Human immunodeficiency virus (HIV) epidemic has undoubtedly increased the incidence of tuberculosis (TB) globally, posing a formidable global health challenge affecting 1.2 million cases. Pulmonary TB assumes utmost significance in the programmatic perspective as it is readily transmissible as well as easily diagnosable. HIV complicates every aspect of pulmonary tuberculosis from diagnosis to treatment, demanding a different approach to effectively tackle both the diseases. In order to control these converging epidemics, it is important to diagnose early, initiate appropriate therapy for both infections, prevent transmission and administer preventive therapy. Liquid culture methods and nucleic acid amplification tests for TB confirmation have replaced conventional solid media, enabling quicker and simultaneous detection of mycobacterium and its drug sensitivity profile Unique problems posed by the syndemic include Acquired rifampicin resistance, drug–drug interactions, malabsorption of drugs and immune reconstitution inflammatory syndrome or paradoxical reaction that complicate dual and concomitant therapy. While the antiretroviral therapy armamentarium is constantly reinforced by discovery of newer and safer drugs every year, only a few drugs for anti tuberculosis treatment have successfully emerged. These include bedaquiline, delamanid and pretomanid which have entered phase III B trials and are also available through conditional access national programmes. The current guidelines by WHO to start Antiretroviral therapy irrespective of CD4+ cell count based on benefits cited by recent trials could go a long way in preventing various complications caused by the deadly duo. This review provides a consolidated gist of the advancements, concepts and updates that have emerged in the management of HIV-associated pulmonary TB for maximizing efficacy, offering latest solutions for tackling drug–drug interactions and remedial measures for immune reconstitution inflammatory syndrome. HIV and tuberculosis (TB) have always been faithful comrades facilitating each other in spreading across the globe. According to recent World Health Organization (WHO) estimates, 9.6 million cases of tuberculosis (TB) occurred all over the world with 12 % (1.2 million) being co-infected with human immunodeficiency virus (HIV); with 37 % of these new TB cases going undiagnosed  [3. B assay] .A study of asymptomatic adults from South Africa, a country with a high prevalence of co-infection found that the rates of positive ELISPOT and ELISA results did not vary significantly by HIV status compared to TST . Due to Blood biomarkers Studies are underway using host RNA gene expression from whole blood that allows for identification of prospective high risk individuals who can potentially progress to active tuberculosis disease  using fluorescent sensors, MB/BacT system (Organon Teknika) using colorimetric sensors, ESP culture system II  using pressure sensors, or redox reagents, such as Alamar blue –22. AmonMolecular techniques Nucleic acid amplification tests (NAAT), are designed to amplify nucleic acid regions specific for M. tuberculosis complex and can be used directly on the clinical samples. NAAT yield rapid results, are highly specific with improved sensitivity compared to smear microscopy. These techniques can detect specific mutations, thus providing information on drug sensitivity as well.3. M.Tb complex and simultaneously identifies drug-resistant strains by detecting the most common single nucleotide polymorphisms associated with resistance  along with add on drugs like pyrazinamide, ethambutol, high dose INH, bedaquiline, delamanid, Amoxycillin clavulanate, para-amino salicylic acid selected preferentially in the order described above . The intEvidence for this regimen originated from the Bangladesh observational cohort study by Van Deun among MDR-TB patients which showed a relapse free cure rate of 84.5 % among 515 patients, but in a virtually HIV-free population . The samPrognosis of MDR-TB in HIV continues to be grave with a death rate of over 50 % once again highlighting the ardent need to reconstitute the fallen immunity by timely ART initiation . Two addXDR-TB or extensively drug resistant defined as MDR-TB (resistance to INH and rifampicin) plus resistance to any fluoroquinolone and one of the second line anti-TB injectable agents  constitutes a formidable medical challenge. Although the absence of rifampicin brings down the occurrence of drug–drug interactions significantly, therapy for XDR-TB