"Are we barking up the wrong tree? Too much emphasis on Cutibacterium acnes and ignoring other pathogens"- a study based on next-generation sequencing of normal and diseased discs.

BACKGROUND: The majority of literature on bacterial flora in the disc stands disadvantaged in utilizing traditional culture methods and targeting a single bacterium, Cutibacterium acnes. PURPOSE: Our objective was to document the diversity in the bacterial flora between normal and degenerated discs for shortlisting potential pathogens using next-generation genomic tools. STUDY DESIGN: Experimental case-control study. METHODS: Researchers employed 16S metagenome sequencing to profile bacterial diversity in magnetic resonance imaging normal healthy discs from brain-dead organ voluntary donors (n=20) and 40 degenerated disc samples harvested during surgery (Modic [MC]=20 and non-Modic [NMC]=20). The V3-V4 region was amplified using universal bacterial primers 341F and 806R, and the libraries were sequenced using Illumina NovoSeq 6000 platform. Statistical significance was set at bacteria with a minimum of 100 operational taxonomic unit (OTU) and present in at least 70% of the samples. The quality check-filtered reads were processed using the QIIME-2 pipeline. The OTU clustering and taxonomic classification were carried out for the merged reads using the Greengenes/SILVA reference database. Validation was done by identification of bacterial metabolites in samples using the liquid chromatography-mass spectrometry approach. RESULTS: Abundant bacteria differing widely in diversity, as evidenced by Alfa and Beta diversity analysis, were present in all control and degenerative samples. The number of bacterial genera was 27 (14-gram-positive: 13-gram-negative) in the control group, 23 (10-gram-positive: 11-gram-negative) in the Modic group, and 16 (11-gram-positive: 5-gram-negative) in the non-Modic group. In the Modic group, gram-negative bacteria OTUs were found to be predominant (more than 50% of the total bacteria identified), whereas in control and non-Modic groups the OTUs of gram-positive bacteria were predominant. Species-level analysis revealed an abundance of opportunistic gram-negative pathogens like Pseudomonas aeruginosa, Sphingomonos paucibacillus, and Ochrobactrum quorumnocens in the discs with Modic changes, more than in non-Modic discs. The presence of bacterial metabolites and quorum-sensing molecules like N-decanoyl-L-homoserine lactone, 6-hydroxynicotinic acid, 2-aminoacetophenone, 4-hydroxy-3-polyprenylbenzoate, PE (16:1(9Z)/18:0) and phthalic acid validated the colonization and cell-cell communication of bacteria in disc ruling out contamination theory. Cutibacterium acnes was not the predominant bacteria in any of the three groups of discs and in fact was in the 16th position in the order of abundance in the control discs (0.72%), seventh position in the Modic discs (1.41%), and 12th position (0.53%) in the non-Modic discs. CONCLUSION: This study identified a predominance of gram-negative bacteria in degenerated discs and highlights that Cutibacterium acnes may not be the only degeneration-causing bacteria. This may be attributed to the environment, diet, and lifestyle habits of the sample population. Though the study does not reveal the exact pathogen, it may pave the way for future studies on the subject. CLINICAL SIGNIFICANCE: These findings invite further investigation into causal relationships of bacterial profile with disc degeneration phenotypes as well as phenotype-driven clinical treatment protocols.

Novel Biomarkers of Health and Degeneration in Human Intervertebral Discs: In-depth Proteomic Analysis of Collagen Framework of Fetal, Healthy, Scoliotic, Degenerate, and Herniated Discs.

STUDY DESIGN: Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states. PURPOSE: To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs. OVERVIEW OF LITERATURE: Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes. METHODS: Forty NP tissues were snap-frozen in liquid nitrogen (-196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each). RESULTS: Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan-collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation. CONCLUSIONS: COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy.

Bacteria in human lumbar discs - subclinical infection or contamination? Metabolomic evidence for colonization, multiplication, and cell-cell cross-talk of bacteria.

