Recent biochemical analysis of p14/SP minutes . It is cThe fact that SP sequences are common to HML-2 type 2 proviruses argues for a vital role of SP in the former viral life cycle. The presented data rule out that HML-2 SP served to back up or modulate Rec's main function – RNA export activity. SPs of other viral proteins also exert functions beyond ER targeting during the viral life cycle. In cases where specific functions could be attributed to the viral SP, most of these activities relate to the maturation and function of the respective viral glycoprotein and affect processes such as Env folding or post-translational modifications (HIV-1 gp120 and Ebola SP) ,74-76, cRem, especially when considering that both have not been characterized in great detail, yet. In the context of human biology, when assuming a causal relationship between expression of HML-2 gene products and germ cell tumor development, active immune evasion mechanisms are likely in place to counteract elimination of HML-2 protein-expressing cells. Tumor cells and virally infected cells often display significantly reduced human leukocyte antigen (HLA) cell surface levels, rendering them prone to attack and lysis by natural killer (NK) cells. Viruses and tumor cells have therefore evolved various strategies to interfere with NK cell recognition and activation [Rem has been reported to interact with B23/nucleophosmin [The data presented thus far do not allow us to conclude of what the former and potentially current biological activity of HML-2 SP consists. The same is true for a comparison of HML-2 SP functions with those of the related MMTV p14/SPtivation . Of notetivation ,84. Antitivation : followitivation . Worth mtivation . Based oophosmin  that hasophosmin . HML-2 Senv gene gives rise to an additional 13 kDa protein by cleavage of the signal peptide from the Env precursor. The HML-2 SP repesents another functional similarity with the related exogenous Mouse Mammary Tumor Virus that likewise encodes a stable signal peptide, adding another case to the concept of several retroviral SPs having biological functions besides ER-targeting. HML-2 SP and HML-2 Rec protein, the latter potentially involved in germ cell tumorigenesis, are very similar in sequence, except for the N-terminus. Our results strongly indicate that HML-2 SP lacks several functional features previously reported for HML-2 Rec. HML-2 SP is therefore expected to exert a different biological function. Besides a distinct function in the (former) retroviral context, HML-2 SP must also be considered in the context of human biology. Given the biological features found for HML-2 SP, and findings for the MMTV SP, one can hypothesize that HML-2 SP is involved in immune evasion of GCT cells. HML-2 SP may also interact with important cellular proteins, thereby playing a significant role in GCT development. Future research must address HML-2 SP in greater detail.The human endogenous retrovirus family HERV-K(HML-2) is exceptional for various reasons, among them its protein coding capacity. We now found that the HERV-K(HML-2) AF074086) [EcoRI sites of phCMV-G. pSP75-RFP, pSP94-RFP and pSP96-RFP plasmids express respectively the N-extension region (aa 1–75), the sequence missing the last two aa (aa 1–94) and the full length sequence (aa 1–96) of HML-2 signal peptide (SP), each fused to monomeric Red Fluorescent Protein (mRFP). SP sequences were amplified by PCR from phCMV-HML2-Env and inserted into the Ecl136II site of pmRFP-N1 multiple cloning site [rec mRNA, was generated by site-directed mutatgenesis  of Rec splice donor and acceptor sites located at aa 85–87 and aa 651–655, respectively, of Env. Integrity of generated plasmids was verified by sequencing. Expression plasmids for HML-2 Rec, pEGFP-wtcORF and pSG5-Rec, were described elsewhere [HeLa and NIH3T3 cells were maintained in Dubelcco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum (Biochrom). phCMV-HML2-Env was derived from phCMV-G, a VSV-G Env expression plasmid  by replaF074086)  and inseing site . phCMV-Elsewhere ,61. pDM1lsewhere ,70 were A rabbit anti-SP antibody was raised against HML-2 Env signal peptide residues 1–19 (MNPSEMQRKAPPRRRRHRN) and was used unpurified at a 1:1,000 dilution for Western blot analysis and 1:200 for immunofluorescence. The following antibodies were used at the indicated concentration: mouse monoclonal anti-nucleophosmin B23  at a 1:1,000 dilution for Western blotting and 1:50 for immunofluorescence; mouse monoclonal anti-nucleolin C23 (clone MS-3) (Santa Cruz Biotechnology) at a 1:50 dilution for immunofluorescence; mouse monoclonal anti-GAPDH (clone 0411) (Santa Cruz Biotechnology) at a 1:200 dilution for Western blot analysis; mouse monoclonal anti-β actin (clone AC-15) (Sigma) at a 1:10,000 dilution for Western blotting. Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (Dianova): goat anti-rabbit IgG  and sheep anti-mouse IgG . Fluorescent secondary antibodies were purchased from Molecular Probes (Invitrogen) and used at a 1:2,000 dilution: Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 568 goat anti-mouse IgG, and Alexa Fluor 568 goat anti-rabbit IgG.Transfected cells were harvested 48 hours post-transfection and lyzed in cold NP40 lysis buffer  supplemented with protease inhibitor cocktail . Samples were boiled in 5× Laemmli sample buffer, separated by 15% SDS-PAGE, and transferred onto Hybond-P membrane (Amersham). Membranes were blocked with Tris Buffered Saline containing 5% skim milk and 0.05% Tween 20 (Sigma) overnight at 4°C. Immunostaining was performed in the same buffer using appropriate first and secondary antibodies. Protein detection was performed by using the ECL Plus detection kit (Amersham) according to the manufacturer's instructions.2, protease inhibitor cocktail). After the final wash, the nuclear pellet was resuspended in 3 ml of S1 solution and layered over 3 ml of S2 solution . The sucrose cushion was centrifuged . The purified nuclear pellet was resuspended in 500 μl of S2 solution and sonicated (3 × 20 sec on ice). The sonicated nuclei suspension was layered over 1 ml of S3 solution  and centrifuged . The upper fraction was collected and used as nucleoplasm fraction; the pellet contained the purified nucleoli. All extracts were adjusted with 2× Laemmli sample buffer, sonicated and boiled before measuring protein concentration.Cell fractionation was adapted from a protocol by Lam and Lamond  with mod5 HeLa cells were seeded on glass coverslips and transfected with 2 μg expression plasmids. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde in Phosphate Buffered Saline (PBS) for 20 min and permeabilised with 0.1% Triton X-100 in PBS for 1 min. Cells were washed once with PBS before being treated with blocking solution (1% goat serum in PBS) for 15 min. All incubations were done at room temperature; washing steps consisted of three PBS washes of 5 min each. Cells were incubated with rabbit anti-SP, anti-nucleophosmin B23, anti-nucleolin C23 and appropriate fluorescent secondary antibodies. Finally, coverslips were mounted using Histoprime . Fluorescent microscopy images were acquired using an LSM 510 confocal laser scanning microscope (Zeiss) and using a 60× oil immersion objective lens. Confocal images were processed using Adobe Photoshop (Adobe Systems).To assay the subcellular localisation of the signal peptide, 2.5 × 106 HeLa cells were grown in 60 mm tissue culture plates. 16 hours post-transfection, cycloheximide  was added to a final concentration of 100 μg/ml to terminate protein synthesis. Cells were then collected every hour until 8 hours later. Cell lysates were prepared as described above and analyzed by Western blot.1 × 106 HeLa cells were grown in 60 mm tissue culture plates. 16 hours post-transfection, cells were incubated for 8 hours in the presence of 10 mM Carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal  diluted in dimethyl sulfoxyde . Mock samples and DMSO-treated cells were co-cultured in parallel as controls. Cell lysates were prepared as described above and analyzed by Western blot.1 × 105 HeLa cells were transfected with 2 μg pGFPcwtORF, pSP75-RFP or pSP94-RFP expression plasmids. Twenty-four hours after transfection, cells were mixed with 1 × 106 NIH3T3 cells and seeded onto 1.8 × 1.8 cm glass coverslips. Twelve hours later, cells were treated with 100 μg/ml CHX for 1 hour, and fusion of cell membranes was induced by addition of 50% (w/v) polyethylene glycol 8000 (Sigma) in DMEM for 2 min at 37°C. Cells were subsequently washed three times with pre-warmed PBS and once with DMEM, before being incubated at 37°C for another 6 hours in the presence of 100 μg/ml CHX. Finally, cells were fixed for immunostaining as described above. Human and mouse nuclei were distinguished by staining with Hoechst 33258 (Sigma) at 0.5 μg/ml in PBS. Fluorescent microscopy images were acquired using an LSM 510 microscope (Zeiss). Images were processed using Adobe Photoshop (Adobe Systems).2.5 × 105 HeLa cells were co-transfected with the CAT reporter plasmid pDM128/K-RRE and either pSG5-Rec, phCMV-HML2-Env, pK7-K-RevΔNES (RecΔNES) or phCMV-EnvΔRec, and pEGFP-N1 as a control for transfection efficiency. The CAT ELISA Kit (Roche) was used according to the manufacturer's instructions. Briefly, 24 hours after transfection, cells were lyzed and CAT-containing cell extracts were added into microplate wells coated with anti-CAT antibody, followed by addition of a digoxigenin-labeled anti-CAT antibody, and by addition of a peroxidase-conjugated anti-digoxigenin antibody. After addition of peroxidase substrate, the absorbance of the colored reaction product was measured at 405 nm (reference wavelength 490 nm) by using a microplate reader ELX-800 (Bio-Tek Instruments). Observed CAT production was normalized by using GFP as an internal control. GFP fluorescence of individual cells was analyzed by flow cytometry using a FACS Scan (Becton Dickinson).The mRNA export function of the HML-2 signal peptide was assessed by quantitative determination of chloramphenicol acetyltransferase (CAT) in a CAT ELISA assay. 5 × 10The authors declare that they have no competing interests.AR, EM, MS and OF planned and performed experiments. AR, OF and JM conceived the study. NML, EM, OF and JM financed the study. AR, OF and JM wrote the paper. All authors read and approved the final manuscript.


