Regenerative Medicine Applied to Musculoskeletal Diseases in Equines: A Systematic Review.

Musculoskeletal injuries in horses have a great economic impact, predominantly affecting tendons, ligaments, and cartilage, which have limited natural regeneration. Cell therapy, which uses mesenchymal stem cells due to their tissue differentiation properties and anti-inflammatory and immunoregulatory effects, aims to restore damaged tissue. In this manuscript, we performed a systematic review using the Parsifal tool, searching the PubMed and Web of Science databases for articles on regenerative medicine for equine musculoskeletal injuries. Our review covers 17 experimental clinical studies categorized by the therapeutic approach used: platelet-rich plasma, conditioned autologous serum, mesenchymal stem cells, and secretome. These therapies reduce healing time, promote regeneration of fibrocartilaginous tissue, improve cellular organization, and improve joint functionality and sustainability. In conclusion, regenerative therapies using platelet-rich plasma, conditioned autologous serum, equine mesenchymal stem cells, and the emerging field of the secretome represent a promising and highly effective approach for the treatment of joint pathologies in horses, implying a valuable advance in equine healthcare.

Specific Gene Expression in Pseudomonas Putida U Shows New Alternatives for Cadaverine and Putrescine Catabolism.

Pseudomonas putida strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase (spuC1 and spuC2 genes) and a ɣ-aminobutyrate aminotransferase (gabT1 and gabT2 genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, spuC1 is upregulated by putrescine, whereas spuC2 seems to be exclusively induced by cadaverine. However, gabT1 increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although gabT2 does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in P. aeruginosa and Escherichia coli concerning P. putida strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism.

Isolation of Environmental Bacteria Able to Degrade Sterols and/or Bile Acids: Determination of Cholesterol Oxidase and Several Hydroxysteroid Dehydrogenase Activities in Rhodococcus, Gordonia, and Pseudomonas putida.

Interest about the isolation and characterization of steroid-catabolizing bacteria has increased over time due to the massive release of these recalcitrant compounds and their deleterious effects or their biotransformation derivatives as endocrine disruptors for wildlife, as well as their potential use in biotechnological approaches for the synthesis of pharmacological compounds. Thus, in this chapter, an isolation protocol to select environmental bacteria able to degrade sterols, bile acids, and androgens is shown. Moreover, procedures for the determination of cholesterol oxidase or different hydroxysteroid dehydrogenase activities in Pseudomonas putida DOC21, Rhodococcus sp. HE24.12, Gordonia sp. HE24.4J and Gordonia sp. HE24.3 are also detailed.

Identification and Characterization of Some Genes, Enzymes, and Metabolic Intermediates Belonging to the Bile Acid Aerobic Catabolic Pathway from Pseudomonas.

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and/or other steroids (e.g., testosterone and 4-androsten-3,17-dione) under aerobic conditions through the 9,10-seco pathway. From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemically defined media containing some cyclopentane-perhydrophenanthrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the route involved in bile acids and androgens, the 9,10-seco pathway in this bacterium. In this manuscript, we describe the most relevant methods used in our lab for the identification of the chromosomal location and nucleotide sequence of the catabolic genes (or gene clusters) encoding the enzymes of this pathway, and the tools useful to establish the role of some of the enzymes that participate in this route.

Extracellular vesicle and soluble fractions of adipose tissue-derived mesenchymal stem cells secretome induce inflammatory cytokines modulation in an in vitro model of discogenic pain.