BACKGROUND CONTEXT: The accumulating evidence associating sub-clinical infection with disc degeneration (DD) and the controversy of contamination versus infection mandates a further understanding of the microbial activity in the disc and host-microbiome interaction. PURPOSE: To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival, and replication in human lumbar intervertebral discs (LIVD). STUDY DESIGN: An observational case-control study. PATIENT SAMPLE: Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for disc degeneration (DD) (cases) were utilized. METHODS: Untargeted metabolite profiling was carried out in six discs (3-controls and 3-cases) after extraction using methanol: acetonitrile: water (2:2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. The biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores of the metabolites were calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. RESULTS: Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and degenerated discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources, and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed distinct clustered patterns between control and disc degene`ration, indicating a strong variation in concentration, peak, and spectral values of the 64 metabolites between controls and cases. After the exclusion of metabolites that were also associated with humans, drugs, and food, 39 metabolites specific to bacteria were isolated. Nine were primary metabolites related to bacterial growth and survival, and the remaining 30 were secondary metabolites related to different environmental stress response activities. The three significant pathways (p<.001) which were predominant in the bacterial metabolites were autoinducer-2 biosynthesis, peptidoglycan biosynthesis, and chorismate pathway. In addition, a significant fold change of >1.0 was found for nine metabolites which included (S)-14-Methyilhexadecanoic acid related to P. acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M. tuberculosis. There was also upregulation of Autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. CONCLUSION: We identified several bacterial-specific metabolites participating in bacterial growth, survival, and cross-talk pathways. These were found in both groups but up-regulated in degenerated discs. The presence of Quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that the bacterial presence may not be mere contamination but could be colonization with a possible role in infection-mediated inflammation in DD. CLINICAL SIGNIFICANCE: Proof of subclinical infection as an initiator of DD and documentation of exact germ and drug sensitivity will change the way millions of patients with non-specific low back pain (NSLBP) are treated across the world.

Comparative metagenomic analysis of human intervertebral disc nucleus pulposus and cartilaginous end plates.

Study design: The diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity. Objectives: To understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP). Background of the study: There is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP. Materials and methods: Sixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP). Results: NP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD. Conclusion: Results of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate-nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.

Are Modic changes 'Primary infective endplatitis'?-insights from multimodal imaging of non-specific low back pain patients and development of a radiological 'Endplate infection probability score'.

PURPOSE: To probe the pathophysiological basis of Modic change (MC) by multimodal imaging rather than by MRI alone. METHODS: Nineteen radiological signs found in mild infections and traumatic endplate fractures were identified by MRI and CT, and by elimination, three signs unique to infection and trauma were distilled. By ranking the Z score, radiological 'Endplate Infection Probability Score' (EIPS) was developed. The score's ability to differentiate infection and traumatic endplate changes (EPC) was validated in a fresh set of 15 patients each, with documented infection and trauma. The EIPS, ESR, CRP, and Numeric Pain Rating Scale (NRS) were then compared between 115 patients with and 80 patients without MC. RESULTS: The EIPS had a confidence of 66.4%, 83% and, 100% for scores of 4, 5 and, 6, respectively, for end plate changes suggesting infection. The mean EIPS was 4.85 ± 1.94 in patients with Modic changes compared to - 0.66 ± 0.49 in patients without Modic changes (p < 0.001). Seventy-eight (67.64%) patients with MC had a score of 6, indicating high infection possibility. There was a difference in the NRS (p < 0.01), ESR (p = 0.05), CRP (p < 0.01), and type of pain (p < 0.01) between patients with and without MC. CONCLUSION: Multimodal imaging showed many radiological signs not easily seen in MRI alone and thus missed in Modic classification. There were distinct radiological differences between EPCs of trauma and infection which allowed the development of an EIPS. The scores showed that 67.64% of our study patients with Modic changes had EPCs resembling infection rather than trauma suggesting the possibility of an infective aetiology and allowing us to propose an alternate theory of 'Primary Endplatitis'.

Can Scoliotic Discs Be Controls for Molecular Studies in Intervertebral Disc Research? Insights From Proteomics.