Antisera to extracts of a variety of bronchogenic carcinoma were raised in rabbits and extensively absorbed with immunoadsorbents prepared with normal lung extracts cyanogen bromide linked to Sepharose 4B, and glutaraldehyde insolubilized normal lung extracts. The antisera were tested by immunodiffusion against a panel of extracts from a variety of bronchogenic carcinoma, foetal lung extracts and pools of normal lung extracts. The results indicate that two distinct antigenic components are associated with bronchogenic carcinoma; one which is present in a high percentage of the tumour extracts tested and appears to have partial identity with a foetal lung component, and one (or more) which is not foetal and appears to have higher cross-reactivity (but not exclusively) with tumours of the same pathological type. Attempts to detect either antibody or antigens relating to these components in the serum of patients with bronchogenic carcinoma by these techniques were unsuccessful. The foetal cross-reacting component was neither carcinoembryonic antigen and alpha1-foetoprotein.


P< 0.001). Moreover, the uPAR levels in the two types of extracts were weakly, though significantly, correlated with each other . In Cox univariate analysis, high cytosolic levels of uPAR were significantly associated with reduced overall survival (OS) and relapse-free survival (RFS). The levels of uPAR in pellet extracts appeared not to be related with patient survival. In multivariate analysis, elevated levels of uPAR measured in cytosols and pellet extracts were found to be independent predictors of poor OS, not RFS. The prediction of poor prognosis on the basis of high uPAR levels emphasizes its important role in plasmin-mediated degradation of extracellular matrix proteins during cancer invasion and metastasis. © 2001 Cancer Research Campaign http://www.bjcancer.comUsing a previously developed enzyme-linked immunosorbent assay (ELISA), the levels of the receptor for urokinase-type plasminogen activator (uPAR) were determined in cytosols and corresponding membrane pellets derived from 878 primary breast tumours. The levels of uPAR in the pellet extracts were more than 3-fold higher than those measured in the cytosols (


Treponema pallidum that may also affect the inner ear. First reported in 1887 by Adam Politzer, luetic endolymphatic hydrops (LEH) is a treatable complication of syphilis which causes a potentially reversible sensorineural hearing loss. Symptoms of LEH include fluctuating hearing loss (often low frequency), tinnitus, and vertigo. Though audiometric parameters have been examined in patients with otosyphilis, few studies have examined the use of otoacoustic emissions (OAEs) as a tool to measure improvement in cochlear function. Here we report an improvement in hearing loss, speech discrimination, and OAEs following treatment of LEH.Syphilis is a preventable and curable multi-organ disease caused by  Since 2000, the incidence of primary and secondary syphilis in the U.S. has been increasing. During 2005-2006, the number of cases reported to CDC increased 12.4% for early latent syphilis (from 8176 to 9186), 9.9% for late and late latent syphilis (from 16 049 to 17 644), and 11.0% for the total number of cases of syphilis  (from 33 288 to 36 935) [Unlike primary idiopathic hydrops, treatment of LEH with steroids and antibiotics often reverses the symptoms. In a study by Lindstrom and Cleich, treatment of otosyphilis with penicillin and steroids caused an improvement in hearing (speech reception threshold and/or speech discrimination in 25%), decrease in tinnitus (71%), and improvement in disequilibrium in 66% of their patients . HoweverA 42-year-old female presented with complaints of hearing loss in her left ear for 2-3 months with occasional bilateral tinnitus. Prior to treatment, standard behavioral audiometry (250–8000 Hz) was obtained , as wellLaboratory testing included collagen profile test, RPR, and fluorescent treponemal antibody absorption (FTA-ABS) test. Results revealed the patient's RPR and fluorescent treponemal antibody absorption test to be positive. Furthermore, a history of syphilis was confirmed. Following a course of 2.4 million units of Benzathine Penicillin G and Prednisone for three weeks, the patient reported improvement in hearing. Repeat audiologic evaluation obtained one month after diagnosis revealed improved hearing , and intSyphilis is a multisystem disease that can affect the inner ear. Patients with LEH may present with tinnitus, fluctuating hearing loss with poor speech discrimination, episodic vertigo, and nystagmus . LEH man OAEs are believed to be a measurement of outer hair cell function and to be a by-product of the cochlear amplifier, a metabolic mechanism that enhances cochlear partition vibration during transmission of sound through the hearing system , 6. RecoIn this patient, there was both an improvement in hearing loss as well as recovery of OAE following treatment. This reversal of OAE loss suggests that injury to outer hair cell function and the cochlear amplifier are reversible entities in this disease. We suggest that OAE testing can be used to monitor response to treatment of LEH. Further studies may be indicated to assess the sensitivity and specificity of OAE testing as a therapeutic measurement.


Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown.The present findings demonstrate that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) rats compared to young (8-mo). Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II increases within the aged arterial wall. To further explore the relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor 1 or CAST. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001.Calpain-1 activation is a pivotal molecular event in the age-associated arterial Ang II/MMP2 signaling cascade that is linked to cytoskeleton protein restructuring, and VSMC migration. Therefore, targeting calpain-1 has the potential to delay or reverse the arterial remodeling that underlies age-associated diseases i.e. atherosclerosis. A growing body of evidence indicates that aging is the major risk factor for the development of the quintessential cardiovascular diseases i.e. atherosclerosis, hypertension, and stroke 2+ activated neutral protease, is a heterodimeric molecule which consists of a large 80 kDa catalytic and a small 30 kDa regulatory subunit 2+ loading status in a dose–dependent manner within living cells Calpain-1, a ubiquitous, cytosolic CaThe consequences of the physiological engagement of calpain-1 with its substrates are important because the spectrum of calpain actions strikingly resemble those that accompany aging of the arterial wall. We hypothesized that calpain-1 activity increases with aging and mediates some of the aforementioned aspects of the age-associated arterial wall proinflammatory state. In the present study, we demonstrate that the levels of transcription, translation, and activation of calpain-1 are significantly up-regulated in rat aorta or early-passage aortic VSMC from old rats compared to their young counterparts. In an attempt to define a specific role for calpain-1 in the arterial aging process, we constructed recombinant adenoviruses harboring cDNA of calpain-1 or its endogenous inhibitor calpastatin, and infected VSMC. We also infused Ang II into rats to determine its effect on calpain-1 levels and activity. The results show that calpain-1 activation appears to be a pivotal event in the age-associated arterial Ang II/MMP2 signaling cascade, including MMP2 activation, cytoskeleton protein restructuring, and VSMC migration. Thus, calpain-1 activation is central to the initiation and progression of proinflammation in the arterial wall with advancing aging.en face medial aortic sections demonstrated that increased calpain-1 protein within old rats was co-localized with α-smooth muscle actin (α-SMA), a marker of VSMC, stained cells ((left panel) three conspicuous spectrin bands, designated as intact (250 kDa) and cleaved spectrin fragments  respectively, are observed. In contrast, only intact spectrin was detected in young rat aortic lysates. Thus, on the basis of cleaved spectrin fragments old rats have a 3.3 fold increase in aortic calpain activity compared to their young counterparts (The quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the transcript level of calpain-1 increased 1.8 fold within the aortic wall in old (30 mo) compared to young (8 mo) rats Fig. 1A;, yellow color, right lower panel). We attempted to reproduce features of arterial aging in specimens from young animals. A 28 day infusion of Ang II into young rats (8mo) increases aortic calpain-1 protein staining , and activity by 1.2 fold  compared to the saline infused (control). Thus calpain-1 activation follows Ang II exposure in young aortae, resembling that of untreated old animals in .Ang II is a known stimulator of calpain-1 expression in cardiomyocytes staining Fig. 2Bin vivo alters both circulating cells and hemodynamic factors −7 mol/L) for 48 hrs. Calpain-1 protein abundance was increased by 1.7 fold  and activity by 1.2 fold , up to the levels of untreated arterial rings from old rats.Ang II infusion 1.7 fold  and acti), protein expression abundance , as well as activity  were up-regulated within old VSMC compared to young cells. Further, qRT-PCR analysis indicated that Ang II induces young VSMC to increase calpain-1 transcription in a dose-dependent manner, achieving the level of untreated old cells . Likewise, treatment of young cells with Ang II increases calpain-1 protein abundance in a dose-dependent fashion, to the level of untreated old cells . Interestingly, the age- and Ang II-associated increases in calpain-1 protein and activity are substantially reduced by -Angiotensin II acetate hydrate (SG), an AT1 receptor antagonist .To determine whether VSMC in vitro (p3-p5), in fact, retain their properties in vivo, aortic VSMC were isolated from young and old animals. Indeed, calpain-1 transcriptome Fig. 4A. Additionally MMP2 activity within untreated young and old VSMC was substantially inhibited by Ci 1 . To confirm a specific role of calpain-1 in Ang II induced MMP2 activation, young VSMC infected with a virus harboring calpastatin (CAST), an ectopic expression of the endogenous calpain inhibitor, were exposed to Ang II. We have previously shown that