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.

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste.

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster.

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.

Cellular pathways acting along the germband and in the amnioserosa may participate in germband retraction of the Drosophila melanogaster embryo.

Germband retraction in Drosophila melanogaster, like most embryonic morphogenetic events in this organism and in higher eukaryotes, is not well understood. We have taken several approaches to study the relationships between previously identified mutations (u-shaped, serpent, hindsight and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, DER/faint little ball. Our observations from genetic, immunohistochemical, and embryo culture experiments suggest that the former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germband retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by DER/faint little ball, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of DER in the germband may be mediated by the cytoskeleton.

Expanding the Mot1 subfamily: 89B helicase encodes a new Drosophila melanogaster SNF2-related protein which binds to multiple sites on polytene chromosomes.

Many proteins of the SNF2 family, which share a similar DNA-dependent ATPase/putative helicase domain, are involved in global transcriptional control and processing of DNA damage. We report here the partial cloning and characterization of 89B helicase, a gene encoding a new Drosophila melanogaster member of the SNF2 family. 89B Helicase protein shows a high degree of homology in its ATPase/helicase domain to the global transcriptional activators SNF2 and Brahma and to the DNA repair proteins ERCC6 and RAD54. It is, however, most strikingly similar to the Saccharomyces cerevisiae protein Mot1, a transcriptional repressor with many target genes for which no homologue has yet been described. 89B helicase is expressed throughout fly development and its large transcript encodes a >200 kDa protein. Staining with anti-89B Helicase antibodies reveals that the protein is present uniformly in early embryos and then becomes localized to the ventral nerve cord and brain. On the polytene chromosomes, 89B Helicase is bound to several hundred specific sites that are randomly distributed. The homology of 89B Helicase to Mot1, its widespread developmental expression and its large number of targets on the polytene chromosomes of larval salivary gland cells suggest that 89B Helicase may play a role in chromosomal metabolism, particularly global transcriptional regulation.

Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs.

The compound eye of Drosophila develops from a uniform layer of epithelial cells in the eye imaginal disc. One intriguing aspect of eye development is the establishment of the correct number and spacing of the photoreceptor clusters which give rise to the mature ommatidia. Ellipse (Elp) has been implicated as playing a role in this process because the Elp dominant gain of function mutation dramatically reduces the number of photoreceptor clusters in the compound eye without affecting the morphology of individual clusters that are formed (Baker and Rubin, 1989). Since Elp represents an allele of the Drosophila EGF receptor (DER) locus, it encodes a protein which is structurally capable of mediating inductive cell-cell interactions. In an effort to better understand the role of the DER locus in ommatidial patterning, we compared the localization of DER protein in eye imaginal discs of wild-type and Elp larvae. The distribution of this receptor is consistent with the notion of its mediating interactions between cells at the initial stages of photoreceptor precluster positioning and differentiation. However, the basis of the Elp gain of function mutation is not ectopic or increased expression of the DER protein. Rather, expression of the Elp form of the EGF receptor homolog in the normal localization leads to changes in the proliferative pattern of cells dividing posterior to the morphogenetic furrow.

Biochemical properties of the Drosophila EGF receptor homolog (DER) protein.

Antibodies recognizing the Drosophila EGF receptor homolog (DER) were used to define the biochemical properties of the protein. We show that DER is a glycosylated protein, with different processing patterns in Drosophila and mammalian cells, and confirm its localization in the plasma membrane with the predicted orientation. The receptor produced in transfected cells displays only a basal activity of in vivo tyrosine kinase autophosphorylation, providing a sensitive assay for ligands which may trigger DER. EGF and TGF alpha, which trigger the mammalian EGF receptor, did not increase the kinase activity of DER.

Localization of the DER/flb protein in embryos: implications on the faint little ball lethal phenotype.

