An immunohistochemical study of cytokeratins distribution of the human adult male and female urethra.

Surgical treatment of diseases affecting long urethral areas represents a challenge in urology. Recent developments of tissue-engineered urethral substitutes represent a hope for patients. However finding an ideal tissue source for urethral reconstruction first requires proper understanding of the native human urethra physiology and a deep knowledge of the histological and molecular features of the native human urethra. Here we present a comprehensive characterization of male and female urethra by histological, histochemical and immunohistochemical methods with a panel of 15 antibodies. The results demonstrated that the histology of the male and female urethra depend on the area where the sample is taken along its length. Proximal areas of male and female urethra have differential expression of the epithelial basal and suprabasal layer markers CK14 and CK10 which distinguished the prostatic/membranous and proximal female urethra from the bulbar/penile and distal female areas of the urethra. The distal male (penile) and female may be further divided by the distinct expression pattern of CK19. On the other hand, the expression of CK5/6 and CK19 also make a distinction of the proximal and distal female urethra. These results should facilitate a more informed selection of donor graft tissues for urethral replacement. Besides, novel bioengineered urethral tissue approaches should take into account the characterization of the different areas of the urethra presented in this work.

Biology of the eyelash hair follicle: an enigma in plain sight.

Because of their crucial impact on our perception of beauty, eyelashes constitute a prime target for the cosmetic industry. However, when compared with other hair shafts and the mini-organs that produce them [eyelash hair follicles (ELHFs)], knowledge on the biology underlying growth and pigmentation of eyelashes is still rudimentary. This is due in part to the extremely restricted availability of human ELHFs for experimental study, underappreciation of their important sensory and protective functions and insufficient interest in understanding why they are distinct from scalp hair follicles (HFs) (e.g. ELHFs produce shorter hair shafts, do not possess an arrector pili muscle, have a shorter hair cycle and undergo greying significantly later than scalp HFs). Here we synthesize the limited current knowledge on the biology of ELHFs, in humans and other species, their role in health and disease, the known similarities with and differences from other HF populations, and their intrinsic interethnic variations. We define major open questions in the biology of these intriguing mini-organs and conclude by proposing future research directions. These include dissecting the molecular and cellular mechanisms that underlie trichomegaly and the development of in vitro models in order to interrogate the distinct molecular controls of ELHF growth, cycling and pigmentation and to probe novel strategies for the therapeutic and cosmetic manipulation of ELHFs beyond prostaglandin receptor stimulation.

The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15.

BACKGROUND: Anti-CD34 antibodies label the bulge region of mouse hair follicles. However, in human hair follicles, CD34 immunoreactivity is found in the outer root sheath below the bulge zone. The immunohistochemical staining of CD34 in catagen and telogen follicles has not been evaluated. AIMS: To characterize the expression of CD34 immunoreactivity at different stages of the hair cycle in human terminal hair follicles, and to compare the immunostaining pattern of CD34 with that of CK15, used here as a marker of the bulge region. METHOD: Serial vertical sections of human hair follicles in anagen, catagen and telogen phases were immunostained with anti-CD34 (QBEnd 10) and anti-CK15 (LHK15 and C8/144B) antibodies. Double-labelling immunofluorescence was also performed. RESULTS: The catagen and telogen follicles studied did not show CD34 immunoreactivity in the outer root sheath. The location of CD34 and CK15 immunoreactivity in anagen follicles reveals a different staining pattern: CD34-positive cells are located in the outer root sheath below the attachment zone of the arrector pili muscle, whereas CK15-positive cells are located in the outer root sheath above the attachment zone of the arrector pili muscle. CONCLUSIONS: Only anagen human hair follicles show CD34 immunoreactivity. CD34 and CK15 recognize different types of cells or cells at different stages of differentiation.

Complete genome sequence of transmissible gastroenteritis coronavirus PUR46-MAD clone and evolution of the purdue virus cluster.

The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the alpha-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman-Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A-E domains of the palm subdomain of nucleic acid polymerases.