BACKGROUND CONTEXT: Mesenchymal stem cells (MSCs) secretome or conditioned medium (CM) is a complex cocktail of different molecules, some of which, particularly those contained in extracellular vesicles, already have proven therapeutic applications. PURPOSE: CM may well represent promising therapy for discogenic pain and the intention of this work is to assess its therapeutic potential using an in vitro model of this condition. STUDY DESIGN: This is an experimental study. METHODS: Our in vitro model comprised nucleus pulposus (NP) and annulus fibrosus (AF) cells inflamed with TNF. To assess the potential therapeutic value of CM and its components, extracellular vesicles (EVs) and soluble culture fraction (SF), cell inflammation took place under 3 different conditions: either in the presence of whole CM, isolated EVs or SF, and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and neurotrophic factors produced in all 3 cases were compared. RESULTS: In the presence of whole CM, both in vitro gene expression by the NP and AF test cells and analysis of their protein content showed high modulatory effects on inflammation and MMP inhibition. The presence of EVs and SF showed similar but much smaller effects, and this was particularly marked in the case of NP cells. CONCLUSIONS: Our results show that, compared to EVs and SF, the presence of whole CM has the greatest positive effect on the modulation of pro-inflammatory and catabolic factors. These observations suggest that CM could protect against inflammation and the resulting intervertebral disc (IVD) degeneration that leads to discogenic pain. CLINICAL SIGNIFICANCE: Many patients' expectations are not met by current non-operative and surgical treatments for discogenic low back pain. We propose the use of the MSCs secretome for assessing its potential as cell-free therapy to treat degenerative disc disease modulating the inflammatory response.

Xerotolerance: A New Property in Exiguobacterium Genus.

The highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255-15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255-15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255-15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance.

Catabolism of biogenic amines in Pseudomonas species.

Biogenic amines (BAs; 2-phenylethylamine, tyramine, dopamine, epinephrine, norepinephrine, octopamine, histamine, tryptamine, serotonin, agmatine, cadaverine, putrescine, spermidine, spermine and certain aliphatic amines) are widely distributed organic molecules that play basic physiological functions in animals, plants and microorganisms. Pseudomonas species can grow in media containing different BAs as carbon and energy sources, a reason why these bacteria are excellent models for studying such catabolic pathways. In this review, we analyse most of the routes used by different species of Pseudomonas (P. putida, P. aeruginosa, P. entomophila and P. fluorescens) to degrade BAs. Analysis of these pathways has led to the identification of a huge number of genes, catabolic enzymes, transport systems and regulators, as well as to understanding of their hierarchy and functional evolution. Knowledge of these pathways has allowed the design and collection of genetically manipulated microbes useful for eliminating BAs from different sources, highlighting the biotechnological applications of these studies.

A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma.

Trichothecenes are sesquiterpene toxins produced by diverse fungi, including some species of Trichoderma that are potential plant disease biocontrol agents. Trichoderma arundinaceum produces the trichothecene harzianum A (HA), which consists of the core trichothecene structure (12,13-epoxytrichothec-9-ene, EPT) with a linear polyketide-derived substituent (octa-2,4,6-trienedioyl) esterified to an oxygen at carbon atom 4. The genes required for biosynthesis of EPT and the eight-carbon polyketide precursor of the octa-2,4,6-trienedioyl substituent, as well as for esterification of the substituent to EPT have been described. However, genes required for conversion of the polyketide (octa-2,4,6-trienoic acid) to octa-2,4,6-trienedioyl-CoA, the immediate precursor of the substituent, have not been described. Here, we identified 91 cytochrome P450 monooxygenase genes in the genome sequence of T. arundinaceum, and provided evidence from gene deletion, complementation, cross-culture feeding, and chemical analyses that one of them (tri23) is required for conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioyl-CoA. The gene was detected in other HA-producing Trichoderma species, but not in species of other fungal genera that produce trichothecenes with an octa-2,4,6-trienoic acid-derived substituent. These findings indicate that tri23 is a trichothecene biosynthetic gene unique to Trichoderma species, which in turn suggests that modification of octa-2,4,6-trienoic acid during trichothecene biosynthesis has evolved independently in some fungi.

Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways.

Steroids are perhydro-1,2-cyclopentanophenanthrene derivatives that are almost exclusively synthesised by eukaryotic organisms. Since the start of the Anthropocene, the presence of these molecules, as well as related synthetic compounds (ethinylestradiol, dexamethasone, and others), has increased in different habitats due to farm and municipal effluents and discharge from the pharmaceutical industry. In addition, the highly hydrophobic nature of these molecules, as well as the absence of functional groups, makes them highly resistant to biodegradation. However, some environmental bacteria are able to modify or mineralise these compounds. Although steroid-metabolising bacteria have been isolated since the beginning of the 20th century, the genetics and catabolic pathways used have only been characterised in model organisms in the last few decades. Here, the metabolic alternatives used by different bacteria to metabolise steroids (e.g., cholesterol, bile acids, testosterone, and other steroid hormones), as well as the organisation and conservation of the genes involved, are reviewed.