STUDY DESIGN: Proteomic analysis of human intervertebral discs. OBJECTIVES: To compare the characters of scoliotic discs and discs from magnetic resonance imaging (MRI)-normal voluntary organ donors controls used in disc research employing proteomics and establish "true controls" that can be utilized for future intervertebral disc (IVD) research. METHODS: Eight MRI-normal discs from 8 brain-dead voluntary organ donors (ND) and 8 scoliotic discs (SD) from 3 patients who underwent anterior surgery for adolescent idiopathic scoliosis were subjected to tandem mass spectrometry, and further analysis was performed. RESULTS: Mass spectrometry identified a total of 235 proteins in ND and 438 proteins in the SD group. Proteins involved in extracellular matrix integrity (Versican, keratins KRT6A, KRT14, KRT5, and KRT 13A1, A-kinase anchor protein 13, coagulation factor XIII A chain, proteoglycan 4) and proteins involved in transcription and DNA repair (Von Willebrand factor A domain-containing 3B, eukaryotic initiation factor 2B, histone H4, leukocyte cell-derived chemotaxin 2) were found to be downregulated in SD. Inflammatory proteins (C3, C1S), and oxidative stress response proteins (peroxiredoxin-2,6, catalase, myeloperoxidase, apolipoprotein E) were found to be upregulated in SD. These changes were reflected at the pathway level also. CONCLUSION: Findings of our study confirm that scoliotic discs have an abundance of inflammatory, oxidative stress response proteins, which are either absent or downregulated in the ND group indicating that scoliotic discs are not pathologically inert. Furthermore, this study has established MRI-normal discs from voluntary organ donors as the "true" control for molecular studies in IVD research.

Modic changes are associated with activation of intense inflammatory and host defense response pathways - molecular insights from proteomic analysis of human intervertebral discs.

BACKGROUND CONTEXT: Patients with modic changes (MC) form a distinct clinical subset with reports of higher intensity of pain, poor clinical and surgical outcomes and higher incidence of recurrence. MC also is an independent risk factor for increased post-operative surgical site infection. PURPOSE: This study aimed to investigate the biological changes at molecular level, in discs with MCs. We also aim to identify biological biomarkers and potential targets for molecular therapy. STUDY DESIGN: Experimental analysis MATERIALS AND METHODS: Nucleus pulposus (NP) from 24 patients undergoing microdiscectomy for disc herniation [14 discs with MC and 10 without modic changes (NMC)] were procured. The overall expression of proteins, biological processes, protein-protein and metabolite interactions were analysed and compared. Host defense response proteins (HDRPs) and immunological pathways activated in patients with MC were documented and analysed. RESULTS: Label-free proteomic approach with stringent filters revealed a total of 208 proteins in MC and 193 in NMC groups. 45 proteins were specific to MC; 30 to NMC and 163 common to both. Downregulated proteins in MC belonged to components of extracellular matrix such as collagens (COL- 6A1, 6A2, 6A3, 11A1, 12A1, and 20A1), and proteoglycans (versican (VCAN), and biglycan (BGN)). Inflammatory molecules [plasminogen (PLG), angiogenin (ANG), fibroblast growth factor-binding protein 2 (FGFBP2), tetranectin (CLEC3B), cartilage acidic protein 1(CRTAC1), kininogen (KNG-1), chitinase-3-like protein 2 (CHI3L2), and ferritin (FTL) were expressed only in the MC group. The significantly altered pathways in MC included Fc Fragment of IgG Receptor IIIa (FCGR3A)-mediated phagocytosis, regulation of Toll-like receptors (TLR) by endogenous ligand, neutrophil and platelet degranulation. 50 HDRPs were identified in the study, 14 of which were specific to MC and included acute phase reactants, antimicrobial peptides, complement cascade proteins, inflammatory molecule and stress response proteins. Metabolite-protein interaction analysis revealed a significant interaction between 19 proteins, specifically involving ubiquitin mediating proteasome degradative pathway and an association with the metabolite-glutamic acid in the MC group. Accumulation of glutamic acid in MC discs was confirmed by quantitative amino acid analysis using High-performance liquid chromatography. CONCLUSION: Our study confirms that MC represents an intense inflammatory status and activation of host defense response and immunological pathways. Downstream effects leading to ubiquitin mediated proteasomal degradation of ECM proteins and the resulting metabolites such as glutamic acid could cause excessive pain and needs further investigation. CLINICAL SIGNIFICANCE: We have documented the expression of inflammatory molecules, immune mechanisms and host defense response proteins which throw molecular insights into the pathological mechanisms of MC. Further, ubiquitin mediated proteasomal degradation and accumulation of glutamate in discs with MC might serve as targets for molecular therapy.