Ang II increases MMP2 activation in the arterial wall in vivo and ex vivo and in isolated VSMC in vitro . Additio. Additio. Additio2+]) are known to increase with aging With aging, alterations of VSMC cytoskeletal proteins such as α-SMA and vimentin occur concomitantly with changes in VSMC migration rates upper panel) and generates a multiple proteolytic fragment pattern , similar to old uninfected or CANP-1 infected cells . Remarkably, infection of old VSMC with an adenovirus harboring CAST indeed inhibits calpain-1 activity (upper panel) and generates a pattern of proteolytic products of vimentin resembling that from the untreated young cells .This characteristic age-associated shift of vimentin fragments suggests that arterial wall calpain-1 may cleave the intact vimentin, generating multiple proteolytic fragments. To test this hypothesis, we also constructed and amplified adenoviruses harboring calpain-1 and over-expressed calpain-1 in VSMC in (0 hr), and then begins to protrude outwardly as the cell becomes attached to the substratum (3 hrs). At 5 hrs, the cell has become attached, as shown by a completed circular border with calpain-1 preferentially localized at the periphery of the cell. At the 8 hr point, the spreading processes have taken place and calpain-1 has continuously expanded peripherally, accompanied by the formation of lammellipodia. Finally, calpain-1 is uniformly distributed once the cell has achieved a fully spread state (24 hrs).The interactions of calpain-1, α-SMA, spectrin, and vimentin that occur concomitantly with changes in SMC migration with advancing age suggest that the dynamic changes in distribution of calpain associated cytoskeletal proteins may play a role in VSMC attachment and spreading, crucial processes of cell migration. We next determined the spatial and temporal location of endogenous calpain-1 in the attachment processes of VSMC by using time-lapse photomicrography. ), is capable of cleaving spectrin ), and α-smooth muscle actin (data not shown), and is also linked to Ang II induced activation of MMP2. We addressed the idea that calpain-1 plays a role in MMP2 activation and cytoskeleton restructuring, crucial events relevant to age-associated increased VSMC migration. Data presented thus far show calpain-1 within VSMC co-localizes with vimentin Fig. 6AThe increased Ang II protein expression within older VSMC is accompanied by an increased gradient of intimal-medial PDGF-BB in the old aorta . Overexpression of calpastatin reduced the migratory capability of old cells, to the level of young cells infected with GFP alone . Further, the increased chemotaxis induced by CANP1 infection in both young and old cells was substantially reduced by GM6001, an MMP inhibitor . In addition, GM6001 substantially inhibited the migration of old cells .To bypass both Ang II and PDGF-BB signaling, we infected young VSMC with an adenovirus harboring calpain-1 (CANP1) or calpastatin (CAST) cDNA. Over-expression of CANP1 enhanced the migratory ability of young VSMC by 1.5-fold (p<0.05), up to the level of the GFP infected old VSMC . Overexp. Overexp. Overexp. First, we show that the levels of transcript, protein and activity of calpain-1 within the aged aortic wall and isolated VSMC increase with advancing age. Second, increased Ang II within the aged aortic wall or VSMC Our study documents multiple novel roles of calpain-1 in age-associated vascular restructuring in ratsex vivo and in vitro. Chronic infusion of Ang II into young rats for 28 days enhances calpain-1 protein abundance and activation in the arterial wall, similar to that of the old animal. Ang II infusion, in vivo, effects changes in multiple factors, i.e. circulating cells and hemodynamics Our results show that increased Ang II within the aged aortic wall or VSMC stimulates the expression and activation of calpain-1 in vivo, o-tetradecanoyl-phorbol-13-acetate response element (TRE), which binds AP-1 2+ influx or redistributing intracellular stored Ca2+, contributing to a transient increase in cytosolic Ca2+The transcriptional activation of calpain involves the promoter/enhancer motif, 12-. These findings are endorsed by previous reports in fibroblasts and leukemic cells Increased MMP2 activity, due in part to age-associated increases in aortic wall MT1MMP and reductions of TIMP2, is a characteristic feature of the aged arterial wall VSMC exhibit a striking plasticity and are able to change from a differentiated contractile phenotype to a more immature, synthetic phenotype with aging In the present study, we further confirm that VSMC migratory capability increases with aging In summary, our results demonstrate an age-associated increase of calpain-1 gene transcripts, protein levels and activity in the old aortic wall; increased Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro; the Ang II mediated age-associated increased MMP2 activity, is blocked by calpain inhibitor 1 or CAST, indicating that calpain activity is required for MMP activation by Ang II treatment. An increase in old VSMC migratory capacity is mimicked in young VSMC by over-expression of calpain-1. Collectively, these findings point to increased calpain-1 activity, as a central component of the exaggerated Ang II signaling pathway, which is involved in age-associated arterial remodeling. Thus, targeting calpain-1 is a potential approach to delay or reverse the age-associated arterial remodeling that is also associated with diseases such as hypertension, atherosclerosis, and stroke.8-month  and 30-month  Fisher 344 X Brown Norway (F344XBN) male rats were obtained from the National Institute on Aging contract colonies . Twenty young rats were randomly allocated into 2 groups, and implanted subcutaneously with osmotic minipumps  in the midscapular region, according to methods described previously All of the animals were sacrificed by an overdose of sodium pentobarbital, and thoracic aortae were immediately removed, isolated, and processed as described previously Aortic rings were explanted and cultured as described previously 5′-CTCGAGCACCATGTCGGAGGAGATC-3′ and the downstream primer is 5′-AAGCTTGGCAGACGAGGAGGGAGG-3′. The rat calpastatin full-length cDNAs were amplified by reverse transcription-PCR (RT-PCR) from 2 µg of total RNA extracted from rat aorta. The primers for the calpastatin cDNA fragment (2003 bp) which added Kpn I and Xba I restriction sites at the 5′ and 3′ ends respectively were as follows: 5′-GGTACCTCTCCCAGAATGAG-3′5′- TCTAGATACCAAAATGCATAAAC-3′. The PCR fragments were ligated to the pGEM-T vector and sequenced. The calpain-1 and calpastatin were then cut by Xho I and Hind III or Kpn I and Xba I, respectively and then ligated to pAdTrack-CMV which has a reporter gene expressing green fluorescent protein. The adenoviral backbone plasmid pAdEasy-1 and shuttle plasmid pAdTrack-CMV were a gift from Dr Bert Vogelstein . The replication-defective recombinant adenovirus encoded calpain-1 (CANP1) or calpastatin (CAST) together with green fluorescent protein were constructed by homologous recombination, as described in a previous study The human calpain-1 full-length cDNA fragment was obtained as described previously Vascular smooth muscle cells were enzymatically isolated and cultured as previously described 1, Gly8]-Angiotensin II acetate hydrate  (100 nmol/L), an AT1 blocker, or together with the calpain inhibitor, Ci 1  for 48 hours in 0.1% FBS for Western blot and Polyacrylamide Gel Electrophoresis (PAGE) gelatin zymographic analysis.Early passage (p3-p5) VSMC were untreated, or pre-treated with Ang II (100 nmol/L) only or together with [SarAdenoviral infection of VSMC was performed with a multiplicity of infections (MOI) of 100. After forty-eight hours of adenoviral infection, total cellular proteins were isolated for Western blot or migration assays.Quantitative Real-time PCR (q-PCR) was performed using the SYBRGreen PCR-based protocol in a 384-well plate format (Applied Biosystems) as described previously −(Ct-Y intersept)/slope value) where Ct represents the threshold cycle value.RNA was extracted from frozen aortae or cultured VSMC using Trizol reagent. RNA (2 µg) was reverse transcribed for 30 minutes at 48°C using random hexonucleotides according to the manufacturer's instructions (Applied Biosystems Foster City CA). Real-time PCR was performed according to the SYBRGreen PCR protocol (Applied Biosystems Foster City CA). Each sample has been tested in quadruplicate. The reaction conditions were: 10 min at 95°C (one cycle); 30 sec at 95°C; 30 sec at 60°C; and 30 sec at 72°C (40 cycles). Gene-specific PCR products were continuously measured by an ABI PRISM 7900 HT Sequence Detection System . The PCR product sizes were verified by agarose gel electrophoresis. Samples were normalized to the expression of the “housekeeping” gene, the 18S rRNA, which did not change with age. Data are expressed using the formula: quantity = 10Immunohistochemistry and immunofluorescence were performed according to the methods described previously .Western blotting of supernatants from thoracic aortae homogenates or VSMC lysate products was performed according to the methods described previously α-II-spectrin. This antibody recognizes intact 280-kD α-II-spectrin and calpain-specific cleaved 150 and 145-kDa fragments. The spectrin fraction is calculated by dividing the cleaved spectrin densitometry by total spectrin densitometry. The total spectrin densitometry is equal to the intact densitometry plus the cleaved densitometry.The calpain activity in rat aorte or cultured VSMC was determined via a measurement of the fraction of calpain-specific spectrin cleaved products (SBDP), as described previously 2, and 15 mM β-mercaptoethanol with slow shaking for 60 min(with 2 changes of buffer). The gel was then further incubated overnight (16–20 hr) at 37°C in the same developing buffer. Finally, the gel was stained with Coomassie blue.In addition, casein zymography of VSMC was performed according to the modified protocol described previously MMP2 activity assays were determined via PAGE gelatin zymography according to the methods described previously 6/mL) were placed in the upper chamber in 200 µL of MM, performed in triplicate. The assay was stopped after 4 hours at 37°C, and cells that had crossed the basement membrane and migrated to the lower side of the filter, were fixed and stained using the HEMA3 system . Four random fields were counted at 400× magnification for each filter.The following VSMC Migration was performed according to the methods described previously All results are expressed as the mean±SEM from at least three independent experiments. Statistical comparisons for multiple group differences were made via an ANOVA, followed by Bonferroni post hoc tests. Two-age group differences were determined via Student's t- tests. A p value of <0.05 was taken as statistical significance.Figure S1Over-expression of calpain-1 or calpastatin by adenovirus infection. A. Fluorescence photomicrographs show that Adenovirus (GFP or CAST) infected into VSMC. GFP expression was visualized after 48 hrs. Western blots of calpastatin from VSMC infected with CAST virus (right panel). B. Adenovirus (GFP or CANP1) infected into VSMC. GFP expression was visualized by fluorescence microscopy after 48 hrs. Western blots of calpain-1 from VSMC infected with CANP1 virus (right panel).(1.24 MB TIF)Click here for additional data file.