Antibodies were raised against the Drosophila EGF receptor homolog (DER) and used for immunohistochemical analyses of Drosophila embryos. We found that DER is localized in a wide array of embryonic tissues, displaying a dynamic pattern of expression. DER appears to be expressed in all cells at the cellular blastoderm and gastrula stages. In extended-germ-band embryos, it is found predominantly in the mesoderm and the head. Finally, in retracted-germ-band embryos, DER immunoreactivity is most pronounced at sites of somatic muscle attachments and along the ventral midline of the CNS. We have thus observed that DER is expressed in the diverse tissues which are affected in the DER faint little ball (flb) embryonic lethal phenotype. The different pattern and extent of expression in each tissue suggests that the disparate aspects of the flb phenotype may result from different mechanisms of DER function. To understand the basis for the CNS phenotype of DER/flb mutants, we have closely followed the collapse of the CNS in mutant embryos. Our observations on the evolution of the final CNS phenotype, in combination with the temporo-spatial pattern of appearance of DER in the ventral neuroepithelium, suggest that this receptor participates in the second phase of neuron-glia interactions, namely in stabilization of the ladder-like CNS scaffolding formed by outgrowth of pioneer axonal processes along the glial pre-pattern.

Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain.

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v-erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine-kinase receptor family.

Laminin-immunoreactive sites are induced by growth-associated triggering factors in injured rabbit optic nerve.

We have previously demonstrated that application of soluble growth-associated triggering factors (GATFs) from regenerating fish optic nerve or neonatal rabbit optic nerve to a non-regenerative crushed adult rabbit optic nerve provokes regeneration-like changes in the adult rabbit. In this study we show that the responses initiated by GATFs also include a change in pattern of appearance of an extracellular matrix component, laminin, known to play a role in neurite outgrowth and elongation. These findings suggest a mechanism whereby GATFs activate the adult rabbit glial cells to produce or to accumulate laminin and thereby allow partial compensation for the low inherent regenerative ability of the adult rabbit optic nerve.

In vitro analysis of corneal development with monoclonal antibodies.

Anti-corneal monoclonal antibodies, highly selective for corneal stromal and corneal epithelial components, were used to immunohistochemically evaluate differentiation of intact embryonic chick corneas grown in organ culture in the presence or absence of an associated lens. It was observed that both epithelial and stromal components of 5-day corneas initiated expression of their antigens, irrespective of the presence of a lens. This was unlike previous results obtained when 5-day lens-less corneas were explanted to the chorioallantoic membrane, a condition under which epithelial differentiation ensued but stromal differentiation did not. Possibly, in organ culture, the filter support may replace the lens as a substratum for cell migration of neural crest-derived pericorneal mesenchymal cells into the primary corneal stroma. In 5-day organ cultures with lenses, cellular migration into the primary corneal stroma seems to be largely inhibited (also unlike previous results on the chorioallantoic membrane), but mesenchymal cells which had accumulated at the periphery of the eye did express the differentiation antigen.

Analysis of corneal development with monoclonal antibodies. I. Differentiation in isolated corneas.

Monoclonal antibodies highly selective for developmentally regulated antigens present in the cornea (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983) have been used to immunohistochemically evaluate differentiation in intact chick corneas cultured on the chorioallantoic membrane (CAM) of host embryos. One antibody is directed against the epithelial cell layer and the other is against the corneal stromal matrix. It has been established that both antigens recognized by the antibodies are expressed de novo in young explanted corneas and that the stromal matrix antigen is a product of the corneal fibroblasts. Thus expression of the antigens can be used as criteria for overt differentiation of the respective cell types. The antibodies have been employed to assess when the corneal epithelial and stromal cells become capable of autonomous differentiation within isolated corneas. To accomplish this, corneas of various ages were explanted with and without adjacent pericorneal tissues. The results indicate that, under the culture conditions employed, corneal stromal differentiation is dependent on the presence of the lens until stage 28 (51/2-6 days of development), which is the time when invasion of the stroma by pericorneal mesenchymal cells is initiated. After stage 28, the stromal matrix antigen was expressed by isolated corneas irrespective of the presence of the lens. Possibly the lens acts by maintaining the integrity of the corneal endothelial monolayer and thus promoting normal migration of pericorneal mesenchymal cells into the primary corneal stroma, where they undergo differentiation. Conversely, differentiation of the corneal epithelium was independent of any pericorneal structure from the earliest stage examined (41/2-5 days of development). It was even independent of overt stromal differentiation, thus suggesting an early and strong determination for this tissue.