Uncovering molecular targets for regenerative therapy in degenerative disc disease: do small leucine-rich proteoglycans hold the key?

BACKGROUND CONTEXT: Small leucine-rich proteoglycans (SLRPs) play an essential role in extracellular matrix (ECM) organization and function. Recently, dysregulation of SLRPs has been implicated in degenerative disc disease (DDD). An in-depth analysis using high-throughput proteomic sequencing might provide valuable information on their implications in health and disease. PURPOSE: To utilize proteomics for analyzing the expression of SLRPs in fetal, healthy adult, and degenerated discs, to identify possible molecular targets to halt or reverse the degenerative process. STUDY DESIGN: Experimental analysis. METHODS: Proteomic signatures of 8 magnetic resonance imaging (MRI) normal lumbar discs (ND) [harvested from brain dead alive organ donors] were compared to 8 fetal disc samples (FD) [harvested from fetal spines devoid of congenital anomalies following spontaneous or medical termination of pregnancy] and 8 degenerate discs (DD) [collected from patients undergoing fusion surgery]. The various functional pathways along with the differential expression of SLRPs and the associated changes in collagens, large proteoglycans (LLRPs), matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been analyzed further using bioinformatics. This project was self-funded by the Ganga Orthopedic Research and Education Foundation. RESULTS: ESI-LC-MS/MS analysis revealed a total of 1,029 proteins in FD, 1,785 proteins in ND, and 1,775 proteins in DD. Fetal disc proteins were engaged mainly in ribosomal pathways (indicating active proliferation and regenerative potential). The healthy adult discs (ND) primarily participated in ECM maintenance and basic metabolic pathways, whereas the unique proteins of DD group were involved in inflammatory (Complement and coagulation cascades, Systemic Lupus Erythematosus and Leukocyte transendothelial migration) pathways and infective (Staphylococcus aureus infection, Prion diseases, Amoebiasis, Pertussis, and Legionellosis) channels which favor the recent concepts of inflammaging and subclinical infection as causes of DDD. Analysis of SLRPs revealed the upregulation of Biglycan in FDs and downregulation of Lumican, Decorin, Prolargin, and Chondroadherin in the DD group. The universal decrease in the abundance of SLRPs in the DD group was associated with an increase in MMPs and a reduction in TIMPs, collagen and LLRP content. CONCLUSIONS: Our study documents the influence of SLRPs in the maintenance of disc health and also the need for future research in using them for disc regeneration. CLINICAL SIGNIFICANCE: The various SLRPs that we identified are all known to have a beneficial influence on ECM integrity and a negative effect on the degenerative process at different stages in the evolution of degeneration. Biglycan, which is abundantly present in a fetus, may be suitable for regenerative therapy, and the other SLRPs like Lumican, Prolargin, Decorin, and Chondroadherin may serve the same purpose and/or as biomarkers.

Part 1: profiling extra cellular matrix core proteome of human fetal nucleus pulposus in search for regenerative targets.

Intervertebral disc degeneration is accompanied by a loss of Extra-cellular matrix (ECM) due to an imbalance in anabolic and catabolic pathways. Identifying ECM proteins with anabolic and/or regenerative potential could be the key to developing regenerative therapies. Since human fetal discs grow and develop rapidly, studying these discs may provide valuable insights on proteins with regenerative potential. This study compares core matrisome of 9 fetal and 7 healthy adult (age 22-79) nucleus pulposus (NP), using a proteomic and bioinformatic approach. Of the 33 upregulated proteins in fetus NP's, 20 of which were involved in ECM assembly pathways: fibromodulin, biglycan, heparan sulfate proteoglycan 2, chondroitin sulfate proteoglycan 4, procollagen C-endopeptidase enhancer and Collagen-type 1a1, 1a2, 6a1, 6a3, 11a1, 11a2, 12a1, 14a1 and 15a1. Moreover, 10 of the upregulated proteins were involved in growth pathways 'PI3L-Akt signaling' and 'regulation of insulin like growth factor transport and uptake.' Thrombospondin 1,3 and 4, tenascin C, matrilin-3, and collagen- type 1a1, 1a2, 6a1, 6a3 and 9a1. Additionally, matrillin-2 and 'Collagen triple helix repeat containing 1' were identified as possible regenerative proteins due to their involvement in 'Regeneration' and 'tissue development' respectively. In conclusion, the consistency of human fetal NP's differs greatly from that of healthy adults. In view of these outcomes, the core matrisome of human fetal discs contains an abundant number of proteins that could potentially show regenerative properties, and their potential should be explored in future machinal experiments.