The medical records were reviewed for clinical variables. During the development of AIPC, intrapatient changes occurred in two opposite directions for each of the four EGFRs: negativity changed to positivity, and vice versa, statistically significant only for the increase of c-erbB-1 expression (P=0.001). The c-erbB-2 expression in the AIPC biopsy was associated with a significantly shorter survival from the time of the AIPC biopsy (P=0.029). Our results support ongoing therapeutic attempts of EGFR inhibition in subgroups of patients with prostate cancer. Further research is needed to understand the function of EGFRs in this malignancy.Novel palliative strategies for patients with androgen-independent prostate cancer (AIPC) include targeting the epidermal growth factor receptor (EGFR) family. The aim of the present study was to investigate intrapatient changes of EGFRs during the development of AIPC. In total, 106 symptomatic AIPC patients were identified in whom prostatic biopsies (adenocarcinoma) were available both before the start of androgen deprivation (PRTR biopsy) and after the development of AIPC (AIPC biopsy). All four known subgroups of the EGFR family were determined by immunohistochemistry (IHC): c-erbB-1 (EGFR), c-erbB-2 (HER2/neu), c-erbB-3 (HER3) and c-erbB-4 (HER4). Moderate to strong membrane-specific staining was recorded semiquantitatively (<10%  Androgen-independent prostate cancer (AIPC) is defined as biochemical and/or clinical disease progression during androgen deprivation with castration levels of serum testosterone. Patients are in need of palliation, and novel strategies focus on the epidermal growth factor receptor (EGFR) family and related intracellular pathways.Based on current knowledge, the EGFR family comprises four members; c-erbB-1 (EGFR), c-erbB-2 (HER2/neu), c-erbB-3 (HER3) and c-erbB-4 (HER4) . All memPrevious observations have demonstrated increased levels of c-erbB-1 immunoreactivity in hormone-independent human prostate cancer cell lines , we identified AIPC patients with an adenocarcinoma-positive biopsy from the prostate before the start of androgen deprivation (pretreatment (PRTR) biopsy) and after the development of symptomatic AIPC (AIPC biopsy). The latter histological material was obtained by palliative surgery due to local problems /transvesical prostatectomy (TV)), or represented a diagnostic biopsy in order to differentiate between a growing prostate cancer and rectal cancer . Patients displaying primary androgen-independence, defined as clinical progression within 3 months after the start of castration therapy, were excluded from the present study. Any concomitant or prior medical and/or surgical treatment was allowed, whereas radiotherapy (RT) to the prostate prior to the AIPC biopsy was a criterion for exclusion. In all, 10 specimens of archival formalin-fixed normal prostate tissue represented a clinical control group.The following information was extracted from the medical records:Initial diagnosis and treatment: Date, serum prostate-specific antigen (PSA) level, extent of the disease categorised as local, regional or distant metastases according to the registration routines of the Cancer Registry of Norway (www.kreftregisteret. no), date of start and type of androgen deprivation.Clinical course and diagnosis of AIPC: Date and type of clinical progression , date and status of last observation (death or 15 October 2001). For all PRTR and AIPC biopsies, we recorded the date and type of biopsy together with the Gleason score, the latter differentiating between Gleason score 7a (grade 3+4) and Gleason score 7b (grade 4+3) (μm thick) were cut from the blocks with the most representative amount of tumour tissue, as determined in haematoxylin and eosin-stained slides. The newly cut sections were mounted on silane-coated slides and dried for 1 h in 56°C followed by 37°C overnight. The sections were deparaffinised, rehydrated and incubated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase. The pretreatment conditions and the primary antibodies against PSA and the EGFRs are presented in Table 1For each eligible patient, the archival paraffin blocks of the PRTR biopsy and of the first available biopsy after diagnosis of AIPC were collected. Serial paraffin sections (5 The results of the immunostaining were reviewed by an experienced pathologist (JMN) and scored semiquantitatively as follows: no staining: −(minus); scattered cells to less than 1% tumour cells positive: +; 1 to <10% tumour cells positive: ++; 10–50% tumour cells positive: +++ and >50% cells positive: ++++. Scores of −, + and ++ were grouped together as ‘immunohistochemistry (IHC)-negative’ as opposed to ‘IHC-positive’ findings in the examination of the clinical impact. For the EGFRs, only moderate to strong cell membrane-specific immunostaining was taken into account.P-values <0.05 were regarded statistically significant.Statistical analyses were performed using the computer-based program SPSS  version 10.0. Individual changes of immunostaining after diagnosis of AIPC were assessed by the McNemar test for related samples. The overall survival was estimated by the Kaplan–Meier method from the date of the AIPC biopsy to the last observation/death applying the logrank test to assess statistical significance. μg/l. Androgen deprivation was started within median 2 months after initial diagnosis (range 0–109). After a median time of 32 months (range 7–167) AIPC was diagnosed in all patients. Distant metastases were recorded for 43 patients after the development of AIPC. Of the 105 PRTR biopsies evaluable for Gleason score, 79% were ⩾7b  fulfilled the eligibility criteria. The median age at initial diagnosis was 70 years (range: 50–86) (Table 2P=0.001). The comparable figures for c-erbB-2 were 33% and 24%, respectively. c-erbB-1 and c-erbB-2 IHC positivity after the development of AIPC is illustrated by Figure 1A–BThe percentage of PSA immunoreactive specimens decreased from 83% (88 of 106 patients) to 69% (73 patients) as the patients developed AIPC . A totalP=0.001). However, nine of 24 patients with c-erbB-1 positivity before hormone treatment lost this immunoreactivity after the development of AIPC. The direction of intrapatient changes for c-erbB-2, c-erbB-3 and c-erbB-4 was less pronounced. Overall, the positivity of EGFRs was not significantly related to Gleason score or PSA immunoreactivity (data not shown). During the development of AIPC, the number of c-erbB-1-positive specimens was doubled in those patients who lost PSA positivity, whereas the number of c-erbB-2 positive specimens decreased from eight to three   (P=0.064).At the time of the last observation, 91 patients (86%) were dead. The median overall survival time from the date of the AIPC biopsy was 20 months (95% CI: 15–26 months). The immunoreactivity of c-erbB-2 determined decreased survival (P=0.054)  Table 5P=0.029  negativity of the PRTR biopsies changed to positivity of the paired AIPC biopsies and (2) positivity of the PRTR biopsies changed to negativity of the paired AIPC biopsies. The development of AIPC was significantly associated with the increase of c-erbB-1 expression, whereas no changes for the other growth factor receptors reached the level of statistical significance. The Gleason score and PSA immunoreactivity were not associated with growth factor receptor expression before or after the development of AIPC. In normal prostate tissue, four out of 10 specimens displayed c-erbB-1 positivity, whereas none were positive for c-erbB-2, c-erbB-3 or c-erbB-4.Previous observations of the expression of c-erbB-1 and c-erbB-2 in prostate cancer tissue and cell lines have demonstrated highly divergent rates. Our results display figures far below the highest reported values of c-erbB-1 and c-erbB-2 expression, but for c-erbB-2 our figures are higher than the results from et al, 80% of the untreated cases of the present study were already Gleason score ⩾7b. Di Lorenzo et al also found a positive correlation between serum PSA and c-erbB-1, which was not confirmed in our study applying IHC-detected PSA. The results of the present study are in agreement with the published observations: expression of c-erbB-1 increases significantly, as the tumour becomes androgen independent. per se is only one of several conditions for this receptor's functionality.Even though high levels of c-erbB-1 can be demonstrated in human prostate cancer, especially after the development of AIPC, its clinical role is not yet clear. This receptor's function is further obscured as it has become clear that internalisation has to take place for its activation . c-erbB-2 is a poor prognosis factor in AIPC patients with local problems. These findings support ongoing attempts to develop new treatment strategies for subgroups of prostate cancer patients with the inhibition of EGFRs and their signalling pathways. The sole immunohistochemical demonstration of membrane-located EGFRs in biopsies may give some indications for these agents’ efficacy in the individual patient. However, these receptors’ final association with response rates has to be determined in larger series of patients with known growth factor receptor status.In summary, we find that during the development of AIPC intrapatient changes occur in two opposite directions for each of the four EGFRs: negativity change to positivity, and vice versa, statistically significant only for the increase of c-erbB-1 expression (