Coronavirus derived expression systems.

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 microg ml(-1)) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome.

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence.

Targeted recombination within the S (spike) gene of transmissible gastroenteritis coronavirus (TGEV) was promoted by passage of helper respiratory virus isolates in cells transfected with a TGEV-derived defective minigenome carrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the formation of true recombinant and pseudorecombinant viruses containing the S proteins of both enteric and respiratory TGEV strains in their envelopes. The recombinants acquired an enteric tropism, and their analysis showed that they were generated by homologous recombination that implied a double crossover in the S gene resulting in replacement of most of the respiratory, attenuated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, virulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codon deletions in a domain of the S gene (nucleotides 217 to 665) previously implicated in the tropism of TGEV. The helper virus, with an original respiratory tropism, was also found in the enteric tract, probably because pseudorecombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demonstrated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.

Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.

The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes, Virology 217: 495-507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated in trans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene beta-glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 microgram/10(6) cells of GUS.

Interference with virus and bacteria replication by the tissue specific expression of antibodies and interfering molecules.

Historically, protection against virus infections has relied on the use of vaccines, but the induction of an immune response requires several days and in certain situations, like in newborn animals that may be infected at birth and die in a few days, there is not sufficient time to elicit a protective immune response. Immediate protection in new born could be provided either by vectors that express virus-interfering molecules in a tissue specific form, or by the production of animals expressing resistance to virus replication. The mucosal surface is the largest body surface susceptible to virus infection that can serve for virus entry. Then, it is of high interest to develop strategies to prevent infections of these areas. Virus growth can be interfered intracellularly, extracellularly or both. The antibodies neutralize virus intra- and extracellularly and their molecular biology is well known. In addition, antibodies efficiently neutralize viruses in the mucosal areas. The autonomy of antibody molecules in virus neutralization makes them functional in cells different from those that produce the antibodies and in the extracellular medium. These properties have identified antibodies as very useful molecules to be expressed by vectors or in transgenic animals to provide resistance to virus infection. A similar role could be played by antimicrobial peptides in the case of bacteria. Intracellular interference with virus growth (intracellular immunity) can be mediated by molecules of very different nature: (i) full length or single chain antibodies; (ii) mutant viral proteins that strongly interfere with the replication of the wild type virus (dominant-negative mutants); (iii) antisense RNA and ribozyme sequences; and (iv) the product of antiviral genes such as the Mx proteins. All these molecules inhibiting virus replication may be used to obtain transgenic animals with resistance to viral infection built in their genomes. We have developed two strategies to target into mucosal areas either antibodies to provide immediate protection, or antigens to elicit immune responses in the enteric or respiratory surfaces in order to prevent virus infection. One strategy is based on the development of expression vectors using coronavirus derived defective RNA minigenomes, and the other relies on the development of transgenic animals providing virus neutralizing antibodies in the milk during lactation. Two types of expression vectors are being engineered based on transmissible gastroenteritis coronavirus (TGEV) defective minigenomes. The first one is a helper virus dependent expression system and the second is based on self-replicating RNAs including the information required to encode the TGEV replicase. The minigenomes expressing the heterologous gene have been improved by using a two-step amplification system based on cytomegalovirus (CMV) and viral promoters. Expression levels around 5 micrograms per 10(6) cells were obtained. The engineered minigenomes will be useful to understand the mechanism of coronavirus replication and for the tissue specific expression of antigen, antibody or virus interfering molecules. To protect from viral infections of the enteric tract, transgenic animals secreting virus neutralizing recombinant antibodies in the milk during lactation have been developed. Neutralizing antibodies with isotypes IgG1 or IgA were produced in the milk with titers of 10(6) in RIA that reduced virus infectivity by one million-fold. The recombinant antibodies recognized a conserved epitope apparently essential for virus replication. Antibody expression levels were transgene transgene copy number independent and were related to the transgene integration site. This strategy may be of general use since it could be applied to protect newborn animals against infections of the enteric tract by viruses or bacteria for which a protective MAb has been identified. Alternatively, the same strategy could be used to target the expression of antibio

The spike protein of transmissible gastroenteritis coronavirus controls the tropism of pseudorecombinant virions engineered using synthetic minigenomes.