Proteomic Signatures of Healthy Intervertebral Discs From Organ Donors: A Comparison With Previous Studies on Discs From Scoliosis, Animals, and Trauma.

OBJECTIVE: To catalog and characterize the proteome of normal human intervertebral disc (IVD). METHODS: Nine magnetic resonance imaging (MRI) normal IVDs were harvested from 9 different brain dead yet alive voluntary organ donors and were subjected to electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) acquisition. RESULTS: A total of 1,116 proteins were identified. Functional enrichment analysis tool DAVID ver. 6.8 categorized: extracellular proteins (38%), intracellular (31%), protein-containing complex (13%), organelle (9%), membrane proteins (6%), supramolecular complex (2%), and 1% in the cell junction. Molecular function revealed: binding activity (42%), catalytic activity (31%), regulatory activity (14%), and structural activity (7%). Molecular transducer, transporter, and transcription regulator activity together contributed to 6%. A comparison of the proteins obtained from this study to others in the literature showed a wide variation in content with only 3% of bovine, 5% of murine, 54% of human scoliotic discs, and 10.2% of discs adjacent to lumbar burst fractures common to our study of organ donors. Between proteins reported in scoliosis and lumbar fracture patients, only 13.51% were common, further signifying the contrast amongst the various MRI normal IVD samples. CONCLUSION: The proteome of "healthy" human IVDs has been defined, and our results show that proteomic data on IVDs obtained from scoliosis, fracture patients, and cadavers lack normal physiological conditions and should not be used as biological controls despite normal MRI findings. This questions the validity of previous studies that have used such discs as controls for analyzing the pathomechanisms of disc degeneration.

Human intervertebral discs harbour a unique microbiome and dysbiosis determines health and disease.

BACKGROUND: To document the role of sub-clinical infections in disc disorders and investigate the existence of microbiome in intervertebral discs (IVD). METHODS: Genomic DNA from 24 lumbar IVDs [8-MRI normal discs (ND) from brain dead yet alive organ donors, 8-disc herniation (DH), 8-disc degeneration (DD)] was subjected to 16SrRNA sequencing for profiling the diversity of human disc microbiome in health and disease. The disc microbiome was further compared to established human gut and skin microbiomes. RESULTS: All healthy MRI normal discs from brain dead yet alive organ donors also had a rich bacterial presence. A total of 424 different species (355-ND, 346-DD, and 322-DH) were detected, with 42.75% OTUs being classified at kingdom level, 44% at the phylum level, 22.62% at genus level, and 5.5% at species level. Varying biodiversity and abundance between healthy and diseased discs were documented with protective bacteria being abundant in normal discs, and putative pathogens abundant in DD and DH. Propionibacterium acnes had a similar but lower abundance to other pathogens in all three groups ND (3.07%), DD (3.88%), DH (1.56%). Fifty-eight bacteria were common between gut and IVD microbiomes, 29 between skin and IVD microbiomes, and six common to gut/skin/IVD. CONCLUSION: Our study challenges the hitherto concept of sterility in healthy IVD and documented a microbiome even in MRI normal healthy discs. The varying abundance of bacteria between ND, DD, and DH documents 'dysbiosis' as a possible etiology of DD. Many known pathogens were identified in greater abundance than Propionibacterium acnes, and there was evidence for the presence of the gut/skin/spine microbiome axis.

Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc.

