Kerryn L. Garrett

Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes

SummaryThe activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precusor cells in muscle regenerating in vivo.

The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides.

Abstract Developed over the past two decades, the antisense strategy has become a technology of recognised therapeutic potential, and many of the problems raised earlier in its application have been solved to varying extents. However, the adequate delivery of antisense oligodeoxynucleotides to individual cells remains an important and inordinately difficult challenge. Synthetic polymers appeared on this scene in the middle 1980s, and there is a surprisingly large variety used or proposed so far as agents for delivery of oligodeoxynucleotides. After discussing the principles of antisense strategy, certain aspects of the ingestion of macromolecules by cells, and the present situation of delivery procedures, this article analyses in detail the attempts to use synthetic polymers as carrier matrices and/or cell membrane permeabilisation agents for delivery of antisense oligodeoxynucleotides. Structural aspects of various polymers, as well as the results, promises and limitations of their use are critically evaluated.

Central Zone Carcinoma of the Prostate Gland: A Distinct Tumor Type With Poor Prognostic Features

PURPOSE The central zone of the prostate gland is a region rarely associated with carcinoma. To our knowledge central zone tumors have not previously been compared to carcinoma originating in the peripheral or transition zone of the prostate gland. MATERIALS AND METHODS All 2,010 radical prostatectomy cases seen at our institution from October 1998 to December 2006 were reviewed to identify tumor zonal origin. Central zone carcinoma was characterized and compared with tumors of other zones. RESULTS Zonal origin was determined in a total of 2,494 tumors in 1,703 cases. Of the tumors 63 (2.5%) were of central zone origin with 59 of the 63 representing the index or main tumor. Comparative analysis of a defined subset of 726 cases showed that central zone cancers were significantly more aggressive than peripheral or transition zone cancers with a far greater risk of extracapsular extension, seminal vesicle invasion and positive surgical margins. Escape from the gland was often via the ejaculatory ducts and seminal vesicles. Kaplan-Meier analysis confirmed that the probability of post-prostatectomy biochemical failure was double that of tumors of the other zones with a far more rapid rate of failure. Multivariate Cox regression analysis identified Gleason grade, positive margins, extracapsular extension, tumor volume and preoperative serum prostate specific antigen as the major contributors to this poor prognosis, rather than specific zonal origin. CONCLUSIONS To our knowledge this study provides the first characterization and comparative analysis of central zone carcinoma, identifying these tumors as a rare but highly aggressive form of prostate carcinoma with a distinct route of spread from the gland that contrasts with tumors of other zones. Preoperative identification is currently hampered by the avoidance of biopsy targeting the central zone. However, if recognized preoperatively, aggressive intervention may possibly improve the currently bleak outlook.

Quantitation of muscle precursor cell activity in skeletal muscle by Northern analysis of MyoD and myogenin expression: Application to dystrophic (mdx) mouse muscle

The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles.

The transcription of MyoD1 and myogenin genes in thymic cells in vivo

The skeletal muscle specific genes MyoD1 and myogenin are closely associated with commitment of cells to the myogenic lineage and differentiation of skeletal muscle precursor cells. The transcription of these genes was studied in the thymus where mononuclear cells termed myoid cells appear to closely resemble skeletal muscle precursors. In thymus from adult SJL/J and BALB/c mice, in situ hybridization with either MyoD1 or myogenin riboprobes showed probe-positive cells concentrated in the medullary region. In neonatal thymus, mRNA for these genes was not detected. These data are the first demonstration in a higher vertebrate of MyoD1 and myogenin expression in a tissue other than skeletal muscle. The sustained expression of MyoD1 and myogenin genes in thymi of adult mice shows that myoid cells are not equivalent to quiescent stem cells of mature skeletal muscle. In addition, studies with antistriational antibodies indicate that myoid cells do not continue to differentiate within the normal murine thymic environment. This arrested differentiation process presents an unusual model for investigating conditions regulating myogenesis in vivo.

Polymerase chain reaction-based identification of Propionibacterium acnes types isolated from the male urinary tract: evaluation of adolescents, normal adults and men with prostatic pathology.

To assess whether colonization of the male urinary tract with Propionibacterium acnes, in particular types IB and II (which are associated with inflammation in radical prostatectomy specimens and might be involved in the development of prostate cancer), is associated with prostate disease, and thus to develop a urine test to detect men at risk of prostate disease.

Preclinical Evaluation of a Phosphorothioate Oligonucleotide in the Retina of Rhesus Monkey

Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF165 genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose- and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application.

Links between Propionibacterium acnes and prostate cancer

Incidental foci of prostate cancer are found at autopsy in 30% of men in their third decade, and by their eighth decade more than 75% have histological evidence of cancer. This unprecedented cancer prevalence points to a ubiquitous causative agent or perhaps an interaction between multiple common carcinogenic cofactors. We propose that one of these carcinogens is Propionibacterium acnes. Several characteristics of prostate cancer suggest the involvement of an infectious agent and we provide evidence that P. acnes is an excellent candidate. We have cultured P. acnes from a substantial proportion of prostate glands containing cancer and shown a significant positive association with prostatic inflammation. P. acnes is well suited to cause persistent, low-grade infection involving a marked inflammatory response and the P. acnes subtypes most frequently associated with prostate cancer become highly prevalent in the urinary tract of males following puberty.

Synthetic hydrogels as carriers in antisense therapy : Preliminary evaluation of an oligodeoxynucleotide covalent conjugate with a copolymer of 1-vinyl-2-pyrrolidinone and 2-hydroxyethyl methacrylate

A major challenge of the antisense therapeutic strategies is the development of improved systems for the delivery of antisense oligodeoxynucleotides (AS ODNs) in order to enhance the cellular uptake, to assure a better efficiency in reaching the target tissue, and to provide sustained delivery over longer periods of time. Because the current methods for delivery (liposomes and cationic polymers) present somedisadvantages, the attention was directed toward the use of neutral polymers as carriers for the AS ODNs. Based on our previous work on synthetic hydrogels for vitreous substitution, we developed a poly[1-vinyl-2-pyrrolidinone-co-(2-hydroxyethyl methacrylate)] hydrogelas a potential carrier for AS ODNs. We have previously demonstrated that such hydrogels are not cytotoxic, and they may have growth-promoting effects on cultured fibroblasts. This copolymer also has the advantage of being injectable. In this study, a specific AS ODN was synthesized and then covalently bound to the copolymer via carbodiimide coupling method. The resulting conjugate was subjected to in vitro release experiments over 46 days in the presence of bovine vitreous humor. Compared with the control (no enzyme present), a significantamount of covalently bound ODN was released from the ODN-hydrogel conjugate, suggesting the possibility of using such systems for the sustained delivery of AS ODNs.

The antibody response to Propionibacterium acnes is an independent predictor of serum prostate-specific antigen levels in biopsy-negative men

To investigate whether the serum titres of Propionibacterium acnes antibodies in patients undergoing prostate biopsy are associated with prostate cancer or markers of prostate disease, including serum prostate‐specific antigen (PSA) levels.