Development of a topical bacteriophage gel targeting Cutibacterium acnes for acne prone skin and results of a phase 1 cosmetic randomized clinical trial.

Background: Topical antibiotics are frequently used to treat acne vulgaris. Their prolonged use, often for longer durations than recommended, has led to antibiotic resistance in Cutibacterium acnes (C. acnes), a bacterium implicated in acne pathophysiology. Bacteriophage (phage), which specifically target C. acnes by a different mechanism of action and do not harm potentially beneficial bacteria, may offer an alternative approach for improvement of the appearance of acne prone skin. Objectives: To identify and characterize C. acnes targeting phage, carry out a comprehensive preclinical safety evaluation of phages selected for further development and examine their safety, tolerability and ability to target facial C. acnes when applied topically in a cosmetic clinical study including participants with mild-to-moderate acne. Methods: Phages were isolated by conventional microbiological methods also used to examine their breadth of host range on different C. acnes strains and specificity to this bacterial species. Safety assessment of three selected phages was carried out by complete genomic analysis to assure the absence of undesired sequences and by ex vivo models employed to evaluate the safety, irritability and potential systemic bioavailability of phage applied topically. A randomized, controlled clinical study assessed safety, tolerability and efficacy in targeting facial C. acnes. Results: Wide host range phages that also target antibiotic resistant C. acnes were identified. Their genomes were shown to be free of undesired genes. The three-phage cocktail, BX001, was not irritant to human skin or ocular tissues in ex vivo models and did not permeate through human epidermis. In a cosmetic clinical study, topically applied BX001 was safe and well tolerated and reduced the facial burden of C. acnes. Conclusions: Combined in silico and ex vivo approaches successfully predicted the observed safety and efficacy of C. acnes targeting phage when these were topically administered in a well-controlled cosmetic clinical study.

Vein expression is induced by the EGF receptor pathway to provide a positive feedback loop in patterning the Drosophila embryonic ventral ectoderm.

The presence of a single EGF receptor in Drosophila is contrasted by multiple ligands activating it. This work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a potent ligand, whereas Vein is an intrinsically weak activating ligand. We show that secreted Spitz emanating from the midline, triggers expression of vein in the ventral-most cell rows, by inducing expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a robust mechanism for patterning the ventral ectoderm.

EGF receptor signaling induces pointed P1 transcription and inactivates Yan protein in the Drosophila embryonic ventral ectoderm.

The induction of different cell fates along the dorsoventral axis of the Drosophila embryo requires a graded activity of the EGF receptor tyrosine kinase (DER). Here we have identified primary and secondary target genes of DER, which mediate the determination of discrete ventral cell fates. High levels of DER activation in the ventralmost cells trigger expression of the transcription factors encoded by ventral nervous system defective (vnd) and pointed P1 (pntPl). Concomitant with the induction of pntP1, high levels of DER activity lead to inactivation of the Yan protein, a transcriptional repressor of Pointed-target genes. These two antagonizing transcription factors subsequently control the expression of secondary target genes such as otd, argos and tartan. The simultaneous effects of the DER pathway on pntP1 induction and Yan inactivation may contribute to the definition of the border of the ventralmost cell fates.

The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm.

The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form was shown to be the limiting event, but the cells in which processing takes place and the mechanism that may generate a gradient of secreted Spitz in the ectoderm were not known. The ectodermal defects in single minded (sim) mutant embryos, in which the midline fails to develop, suggested that the midline cells contribute to patterning of the ventral ectoderm. This work shows that the midline provides the site for Spitz expression and processing. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rho or Star mutant embryos could be rescued by inducing the expression of the respective normal genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gave rise to ventralization of the embryo, increased expression of secreted Spitz in the midline did not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm.

Argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop.

Argos is a secreted molecule with an atypical EGF motif. It was recently shown to function as an inhibitor of the signaling triggered by the Drosophila EGF receptor (DER). In this work, we determine the contribution of Argos to the establishment of cell fates in the embryonic ventral ectoderm. Graded activation of DER is essential for patterning the ventral ectoderm. argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the DER pathway. In the embryonic ventral ectoderm, argos is expressed in the ventralmost row of cells. We show that argos expression in the ventral ectoderm is induced by the DER pathway: argos is not expressed in DER mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the DER pathway. argos expression appears to be triggered directly by the DER pathway, since induction can also be observed in cell culture, following activation of DER by its ligand, Spitz. Argos therefore functions in a sequential manner, to restrict the duration and level of DER signaling. This type of inhibitory feedback loop may represent a general paradigm for signaling pathways inducing diverse cell fates within a population of non-committed cells.

Cloning and characterization of the human GnRH receptor.

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

IL-3 facilitates lymphocyte hexose transport by enhancing the intrinsic activity of the transport system.

Interleukin 3 (IL-3) promotes the survival and proliferation of hematopoietic cells of various lineages in culture. Like most other hematopoietic colony stimulating factors, its mode of action is unknown. However, binding of the lymphokine induces protein tyrosine phosphorylation and enhanced glucose transport in some myeloid progenitor cells. We have studied the hexose uptake following IL-3 stimulation in IL-3-dependent pro-B cells. IL-3 facilitated the uptake of 2-deoxyglucose within 15 min. Kinetic analysis of the 2-deoxyglucose uptake attributed the enhanced transport to improved transporter function, while other hormones or cytokines affect glucose transport primarily via the number of cell surface transporters.