The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known.We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children.SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups.Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease. With each litre of air people inhale, the lung is exposed on average to at least one microorganism, in conjunction with particulates, antigens and possibly noxious gases . In the SP-B is synthesized by alveolar type II cells to the mature 8 kDa protein and secreted into the alveolar space, where it is mainly found as a dimer . BronchoThe main functions of SP-B are to increase the adsorption rate of phospholipids at the air-water interface and an involvement in the formation of tubular myelin together with SP-A and calcium ,6. ApartThere are some data available on the levels of SP-B in the alveolar space, but almost no data on the molecular organization of SP-B and of pro-SP-B during various disease states . ChildreSP-C is exclusively synthesized by alveolar type II pneumocytes and secreted into the alveolar space . It stabSP-C inserted into lipid vesicles interacts with a 12 residue non-transmembrane domaine with bacterial lipopolysaccharide; this enhances the association and the binding efficacy between lipopolysaccharide and CD14, a receptor on phagocytes ,23.Recently in various animal models of lung injury the regulation of SP-C expression by inflammation has been highlighted. SP-C concentrations were reduced in bleomycin induced lung fibrosis, hyperoxia, Aspergillus fumigatus and Pneumocystis jiroveci infections and a model of asthma, suggesting down-regulation of SP-C expression by pulmonary infection and inflammation. Unfortunately, there are not many ex vivo data available on the concentration and the molecular organization of SP-C in bronchoalveolar lavage of humans.In adults, SP-C content in large aggregate surfactant remained unchanged in pneumocystis pneumonia, but decreased significantly in ARDS and other pneumonia . ChildreThe major goal of this study is therefore to investigate the concentrations and molecular forms of SP-B and SP-C and their pro-forms in the alveolar space under different infectious and inflammatory disease conditions.Bronchoalveolar lavage fluid (BALF) from children and young adults with different chronic and acute inflammatory airway diseases including cystic fibrosis (CF), chronic bronchitis, acute pneumonia and a comparison group of subjects without lung disease (controls) was investigated.CF was diagnosed by repeated sweat tests with elevated chloride concentrations together with the characteristic clinical presentation. 14 of 21 children were deltaF508 homozygous and 4 heterozygous. In 3 patients no mutation was detected by routine screen for the 30 most prevalent mutations in Germany. All patients were clinically stable, none had evidence for allergic bronchopulmonary aspergillosis or advanced hepatic disease. Some children with CF were part of the "bronchoalveolar lavage for the evaluation of anti-inflammatory treatment" (BEAT) study, a multicenter study to evaluate the effect of treatment with rhDNase on endobronchial inflammation, and were recruited in the Berlin and Munich centres . Others The 15 children in the chronic bronchitis group suffered from bronchitis without obstruction over a period of at least 3 months and endobronchial inflammation was diagnosed visually by bronchoscopy. Cystic fibrosis, primary ciliary dyskinesia, cellular or humoral immune deficiencies, gastro-oesophageal reflux disease and anatomic anomalies of the airways were excluded if clinically indicated. None of them had a pulmonary infiltrate in the x-ray or were diagnosed as having bronchial asthma (obstructive bronchitis).Inclusion criteria for the 14 children in the pneumonia group were clinical symptoms (cough and/or fever) and a pulmonary infiltrate in x-ray and/or computer tomography. Exclusion criteria were interstitial pneumonia, immune deficiencies and malignancies as a reason for the pneumonia.The 14 children of the control group did not have any illness with pulmonary affection. Here BAL was performed during anesthesia for elective surgery for minor conditions .Clinical details of the children, the cellular results, total protein content and the microbiology results of the bronchoalveolar lavage are given in Table The study was approved by the ethics committee at the University of Munich. Written informed consent was obtained from the parents and/or the patients with appropriate age.BAL was performed using a 3.5 mm or 4.9 mm flexible bronchoscope in "wedge position" in a subsegment of the right middle lobe or the lingula. 4 × 1 ml/kg body weight saline (0.9%) at body temperature was instilled and immediately withdrawn by suction. The first aliquot of the recovered lavage fluid was treated separately. Fractions 2–4 were pooled and cleaned by gauze-filtration. The total cell count was measured by a haemocytometer and the differential cell count of the BALF by cytocentrifugation. The supernatant was used for the following analysis of total protein content and surfactant proteins.Total protein content was determined according to the method of Bradford 1976  with theStatistical analysis was performed with Graph Pad Prism Version 4.01  and Microsoft Excel 2000 , using Kruskal Wallis Anova for non-parametric results (given as median and 25. and 75. percentile) or Fisher's exact test for frequency distributions, corrected according to Bonferroni for multiple comparisons. Correlation coefficients were determined according to Spearman. A p-value of less than 0.05 was considered as being significant.All 64 patients had SP-B in their lavages Table . SP-B waThe major physiologic form of pro-SP-B with a molecular weight of 24–25 kDa was found in the lavages of all 21 patients with CF, 14 of 15 children with chronic bronchitis, 12 of 14 patients with pneumonia and 13 of 14 children and young adults from the control group  Fig. .The level of SP-B was not associated with the degree of neutrophilic inflammation or the presence or absence of bacteria in BALF Fig. , Fig. 3.Quantitatively there were no significant differences between the four groups of patients investigated Fig. . Because61 of 64 patients had SP-C in their lavages Table . In the Pro-forms of SP-C were not found in any of the analysed samples. Only the Western blots of the samples CF 20-1 and CF 21-1 showed a reaction with the pro-SP-C-antibody at a molecular weight between 3 and 4 kDa; this was not observed in the blots of the samples CF 20-2 and CF 21-2, taken later Fig. . It is lBetween the four evaluated groups no significant differences were found in the quantity of SP-B and SP-C Fig. . When coSimilarly as for SP-B, SP-C concentration in BALF was not related to the degree of neutrophilic inflammation or the presence or absence of bacteria in BALF Fig. , Fig. 3.In addition to expressing the quantitative results per ml of lavage fluid, we also expressed all results per mg of lavage protein. Equal results to those mentioned above were obtained.Here we present the results of the first systematic comparison of the molecular organisation and concentration of SP-B or SP-C and their pro-forms in BALF in children with cystic fibrosis, chronic bronchitis and pneumonia in comparison to controls. SP-B was detected regularly at molecular weights of 8 kDa in monomeric respectively 16–18 kDa in dimeric form, additional partly at approximately 30 kDa under non reducing conditions, suggesting the presence of oligomers in smaller amounts. SP-C was mainly detected at molecular weights of approximately 4.2 kDa. Some western blots also showed a band at approximately 7 kDa, which could either be caused by SP-C-dimers or covalent bonds between SP-C and other thioles ,28. Pro-This result is surprising, especially for SP-C, as recently in various animal models of lung injury the regulation of SP-C expression by inflammation has been highlighted . AlthougIn adults, SP-C content in large aggregate surfactant remained unchanged in pneumocystis pneumonia, but decreased significantly in ARDS and other pneumonia . This maAnother issue concerns the distribution of SP-B and SP-C in the alveolar surfactant. Due to shortage of material the lavages were not separated into large surfactant aggregates and small surfactant aggregates. However from previous measurements we know that SP-C is mainly associated with the large aggregates, whereas SP-B is also present in the small aggregates, even to a larger extent as might be anticipated . We did also not perform surface tension measurements of the surfactant in the lavages. From previous studies it is known that the impairment in biophysical activity associated with airway inflammation was not associated with the concentrations of SP-B and SP-C .Apart from the loss of SP-C in the samples of three patients with very high amounts of neutrophile granulocytes in their lavages, which was probably due to proteolysis, no significant correlation could be observed between the level of neutrophilic inflammation and the amounts of SP-B and SP-C in the lavage samples. In conclusion our data do not support, if not contradict, the notion of down-regulation of SP-B and SP-C during acute and chronic bronchial infection and inflammation in the investigated human lung diseases.The author(s) declare that they have no competing interests.OT carried out the laboratory research and the patients characterisation for the classification of the different patient groups and contributed to the manuscript. PL coordinated the study, participated in writing of the manuscript and assisted in performing the statistical analysis. KP recruited the CF-patients and helped with study design. MW assisted in the patients' characterisation, in laboratory analysis and interpretation of results. TW assisted in the patients' characterisation. MG conceived and supervised the study as head of the lung research group, participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To investigate if noradrenaline (NA) and 5-hydroxyptamine (5-HT) drugs induce responses of isolated control and varicose veins are altered by removal of the endothelium. Specimens of the great saphenous vein (GSV) were obtained from 12 subjects