is confounded by other adverse reactions like QTC prolongation, anemia, psychiatric effects, nephrotoxicity and gastrointestinal intolerance in addition to increased pill burden . TreatmeBedaquiline (BDQ), one of the newer drugs conditionally approved by the FDA recently, has been a useful adjunct in drug resistant-TB therapy, especially when choice is limited. It is a diarylquinoline derivative that inhibits the mycobacterial ATP synthetase . It inhiBedaquiline concentration is increased with co-administered lopinavir/ritonavir by 22 % due to reduced clearance, while BDQ concentration is reduced by 50 % with efavirenz and these combinations are better avoided. Nevirapine has no interaction with BDQ and can be given safely . Delaman3 suggesting that preservation of adequate immunity could potentially improve TB outcomes in HIV emphasizing the need for early ART initiation [In the pre-HAART era, a number of studies have shown considerably lower cure rates, higher mortality and recurrence rates of TB after standard ATT in HIV co-infected patients compared to sero-negative individuals –117. Theitiation . Treatmeitiation  and the itiation  to recomitiation . Timely itiation , 120. Puitiation . A studyitiation .Tenofovir, emtricitabine/lamivudine along with efavirenz as a single pill once a day is most ideal . That efRecently discovered drugs in the pipeline include tenofovir alafenamide (GS 7340), a prodrug of tenofovir, that gets converted at the site of lymphoid involvement and liver making it more potent, with greater tissue infiltration at a dosage ten times less than the conventional tenofovir and is currently evaluated in phase III trials in a fixed dose combination. There was significant improvement in estimated glomerular filtration rate, bone density and reduced proteinuria compared to conventional tenofovir disoproxil fumarate . Doluteg3 need to start ART at the earliest with counselling on early recognition of signs and symptoms of IRIS and toxicity, a permissible delay of ART initiation among patients with higher CD4 up to 12 weeks after starting ATT helps in reducing drug interactions between rifampicin and NNRTIs, cumulative drug toxicities , pill burden and the incidence of immune reconstitution inflammatory syndrome without any compromise on survival or predisposition to newer opportunistic infections [Evidences from the START  and the fections .The CAMELIA study enrolling 661 patients with a median follow-up of 25 months reflected similar findings that were deduced from patients in the lower range of CD4 in the SAPIT and STRIDES study. Early ART initiation by 2 weeks post ATT reduced mortality significantly compared to late initiation of ART at 8 weeks post ATT . However, the risk of IRIS was 2.5 times more in the early ART group .Current WHO and ART guidelines in India also recommend ART between 2 and 8 weeks of starting ATT on similar principles , 142.Adverse reactions occur more often among HIV-infected patients with TB on concurrent medication than among TB without HIV (serious ADR—27 vs 13 %), occurring mostly in the first 2 months of treatment , 144. HeIsolated hyperbilirubinemia is very common with use of atazanavir but is more of a cosmetic disfigurement that may stigmatise the patient. Zinc sulphate supplementation may be useful in management of ATV-related HBR in selected patients .3 with compromise on survival, . However, among patients with a CD4 of less than or equal to 50 cells/mm, immediate initiation of ART is the rule with proper counselling for IRIS and close monitoring for cumulative/additive toxicity that will help to maintain adherenceThe ideal co-administered ART regimen with ATT is a single pill of tenofovir (300 mg) along with emtricitabine/lamivudine (300 mg) and efavirenz (600 mg) initiated between 2 and 12 weeks after ATT initiation, provided the patients’ CD4 cell count is above 50 cells/mmTuberculosis immune reconstitution inflammatory syndrome (TB-IRIS) is the paradoxical worsening of symptoms and signs of TB after starting ART (rarely with ATT itself), despite a favorable immunological recovery and effective virological suppression . IncidenThe most consistent risk factors are very low levels of CD4 cell count, CD4/CD8 ratio, hemoglobin, weight, presence of disseminated disease, shorter ATT-ART time interval, extra pulmonary foci and other opportunistic infections at the time of ART initiation –152. IntFunctional restoration of immunocompetent cells (CD4) causing a cytokine outburst with an overriding Th1 over Th2 response, apart from