The minimum sequence required for the replication and packaging of transmissible gastroenteritis virus (TGEV)-derived minigenomes has been determined. To this end, cDNAs encoding defective RNAs have been cloned and used to express heterologous spike proteins, to determine the influence of the peplomer protein in the control of TGEV tropism. A TGEV defective interfering RNA of 9.7 kb (DI-C) was isolated, and a cDNA complementary to DI-C RNA was cloned under the control of T7 promoter. In vitro transcribed DI-C RNA was replicated in trans upon transfection of helper virus-infected cells. A collection of DI-C deletion mutants (TGEV minigenomes) was generated and tested for their ability to be replicated and packaged. The size of the smallest minigenome replicated in trans was 3.3 kb. The rescue system was used to express the spike protein of an enteric TGEV isolate (C11) using as helper virus a TGEV strain (C8) that replicates very little in the gut. A mixture of two pseudorecombinant viruses containing either the helper virus genome or the minigenome was obtained. These pseudorecombinants display in the surface the S proteins from the enteric and the attenuated virus, and showed 10(4)-fold increase in their gut replication levels as compared to the helper isolate (C8). In addition, the pseudorecombinant virus increased its enteric pathogenicity as compared to the C8 isolate.

Progress towards the construction of a transmissible gastroenteritis coronavirus self-replicating RNA using a two-layer expression system.

Three transmissible gastroenteritis coronavirus (TGEV) defective interfering RNAs of 21, 10.6 and 9.7 kb (DI-A, DI-B and DI-C, respectively) were isolated. Dilution experiments showed that the largest DI RNA, DI-A, is a self-replicating RNA (replicon), and thus codes for a functional RNA polymerase and all the necessary replication signals. In order to engineer a cDNA encoding the RNA replicon a strategy based on the cloning of DI-C cDNA, followed by the insertion of the sequences required to complete the DI-A sequence has been developed. A cDNA complementary to DI-C RNA was cloned under the control of the CMV promoter (pDI-C-CMV) and rescued with a helper virus. In the ORF 1a of polymerase gene pDI-C-CMV contained a 10 kb deletion and in ORF 1b a 1.1 kb deletion. The consensus sequence corresponding to the deleted regions was cloned, and the deletions in pDI-C-CMV were replaced to yield a complete cDNA clone of DI-A, pDI-A-21-CMV, containing a full-length TGEV polymerase, driven by a CMV promoter. Expression of a functional TGEV polymerase is being investigated.

Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity.

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5'-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3' end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3' end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

Structure and encapsidation of transmissible gastroenteritis coronavirus (TGEV) defective interfering genomes.

Serial undiluted passages were performed with the PUR46 strain of TGEV in swine testis (ST) cells. Total cellular RNA was analyzed at different passages after orthophosphate metabolic labeling. Three new defective RNA species of 24, 10.5, and 9.5 kb (DI-A, DI-B, and DI-C respectively) were detected at passage 30, which were highly stable and significantly interfered with helper mRNA synthesis in subsequent passages. By Northern hybridization DIs A, B, and C were detected in purified virions at amounts similar to those of helper RNA. Standard and defective TGEV virions could be sorted in sucrose gradients, indicating that defective and full-length genomes are independently packaged. cDNA synthesis of DI-B and DI-C RNAs was performed by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments in each case. Cloning and sequencing of the DI-C PCR products showed that the smallest DI particle comprises 9.5 kb and has 4 discontinuous regions of the genome. It contains 2.1 kb from the 5'-end of the genome, about 7 kb from gene 1b, the first 24 nucleotides of the S gene, 12 nucleotides of ORF 7, and the 0.4 kb of the UTR at the 3'-end.