with primary varicose veins and 12 subjects from donor vessels at cardiac surgery. A total of 10 normal healthy volunteers were selected for comparison. The diameter changes of GSV during the resting phase, at the end of 5 minutes occlusion, and then every 30 seconds post deflation for five minutes were measured using B-mode ultrasound. Post-surgery the vein sample was collected in a tube of Krebs-Henseleit solution. The repeated measure ANOVA test for the diameter, percent, and difference changes of GSV diameter from maximum diameter at different time intervals showed significance difference within and between all groups. NA and 5-HT produced concentration-dependent contractions of control and varicose saphenous vein segments. There was no significant difference in the potency of NA and for 5-HT, but the maximum response, normalized for tissue weight, was less in varicose vein segments. Removal of the endothelium had no effect on the potency of NA or 5-HT but significantly (p<0.05) reduced the maximum response to NA and 5-HT in varicose vein segments but not to 5-HT in control veins. The venous endothelial damage may cause vascular smooth muscle contractions dysfunction that favours dilatation and secondary valvular insufficiency. There is some controversy regarding the primary pathology that leads to the development of venous incompetence. Various theories, the valvular hypothesis  and the Patients with varicose veins exhibit increased threshold autonomic nerve function to temperature and vibration stimuli . ReducedInvestigators have concentrated on the collagen, elastic, and muscle fiber components of the vein wall and their effect on the overall physical properties of the conduit . Abnormain vivo phase of the study was to examine vein function by measuring diameter changes of the great saphenous vein (GSV) using duplex ultrasound (DUS) before, during, and after venous occlusion in control, normal, and varicose veins subjects. In the second in vitro phase of the study the aim was to investigate if noradrenaline (NA) and 5-hydroxytryptamin (5-HT) induce responses of isolated control and varicose veins that were altered by removal of the endothelium.The aim of the Subjects were recruited from the general population prior to varicose vein surgery (varicose vein group) and undergoing coronary artery bypass graft (CABG) but without varicose veins (control group). A total of 24 venous segments specimens were studied. Varicose vein subjects undergoing surgery for stripping the great saphenous vein were asked to be off all venotropic drugs including Daflon 500 mg. Control subjects undergoing CABG were required to have no varicose vein or any evidence of venous insufficiency. Ten normal volunteers without any history of diseases or medications were matched for age and sex for comparison.in vitro part of the study. The study was approved by the Human Research and Ethics Committee from Kuwait University, Faculty of Allied Health Sciences and Ministry of Health, Mubarak Al-Kabeer Hospital at Kuwait. All subjects were asked to sign the consent form.Subjects were asked to complete a questionnaire administered by the author that included personal and occupational details, relevant medical and family history, and possible risk factors for venous disease. They underwent duplex ultrasound to diagnose venous reflux and the great saphenous vein, above knee, was assessed for diameter changes in response to occlusion-release stimulation, and was marked one day before surgery by permanent pen, so that the vein segment could be harvested for All subjects were assessed in temperature-controlled (22±2°C) environment using a Philips HDI 5000 ATL  ultrasound machine equipped with linear 7-4 MHz transducer. Venous reflux was elucidated by manual compression distal to venous segments under examination. Venous reflux duration more than one second is considered abnormal . FirstlySubject was then placed in a supine position with the leg to be investigated elevated 30cm at the heel. The knee was slightly flexed and externally rotated while ensuring adequate relaxation of the leg. After 20 minutes rest the diameter of the great saphenous vein 4 cm above the kneecap was measured and then venous occlusion was induced by inflation of a pneumatic cuff (17 cm) placed around the upper-mid thigh to a pressure of 80 mmHg until the maximum diameter is achieved. When there was no further changes in the diameter (approximately 5 minutes), the cuff was deflated. The diameter of the vein was measured every 30 seconds for the first two minutes and then every one minute for five minutes. The great saphenous vein diameter was measured with B-mode ultrasound during quite respiration. In applying the transducer against the cutaneous surface, minimal pressure was used to guarantee satisfactory saphenous vein imaging, while avoiding undesirable compression that could have modified vein diameter assessment. This was easily obtained in every case, since vein compression is clearly visible on the ultrasound image. In measuring vein diameter the axis parallel to the skin was preferred as it depends more on the flattening of the saphenous vein . Venous in vivo venous responses to occlusion and reperfusion, surface skin markings were made 4 cm about the knee in 12 of the legs exhibiting varicose veins and in 12 of the normal legs of control subjects awaiting CABG surgery. At surgery the marked great saphenous vein segments were harvested for subsequent in vitro pharmacological studies.Following studies of The specimens were harvested at operations for radical removal of varicose veins under pentobarbital-halothane or spinal anesthesia. Moreover, the anesthesia for control: di-azepam 100 μ/kg, papaveretum 120 μg/kg, fentanyl 40 μg/kg and pancuronium bromide 100 μg/kg.2 2.5 mmol/L; MgSo4 1.2 mmol/L; NaHCO3 22.1 mmol/L; KH2PO4 1.1 mmol/L and glucose 11.0 mmol/L) maintained at pH 7.4, at a temperature 37°C and gassed with 95% oxygen and 5% carbon dioxide.After excision the vein samples were put into cool Krebs-Henseleit solution and send for testing immediately or kept in a fridge at 4°C for testing within 1-4 hours of sample collection. The specimen was cleaned of connective tissue and fat and cut into two rings, each 4 mm in length. During this procedure great care was taken to avoid unintentional rubbing of the intimal surface so as to maintain the integrity of the endothelial layer and meanwhile not damaging the muscular layer of the vessel. One ring served as control and the other was de-endothelialized by inserting the tip of a small forceps and gently rolling the ring for 15 seconds according to the method of De May and Vanhoutte . The pre2 values, where pD2 is the negative logarithm of the agonist concentration producing 50% (EC50) of the maximum response. EC50 and maximal contraction were determined from dose-response curves.The vein rings were attached to the bottom of the organ bath and the upper end connected to dynamometer UF-1 force transducer. Isometric contractions were continuously recorded on a Lectromed 4-channel polygraph (MultiTrace 4P). Before the experiments were begun a pretension of 2 gram was applied to the preparation by adjustment of a micrometer screw. The system was allowed to equilibrate for 30-40 minutes. After the period of equilibration, KCl (80 mM) was added to the bath to test for tissue viability. This concentration of KCl was repeated after 30 minutes. The tissues were then washed repeatedly over the next 30 minutes period. Thereafter, agonist (NA or 5-HT) concentration-response curves were established by adding, cumulatively, increasing concentrations (0.5 log unit increments) of each agonist to the bath and allowing the response to each concentration reach a peak before adding the next concentration. In all cases, two consecutive concentration-response curves were separated determined by a rest period of 60 minutes. The second concentration-response curve was used as the control  was used to determine the significance of difference between control, normal, and varicose veins . Cross-tabulation analysis (Chi-Square test) was used to test for the significance of the differences among groups in return of vein diameters to<10% of resting baseline.t-test. The difference was assumed to be significant when p<0.05. The relationship between duplex ultrasound and pharmacological parameters were carried out using Pearson correlation coefficients test.Data was recorded as mean of ‘n’ experiments where n is the number of samples from ‘N’ subjects. Graphs were plotted and analyzed using Graphpad Prism software. Differences between mean values were tested for significance using paired Student’s 2). Control great saphenous vein specimens were obtained from donor vessels of the great saphenous vein above knee at cardiac surgery from 12 subjects  and with BMI 29.9±4.5 (mean±SD kg/m2). Table Specimens of the great saphenous vein were obtained from 12 subjects with primary varicose veins above knee , and with body mass index (BMI) 27.1±4.5 . In addition, the cross-tabulation analysis were carried out for 20% and 30% and found to be significant but not at 30% for all groups. It is apparent that the varicose vein segments dilate more and return more slowly to original resting diameter than the control and normal venous segments.The repeated measure ANOVA test for the diameter, percentage, and difference changes of great saphenous vein diameter from maximum diameter at different time interval showed significance difference (p <0.05) within and between control, normal and varicose vein venous segments. Data are represented in Table a,b) and Table NA and 5-HT produced concentration-dependent contractions of control and varicose saphenous vein segments. There was no significant difference in the potency of NA  and for 5-HT  between the two groups but the maximum response, normalized for tissue weight, was less in varicose vein segments for both NA and 5-HT. Results are represented in Fig. a,b and Ta,b) and Table Removal of the endothelium significantly (p<0.05) reduced the maximum response to NA in control veins. The maximum response to NA in varicose vein segments was not statistically significant after endothelial denudation. Removal of the endothelium had no effect on the potency of 5-HT in control vein segments but significantly (p<0.05) reduced the potency of 5-HT