Histamine catabolism in Pseudomonas putida U: identification of the genes, catabolic enzymes and regulators.

In this study, the catabolic pathway required for the degradation of the biogenic amine histamine (Hin) was genetically and biochemically characterized in Pseudomonas putida U. The 11 proteins (HinABCDGHFLIJK) that participate in this pathway are encoded by genes belonging to three loci hin1, hin2 and hin3 and by the gene hinK. The enzymes HinABCD catalyze the transport and oxidative deamination of histamine to 4-imidazoleacetic acid (ImAA). This reaction is coupled to those of other well-known enzymatic systems (DadXAR and CoxBA-C) that ensure both the recovery of the pyruvate required for Hin deamination and the genesis of the energy needed for Hin uptake. The proteins HinGHFLKIJ catalyze the sequential transformation of ImAA to fumaric acid via N2 -formylisoasparagine, formylaspartic acid and aspartic acid. The identified Hin pathway encompasses all the genes and proteins (transporters, energizing systems, catabolic enzymes and regulators) needed for the biological degradation of Hin. Our work was facilitated by the design and isolation of genetically engineered strains that degrade Hin or ImAA and of mutants that accumulate Ala, Asp and Hin catabolites. The implications of this research with respect to potential biotechnological applications are discussed.

Identification and Characterization of the Genes and Enzymes Belonging to the Bile Acid Catabolic Pathway in Pseudomonas.

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and other steroids (e.g., testosterone and 4-androsten-3,17-dione). From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemical defined media containing some cyclopentane-perhydro-phenantrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the 9,10-seco pathway, the route involved in the aerobic assimilation of steroids. In this manuscript, we describe the most relevant methods required for (1) isolation and characterization of these species; (2) determining the chromosomal location, nucleotide sequence, and functional analysis of the catabolic genes (or gene clusters) encoding the enzymes from this pathway; and (3) the tools employed to establish the role of some of the proteins that participate in this route.

Assessment of regeneration in meniscal lesions by use of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation on a scaffold. Horses were allocated into 2 groups. In one group, defects in the medial meniscus were treated with MSCs derived from BM, whereas in the other group, defects were treated with MSCs derived from AT. Defects were created in the contralateral stifle joint but were not treated (control samples). RESULTS Both types of MSCs had universal stem cell characteristics. For in vivo testing, at 12 months after treatment, treated defects were regenerated with fibrocartilaginous tissue, whereas untreated defects were partially repaired or not repaired. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSCs derived from AT could be a good alternative to MSCs derived from BM for use in regenerative treatments. Results also were promising for a stem cell-based implant for use in regeneration in meniscal lesions. IMPACT FOR HUMAN MEDICINE Because of similarities in joint disease between horses and humans, these results could have applications in humans.

Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

The phasin PhaF controls bacterial shape and size in a network-forming strain of Pseudomonas putida.

Pseudomonas putida N, a poly-3-hydroxyalkonate (PHA)-producing bacterium, showing ampicillin resistance, is an unusual strain. In the presence of this antibiotic, it grows as giant cells (25-50µm) forming complex networks inter-connected by micro-tubular structures. The transformation of this bacterium with a plasmid containing the gene phaF, which encodes a phasin involved in the molecular architecture of the PHA-granules, (i) restores the wild-type phenotype by reducing both bacterial size and length (coco-bacilli ranging between 0.5 and 3µm), and (ii) increases ampicillin resistance by more than 100-fold.

The loss of function of PhaC1 is a survival mechanism that counteracts the stress caused by the overproduction of poly-3-hydroxyalkanoates in Pseudomonas putidaΔfadBA.