increase in number and their redistribution to the site of the lesion, is the primary mechanism of IRIS causation . The majFever with rigor or chills  is the commonest and consistent symptom of TB-IRIS, with lymph node enlargement being the commonest manifestation . SymptomIt is important for physicians to remember that the onset of this syndrome is linked temporally to ART initiation, ART substitution (from I line to II line suppressing viremia) and ART interruption followed by re-initiation . A stronWhen IRIS occurs at an extrapulmonary location, appropriate investigation can be performed and tissue specimen obtained wherever possible, and sent for bacteriological staining and culture in addition to routine histopathology. A negative culture that succeeds a pre-existing or baseline positive culture (that was sensitive to specific drug therapy) confirms the diagnosis of IRIS straightaway. However, this criteria of culture negativity cannot be applied for diagnosing unmasking IRIS or in paradoxical IRIS when the ATT-IRIS interval is too short. Hypercalcemia is a unique accompaniment of TB-IRIS, attributed to the calcium deposition through increased secretions of 1.25 dihydoxy cholecalciferol by activated macrophages and CD4 T cells .Drug resistant TB is the closest mimic that requires to be ruled out before steroids are administered as they form the cornerstone of IRIS therapy currently . The phe3, the initiation of ART can be delayed up to 8 weeks with close monitoring that reduces toxicity and IRIS but does not compromise on survival [Intensive screening for TB and other opportunistic infections along with INH and cotrimaxozole prophylaxis reduces IRIS incidence , 167–169Anti-inflammatory drugs especially steroids form the backbone therapy for TB-IRIS, even though non-steroidal anti-inflammatory agents could prove to be a useful initial therapy for milder and localized cases of IRIS , 170. A HIV-associated pulmonary TB mandates a committed approach that encompasses both effective as well as enduring therapy originating from newer drug combinations, evolving ideas and emerging concepts from clinical trials globally, which if implemented in a proper and coordinated manner could not only save millions of lives but also offer a better quality of life to patients suffering from this coinfection.


Early onset infection (EOI) in preterm infants <32 weeks gestational age (GA) is associated with a high mortality rate and the development of severe acute and long-term complications. The pathophysiology of EOI is not fully understood and clinical and laboratory signs of early onset infections in this patient cohort are often not conclusive. Thus, the aim of this study was to identify signatures characterizing preterm infants with EOI by using genome-wide gene expression (GWGE) analyses from umbilical arterial blood of preterm infants. This prospective cohort study was conducted in preterm infants <32 weeks GA. GWGE analyses using CodeLink human microarrays were performed from umbilical arterial blood of preterm infants with and without EOI. GWGE analyses revealed differential expression of 292 genes in preterm infants with EOI as compared to infants without EOI. Infants with EOI could be further differentiated into two subclasses and were distinguished by the magnitude of the expression of genes involved in both neutrophil and T cell activation. A hallmark activity for both subclasses of EOI was a common suppression of genes involved in natural killer (NK) cell function, which was independent from NK cell numbers. Significant results were recapitulated in an independent validation cohort. Gene expression profiling may enable early and more precise diagnosis of EOI in preterm infants.Gene expression (GE) profiling at birth characterizes preterm infants with EOI.GE analysis indicates dysregulation of NK cell activity.NK cell activity at birth may be a useful marker to improve early diagnosis of EOI.The online version of this article (doi:10.1007/s00109-016-1466-4) contains supplementary material, which is available to authorized users. Preterm birth (PTB) remains a major problem in perinatal medicine and accounts for 75 % of the perinatal mortality and 50 % of the perinatal morbidity . Early oIn this prospective cohort study, we applied gene expression profiling from umbilical arterial blood of preterm infants <32 weeks of gestational age to provide comprehensive biological information and identify biological pathways relevant for the development of EOI in order to enable the identification