in varicose vein segments . Results are represented in Fig. a,b and Ta,b) and  and . Whilst The main findings of the ultrasound component of the study were that the control and normal group showed almost identical responses. This suggests that the venous wall was functioning normally in the control group. In contrast, in the varicose vein group the vein dilated more and returned more slowly to its original diameter. A weaker maximal contraction in varicose veins when compared to control veins was expected and this was confirmed in this study.The findings of the present study must be taken in context with the subjects investigated and the methods of investigations. The former is important because the control group subjects had underlying risks for cardiovascular diseases as from Table The pharmacological study confirmed the findings of Thulesius  in that The reason for reduced contraction of varicose veins may involve disordered smooth muscle responsiveness and/or endothelial dysfunction. The reduced contractile responses to NA suggest the former; the responses of GSV segments from subjects with varicose veins to 5-HT, showing a further decrease in maximum contraction after endothelial denudation, in contrast to the control vein responses, suggest the latter. It is possible that the significant fall in 5-HT induced maximum contraction of the varicose vein rings after endothelial denudation may reflect an endogenous endothelial-dependent venoconstrictor influence in these rings, perhaps due to en-dothelin.2 and prostaglandin E2 synthesis, and with an increased interaction among leukocytes, platelets and endothelial cells, is observed in varicose veins [in vitro contractility of the same segments, suggest that endo-thelial dysfunction may be an important aetiological factor in the pathogenesis of primary varicose veins and CVI development.By correlating the ultrasound findings with the pharmacological findings, it might be considered that dilatation of the varicose vein segments with slow return to initial resting diameter represents a contraction dysfunction of the venous wall initiated most probably by the endothelium due to deficient EDCF or due to a release of EDRF in a healthy vascular smooth muscle fibres in the venous wall of the varicose vein group. The results of a previous study  clearly se veins , 25. Thiin vitro veno-constrictor responses to be inversely related to the in vivo functional responses (time of return of vein within 10% of its resting diameter), with (r) value of -0.32 for all data and -0.40 for 5-HT responses, these do not reach statistical significance. The significant variability of responses suggests that greater numbers need to be studied to further explore this relationship.Whilst there was a trend for the maximum Finally, in order to come up with a proper management of varicosis, it is essential to understand the basic mechanisms leading to the development of such a disease in people at various ages. The prevailing view of the aetiology of varicose veins revolves around the trigger(s), as well as the cascade of events. A better understanding of the nature of the triggers(s) and of the progressive changes occurring in the vessel wall of the varicose vein, obtained by combining structural and functional studies, including electron microscopy as well as pharmacological techniques, may lead to not only a better treatment, but also prevention of the disease. Also studies are necessary to explore haematological as well as biochemical markers in varicose veins subjects, and how these change with disease development. In view of that en-dothelial damage may cause vascular smooth muscle dysfunction that favours dilatation and secondary valvular insufficiency, it could equally well be that valvular insufficiency leads to dilatation which causes endothelial damage. Our present studies do not allow differentiation between these two possibilities and further studies are needed to resolve such issue. In addition the role of calcium storage in vascular smooth muscle in the wall of the veins warrant more study.Varicose venous segments dilated more during occlusion hyperemia and returned slowly to their original resting diameter when compared to control and normal venous segments. This might be attributed to venous wall dysfunction. Venous endothelial damage may cause vascular smooth muscle contractile dysfunction that favours dilatation and secondary valvular insufficiency with deficient cusps apposition or valvular insufficiency leads to dilatation which causes endothelial damage. This may be the primary factor in the development of varicose veins and CVI.


To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf.The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling. Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia (Mitf) were also used in overexpression studies. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina.The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation by acting downstream of FGFR/MEK/Erk signaling, likely by inhibiting the increase in Pax6 protein in the RPE.FGF2 stimulates Pax6 expression during induction of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated after removal of the retina even in the absence of FGF2. This Mitf downregulation is not accompanied by Pax6 upregulation, demonstrating that FGF2 stimulated Pax6 upregulation is required for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation. Regeneration of the retina occurs through the process of transdifferentiation in chick embryos and is limited to a small window of development between E3.5 and E4.5. Transdifferentiation is the process by which RPE cells lose their normal characteristics, becoming "stem cell-like." Retina removal is characterized by a thickening of the RPE, and upon addition of ectopic fibroblast growth factor 2 (FGF2), the RPE proliferates and gives rise to a neuroepithelium, which will subsequently differentiate into all of the different cell types of the retina -3.Transdifferentiation of the RPE in the embryonic chick has been well characterized ,4-6 and Other stimulators of transdifferentiation analyzed during eye development include transcription factors such as Ceh-10 homeodomain-containing homolog (Chx10) and paired box 6 (Pax6), which are essential for retina development -17. One Pax6 is required for transdifferentiation of chick RPE cells into retina cells in vitro. During this in vitro transdifferentiation, Pax6 expression is upregulated while Mitf expression is downregulated. Similarly, overexpression of Mitf can inhibit the expression of Pax6 in cultured RPE . These oRecently, it was shown that Pax6 is able to induce RPE transdifferentiation when overexpressed in chick eyes at embryonic stages of development as late as E14 . Thus, eHowever, it remains unclear why the RPE is capable of responding to FGF signals at E4 and not E5. It is the purpose of this work to closely examine early events of FGF-stimulated transdifferentiation in E4 RPE to lay the ground work for understanding why this process is so temporally restricted during development. Specifically, we show that during retina regeneration FGF acts through FGF receptors to stimulate Erk phosphorylation, and that this phosphorylation event is dependant on MEK activity, demonstrating that transdifferentiation occurs through the FGF-FGFR-MEK-Erk pathway. Furthermore, we show that one of the earliest events that takes place in RPE exposed to FGF2 is an upregulation of Pax6 protein as well as an increase in cell proliferation demonstrated by BrdU incorporation. We also show that Pax6 upregulation is not necessary for Mitf downregulation in the RPE during induction of transdifferentiation, but that Mitf overexpression is able to inhibit the early inductive events characteristic of transdifferentiation.White Leghorn fertilized eggs were purchased from the Ohio State University, Columbus, OH and incubated in a humidified incubator at 38 °C.A window was made in the egg shell using forceps, and microsurgical removal of the retina was carried out at E4 following guidelines previously described in references ,4,5.Heparin-coated polyacrylamide beads  were washed 3 times in 1x PBS. FGF2  was resuspended in 1x PBS at a concentration of 1 μg/μl. Heparin beads were then incubated with FGF2 for at least 2 h before use.DD and Rcas-GFP (control) were prepared using a procedure previously described in reference . It . It 1]. Our observations that Mitf is downregulated after retina removal are intriguing, and the specific mechanism by which removing the retina leads to Mitf downregulation deserves attention in the future.For the first time, we show that FGF2-induced transdifferentiation (in eyes undergoing retina regeneration) require FGF signaling through FGF receptors as well as the downstream effectors MEK and Erk. These signaling events ultimately lead to an increase in Pax6 protein, which is sufficient to induce transdifferentiation of the RPE. Results from Azuma et al.  corroborAfter retinectomy at E4, the increase in Pax6 does not need to overcome the Mitf protein in the RPE, since, as already discussed, it is spontaneously downregulated with retina removal. However, in the presence of ectopic Mitf expression using Rcas-Mitf, FGF-induced transdifferentiation is inhibited.Xenopus laevis eyes results in induction of ectopic RPE [A reciprocal relationship has been demonstrated between Mitf and Pax6. That is, when Mitf is inhibited, Pax6 levels in RPE increase and when Pax6 levels are ectopically increased, Mitf levels are decreased ,28. Thisopic RPE .Moreover, it has been shown that addition of FGF-coated beads near the optic vesicle leads to a rapid downregulation of Mitf in the developing mouse RPE at E9.5 . BecauseIn this study we show that Mitf is spontaneously downregulated in the RPE after retina removal at E4, and this decrease in Mitf protein is not sufficient for transdifferentiation to occur. Furthermore, ectopic FGF2 is required to drive Pax6 expression and induce transdifferentiation. The increased Pax6 expression is associated with increased BrdU incorporation. Finally, we show that the FGF/FGFR/MEK/Erk signaling cascade leads to an increase in Pax6 levels in the RPE, and that ectopic Mitf expression is sufficient to inhibit Pax6 expression but does not interfere with the activation of the FGF downstream effector Erk.