The poly-3-hydroxylkanoate (PHA)-overproducing mutant Pseudomonas putida U ΔfadBA (PpΔfadBA) lacks the genes encoding the main ß-oxidation pathway (FadBA). This strain accumulates enormous amounts of bioplastics when cultured in chemically defined media containing PHA precursors (different n-alkanoic or n-aryl-alkanoic acids) and an additional carbon source. In medium containing glucose or 4-hydroxy-phenylacetate, the mutant does not accumulate PHAs and grows just as the wild type (P. putida U). However, when the carbon source is octanoate, growth is severely impaired, suggesting that in PpΔfadBA, the metabolic imbalance resulting from a lower rate of ß-oxidation, together with the accumulation of bioplastics, causes severe physiological stress. Here, we show that PpΔfadBA efficiently counteracts this latter effect via a survival mechanism involving the introduction of spontaneous mutations that block PHA accumulation. Surprisingly, genetic analyses of the whole pha cluster revealed that these mutations occurred only in the gene encoding one of the polymerases (phaC1) and that the loss of PhaC1 function was enough to prevent PHA synthesis. The influence of these mutations on the structure of PhaC1 and the existence of a protein-protein (PhaC1-PhaC2) interaction that explains the functionality of the polymerization system are discussed herein.

Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida†DOC21.

Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

The tick saliva immunosuppressor, Salp15, contributes to Th17-induced pathology during Experimental Autoimmune Encephalomyelitis.

Salp15 is a tick saliva protein that inhibits CD4(+) T cell differentiation through its interaction with CD4. The protein inhibits early signaling events during T cell activation and IL-2 production. Because murine Experimental Autoimmune Encephalomyelitis development is mediated by central nervous system-infiltrating CD4(+) T cells that are specific for myelin-associated proteins, we sought to determine whether the treatment of mice with Salp15 during EAE induction would prevent the generation of proinflammatory T cell responses and the development of the disease. Surprisingly, Salp15-treated mice developed more severe EAE than control animals. The treatment of EAE-induced mice with the tick saliva protein did not result in increased infiltration of T cells to the central nervous system, indicating that Salp15 had not affected the permeability of the blood-brain barrier. Salp15 treatment did not affect the development of antibody responses against the eliciting peptide or the presence of IFNγ in the sera. The treatment with Salp15 resulted, however, in the increased differentiation of Th17 cells in vivo, as evidenced by higher IL-17 production from PLP(139-151)-specific CD4(+) T cells isolated from the central nervous system and the periphery. In vitro, Salp15 was able to induce the differentiation of Th17 cells in the presence of IL-6 and the absence of TGFß These results suggest that a conductive milieu for the differentiation of Th17 cells can be achieved by restriction of the production of IL-2 during T cell differentiation, a role that may be performed by TGFß and other immunosuppressive agents.

The 3,4-dihydroxyphenylacetic acid catabolon, a catabolic unit for degradation of biogenic amines tyramine and dopamine in Pseudomonas putida U.

Degradation of tyramine and dopamine by Pseudomonas putida U involves the participation of twenty one proteins organized in two coupled catabolic pathways, Tyn (tynABFEC tynG tynR tynD, 12 338 bp) and Hpa (hpaR hpaBC hpaHI hpaX hpaG1G2EDF hpaA hpaY, 12 722 bp). The Tyn pathway catalyses the conversion of tyramine and dopamine into 4-hydroxyphenylacetic acid (4HPA) and 3,4-dihydroxyphenylacetic acid (3,4HPA) respectively. Together, the Tyn and Hpa pathways constitute a complex catabolic unit (the 3,4HPA catabolon) in which 3,4HPA is the central intermediate. The genes encoding Tyn proteins are organized in four consecutive transcriptional units (tynABFEC, tynG, tynR and tynD), whereas those encoding Hpa proteins constitute consecutive operons (hpaBC, hpaG1G2EDF, hpaX, hpaHI) and three independent units (hpaA, hpaR and hpaY). Genetic engineering approaches were used to clone tyn and hpa genes and then express them, either individually or in tandem, in plasmids and/or bacterial chromosomes, resulting in recombinant bacterial strains able to eliminate tyramine and dopamine from different media. These results enlarge our biochemical and genetic knowledge of the microbial catabolic routes involved in the degradation of aromatic bioamines. Furthermore, they provide potent biotechnological tools to be used in food processing and fermentation as well as new strategies that could be used for pharmacological and gene therapeutic applications in the near future.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid in Pseudomonas putida U.

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

The Ixodes scapularis salivary protein, salp15, prevents the association of HIV-1 gp120 and CD4.

Ixodes scapularis salivary protein, Salp15, inhibits CD4(+) T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the envelope glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to define exposed gp120 epitopes for the generation of neutralizing vaccines.