STUDY DESIGN: Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE: To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE: Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS: IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS: A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS: This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

Inflammaging determines health and disease in lumbar discs-evidence from differing proteomic signatures of healthy, aging, and degenerating discs.

BACKGROUND CONTEXT: The true understanding of aging and disc degeneration (DD) is still elusive. MRI has not helped our attempts to understand the health and disease status of the discs as it reflects mainly the end morphologic changes and not the changes at a molecular level. Understanding degeneration at a molecular level through proteomics might allow differentiation from normal aging and also aid in the development of biomarkers for early diagnosis and preventive therapies. PURPOSE: To utilize proteomics to understand the molecular basis of healthy, aging, and degenerating discs and conclusively differentiate normal aging and degeneration. STUDY DESIGN: Proteomic analysis of human intervertebral disc samples. METHODS: L4-L5 disc samples from three groups were acquired and subjected to proteomic analysis. Samples from individuals aged in the second, third, and fourth decades were used to represent young healthy discs (Group A). Those from MRI normal donors aged in the fifth, sixth, and seventh decades represented normal aging (Group B). Five degenerated discs obtained from patients at surgery represented degeneration (Group C). The entire proteome map and alteration in protein expressions were further analyzed using bioinformatics analysis. This was a self-funded project. RESULTS: There were 84 common proteins. Specific proteins numbered 225 in A, 315 in B, and 283 in C. By gene ontology biological process identification, Group A predominated with extracellular matrix organization, cytoskeletal structural and normal metabolic proteins. Group B differed in having additional basal expression of immune response, complement inhibitors, and senescence proteins. Group C was different, with upregulation of proteins associated with oxidative stress response, positive regulators of apoptosis, innate immune response, complement activation and defense response to gram positive bacteria indicating ongoing inflammaging. CONCLUSIONS: Our study documented diverse proteome signatures between the young, aging and degenerating discs. Inflammaging was the main differentiator between normal biological aging and DD. CLINICAL SIGNIFICANCE: Multiple inflammatory molecules unique to DD were identified, allowing the possibility of developing specific biomarkers for early diagnosis and thereby provide evidence-based metrics for preventive measures rather than surgical intervention and also to monitor progress of the disease.

ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis.

STUDY DESIGN: Proteomic and 16S rDNA analysis of disc tissues obtained in vivo. OBJECTIVE: To address the controversy of infection as an aetiology for disc disorders through protein profiling. There is raging controversy over the presence of bacteria in human lumbar discs in vivo, and if they represent contamination or infection. Proteomics can provide valuable insight by identifying proteins signifying bacterial presence and, also host defence response proteins (HDRPs), which will confirm infection. METHODS: 22 discs (15-disc herniations (DH), 5-degenerate (DD), 2-normal in MRI (NM) were harvested intraoperatively and immediately snap frozen. Samples were pooled into three groups and proteins extracted were analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Post identification, data analysis was performed using Uniprotdb, Pantherdb, Proteome discoverer and STRING network. Authentication for bacterial presence was performed by PCR amplification of 16S rDNA. RESULTS: LC-MS/MS analysis using Orbitrap showed 1103 proteins in DH group, compared to 394 in NM and 564 in DD. 73 bacterial specific proteins were identified (56 specific for Propionibacterium acnes; 17 for Staphylococcus epidermidis). In addition, 67 infection-specific HDRPs, unique or upregulated, such as Defensin, Lysozyme, Dermcidin, Cathepsin-G, Prolactin-Induced Protein, and Phospholipase-A2, were identified confirming presence of infection. Species-specific primers for P. acnes exhibited amplicons at 946 bp (16S rDNA) and 515 bp (Lipase) confirming presence of P. acnes in both NM discs, 11 of 15 DH discs, and all five DD discs. Bioinformatic search for protein-protein interactions (STRING) documented 169 proteins with close interactions (protein clustering co-efficient 0.7) between host response and degenerative proteins implying that infection may initiate degradation through Ubiquitin C. CONCLUSION: Our study demonstrates bacterial specific proteins and host defence proteins to infection which strengthen the hypothesis of infection as a possible initiator of disc disease. These results can lead to a paradigm shift in our understanding and management of disc disorders.