Analysis of corneal development with monoclonal antibodies. II. Tissue autonomy in cornea-skin recombinants.

Developmental autonomy of corneal epithelial and stromal components was assessed by their subsequent differentiation after recombination with feather-forming thigh dermis and epidermis, respectively. Work by others has shown that feather-forming dermis exhibits strong inductive ability when used in such epithelial-mesenchymal recombinations. After culture of the recombinants on the chorioallantoic membrane (CAM) of host embryos, differentiation as "cornea" was assessed immunohistochemically using the anti-corneal stromal matrix and anti-corneal epithelial antibodies described previously (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983). Feather initiation and outgrowth and keratin synthesis served as markers for differentiation as skin. It has been found that corneal epithelia from 5-day embryos, when grown in association with feather-forming dermis from the thigh, will participate in feather formation. In such recombinants, when the corneal epithelium became incorporated into feathers it failed to express the corneal epithelial antigen, but in regions of the recombinant where feathers did not form, de novo expression of the antigen was sometimes detected. The limited liability of the epithelium is not present in corneal epithelia taken from embryos a day or two older. When such epithelia were used for making the recombinants, no feathers were formed and the corneal epithelial antigen was extensively produced. Thus epithelial determination occurs long before the epithelium would begin to overtly differentiate and express the epithelial antigen in vivo (about 12 days of development). In reciprocal recombinations of corneal stromas with feather-forming epidermis, the stromas proceeded to express the corneal stromal matrix specific antigen de novo after culture on the CAM. They did not, however, redirect differentiation of the epidermis which never expressed the corneal epithelial antigen and in some cases went on to keratinize. These results indicate that development of both the corneal epithelial and stromal components becomes autonomous at least several days before these tissues overtly differentiate. This suggests that the component tissues of the cornea may not interact in a manner typical of those of other organs which, in general, are thought to require continual interaction of their epithelial and mesenchymal components for normal development.

Monoclonal antibodies against developmentally regulated corneal antigens.

The chick cornea is comprised of three cellular layers, each associated with a discrete extracellular matrix. The absence of specific markers for these cellular and acellular components has made it difficult to investigate the cell-cell and cell-matrix interactions which occur during development of this organ. We have approached this problem by producing monoclonal antibodies to species-specific, developmentally regulated antigens of the chick cornea. By immunofluorescence staining patterns the antibodies fall into three distinct groups. One group is directed against the corneal extracellular matrix. At 9 days of embryonic development staining by these antibodies is detected at the endothelial surface (in Descemet's membrane), and in the posterior part of the stroma. During development it progresses anteriorly throughout the entire width of the corneal stroma and Bowman's membrane until, by 14 days, it is found in all three specialized extracellular matrices of the cornea. Throughout most of development these antibodies do not recognize any other ocular or nonocular tissue examined. Late in development they begin to lightly stain nerve bundles. A second group of antibodies is highly selective for the corneal epithelial cell layer. These begin to stain at 12 to 13 days of development and cause very bright fluorescence by 14 days. A third group stains the extracellular matrix of the cornea in a manner spatially and temporally identical to that of the first group, but in addition recognizes certain basement membranes. The possible relationship of the antigens recognized by these groups of antibodies to developmental events occurring at the time of their appearance, and the potential use of all three antibody groups in studying corneal development are discussed.

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.