The epidermal growth factor receptor (EGFR) family plays an important role in breast carcinogenesis. Much interest has been focused recently on its members because of their potential role as prognostic indicators in breast cancer and their involvement in cancer therapy. We have evaluated more than 1500 cases of invasive breast carcinoma immunohistochemically using tissue microarray technology to examine the expression of EGFR family receptor proteins. We have found that 20.1 and 31.8% of cases were positive for EGFR and c-erbB-2, respectively, and 45 and 45.1% of tumours overexpressed for c-erbB-3 and c-erbB-4, respectively. The expression of either EGFR or c-erbB-2 was associated with other bad prognostic features and with poor outcome. Neither c-erbB-3 nor c-erbB-4 had any association with survival. c-erbB-2 had an independent prognostic effect on overall and disease-free survival (DFS) in all cases, as well as in the subset of breast carcinoma patients with nodal metastases. Several hetero- and homodimeric combinations have been reported between the EGFR members. Those dimers can evoke diverse signal transduction pathways with variable cellular responses. We stratified cases according to their co-expression of receptors into distinct groups with different receptor-positive combinations. Patients whose tumours co-expressed c-erbB-2 and c-erbB-3, as well as those whose tumours co-expressed EGFR, c-erbB-2 and c-erbB-4 showed an unfavourable outcome compared with other groups, while combined c-erbB-3 and c-erbB-4 expression was associated with a better outcome. In cases showing expression of one family member only (homodimers), we found a significant association between c-erbB-4 homodimer-expressing tumours and better DFS. In contrast, patients with c-erbB-2 homodimer-expressing tumours had a significant poorer DFS compared with other cases. These data imply that the combined profile expression patterns of the four receptor family members together provide more accurate information on the tumour behaviour than studying the expression of each receptor individually. The epidermal growth factor receptor (EGFR) tyrosine kinase family consists of four members: EGFR, c-erbB-2, c-erbB-3 and c-erbB-4 . All shaRelatively less information has been reported about the other two members: c-erbB-3 and c-erbB-4. Significant correlations have been found between c-erbB-3 overexpression and tumour size . Mean survival was 62 months (range 1–192 months). Information on local, regional and distant recurrence and survival is maintained on a prospective basis. Patients are followed up at 3-month intervals initially, then 6 monthly and then annually, for a median period of 58 months. The disease-free interval was defined as the interval (in months) from the date of the primary surgical treatment to the first loco-regional or distant recurrence. The overall survival was taken as the time (in months) from the date of the primary surgical treatment to the time of death. Oestrogen receptor status was estimated immunohistochemically in 1805 of the tumours; 553 (30.6%) were negative for ER expression, while 1252 (69.4%) carcinomas were ER positive.Data for histological tumour type, grade and staging are summarised in Breast cancer tissue microarrays were prepared as described previously , c-erbB-2 , c-erbB-3 , c-erbB-4  and ER . Sections were incubated with the biotin-labelled secondary antibody (diluted 1 : 100) for 30 min, then in avidin–biotin complex (diluted 1 : 100) for a further 45 min. 3-3′Diaminobenzidine tetrahydrochloride was used as the chromogen.Immunohistochemical staining for the sections was performed according to the avidin–biotin complex method. Tissue sections of 3 Positive and negative controls were included in each staining run. Positive controls were myoepithelial cells of normal duct in normal mammary gland for EGFR, renal tissue  for c-erbB-3 and known positive cases of breast carcinoma for c-erbB-2, c-erbB-4 and ER. Negative controls were obtained by omitting the primary antibodies.The modified histochemical score (H-score) (in situ were excluded from assessment. If one core was uninformative (either lost or contained no tumour tissues), the overall score applied was that of the remaining core. Previous studies have validated the use of one core to study the expression of tumour markers having heterogeneous distribution . For ER, the cutoff point was taken at an H-score of 20.χ2 test. A P-value of <0.05 was considered to reflect a significant relationship. Survival curves were calculated by the Kaplan–Meier method. The differences between survivals were estimated by log-rank test. Multivariate Cox regression analysis was used to evaluate whether there was any independent prognostic effect of the variables on disease-free interval or overall survival.Association between the immunohistochemical findings and different clinicopathological parameters was evaluated by χ2=7.01, P=0.008); 54.7% of HER1-positive cases are overexpressing to HER3 and 23.9% of HER3-overexpressing tumours are HER1 positive ; 55.8% of HER1-positive tumours are overexpressing to HER4 and 24.3% of HER4 overexpressing cancers are positive to HER1 ; 50.1% of HER2-positive tumours are overexpressing to HER3 and 34.9% of HER3-overexpressing cases are HER2 positive ; 49.5% of HER2-positive tumours are overexpressing to HER4 and 34.6% of HER4-overexpressing tumours are positive to HER2 ; 64.9% of HER3-overexpressing tumours are also overexpressing for HER4 and 63.7% of HER4-overexpressing tumours are also overexpressing for HER3 .Significant positive associations were found between all EGFR family members (P=0.0265) and overall survival (P=0.0035). c-erbB-2 was also significantly correlated with poorer overall survival (P=0.0006) and DFS (P=0.0001)  or overall survival .Survival analyses were performed comparing the expression of the four markers in relation to both DFS and OS. We found that EGFR expression was significantly associated with shorter disease-free survival  and OS (P=0.2501), we found that OS was worse in patients with tumours expressing c-erbB-2/c-erbB-3 heterodimers compared to those with tumours expressing c-erbB-2/c-erbB-2 homodimer  histological grade, high NPI score, negative ER status, larger tumour size, the development of distant metastases and death. Previous studies have reported the same relationships with tumour grade  compared with other dimers. We have no explanation for this finding; however, cell line studies have shown that NDF induced mitogenesis in cells expressing c-erbB-3 or c-erbB-4 but not transformation, which was induced only when either EGFR or c-erbB-2 were coexpressed with c-erbB-3 or c-erbB-4 (In the present study, c-erbB-2 homodimers were significantly associated with a poorer DFS. In spite of being a ligandless receptor, cell line studies have shown that its overexpression induced homodimerisation that was sufficient to induce growth, malignant transformation (The diversity identified in our study, in terms of survival, between different heterodimers and homodimers may be useful in subgrouping of breast cancer patients with significantly differing outcome. These data suggest that the predictive value of EGFR family overexpression may be optimised by combining information about the expression of all of the family members, rather than the assessment of a single receptor in isolation.There are other essential components of the EGFR family network, which have not been studied here; 10 or more ligands and the proteins that are involved in EGFR family-induced pathways. The receptors are conduits for the ligand-activated signalling pathways and, although their expression levels clearly do contain useful information, the precision of the analysis can only be improved in the future by using antibodies for detection of such proteins.