of early diagnostic markers.Newborn infants <32 weeks gestational age (GA) were prospectively included in this study.Depending on the availability of diagnostic criteria at birth, infants were pro- or retrospectively excluded when one of the following diagnoses was present: premature rupture of membranes ≥3 weeks prior to birth leading to oligo- or anhydramnios, severe congenital malformations, diagnosis of severe metabolic disorders, prepartum treatment of the mother with cytostatic or immunosuppressive medication other than for lung maturation, and postnatal treatment with corticosteroids in a dose ≥1 mg/kg body weight. Analysis of C-reactive protein (CRP), whole white blood count (WBC), and microbiological examination of blood cultures, swabs, urine, and stool samples were carried out in the first 72 h of life. Patients were clinically re-evaluated in short intervals and continuously monitored for vital signs, i.e., heart rate, blood pressure, microcirculation, and breathing pattern.P values were calculated using Fisher’s exact test for qualitative parameters and Wilcoxon U test for quantitative parameters.Patients were allocated to one of the two following groups: (I) EOI and (II) non-EOI (no signs of infection in the first 72 h of life). EOI was diagnosed if the infant showed both a pathologic ratio of immature to total granulocytes  and/or a pathologic white blood cell count paralleled or followed by an increase in CRP ≥ 6 mg/l in the first 72 h of life –14. ThesThe comprehensive monitoring of the perinatal course is further defined in Supplemental Materials and Methods. The study has been approved by the legal ethical committee .Blood for standard laboratory analyses including WBC and blood samples for transcriptome analyses were obtained from an indwelling umbilical artery catheter immediately after birth. WBCs were repeated upon clinical indication in the later postnatal course as a possible indicator of developing  infections. Details of the microarray experiments and data analysis can be found in Supplemental Materials and Methods.Briefly, 250–300 μl of umbilical arterial blood was obtained immediately after birth from an indwelling umbilical artery catheter and directly transferred to 750–900 μl of the PAXgene Blood RNA System . RNA isolation was performed according to the manufacturer’s recommendations . RNA was hybridized on CodeLink UniSet Human 10 K Bioarrays  using the CodeLink Expression Assay Kit  and samples processed using CodeLink Expression Software V4.1 .In order to account for confounding effects of WBCs on the transcriptome pattern, we evaluated differences between EOI and non-EOI preterm infants in their differential WBCs at birth by using the Wilcoxon rank-sum test. Missing data from WBC counts resulting from technical problems or limited sample size were imputed based on a model using a regularized iterative principal component analysis algorithm  taking it statistics in studies with small sample sizes, this method was chosen for primary analysis [The gene expression dataset was normalized using quantile normalization in R . For staanalysis . A falseTo support the results derived from Rank Products and to account for potential hidden confounders affecting gene expression analysis, the data were first corrected for variables that significantly correlated with structural differences between the groups, i.e., the EOI and non-EOI cohort Table . These cSubsequent statistical analyses were conducted using the software tools “dChip” for hierarchical clustering, “DAVID” for gene ontology, and functional annotation clustering following the software recommendations .PCA as a mathematical vector space transformation allows for the reduction of multidimensional data sets to lower dimensions (principle components) accounting for the variability of the data set . PCA wasP value derived from analysis of variances (ANOVA), pairwise Student’s t test with Benjamini-Hochberg correction for group A genes as well as the non-parametric Kruskal-Wallis test and the non-parametric pairwise Wilcoxon ranks sum test with Benjamini-Hochberg correction for group B genes. The complete data set is available at the Gene Expression Omnibus (GEO) database under the accession number GSE5760.To compare disease-dependent differences in the magnitude of gene expression in preterm infants, we used an aggregate measure designated DLI as described previously . The DLIn = 20) < 32 weeks of GA included and characterized exactly as described above. Hematopoietic cell staining was performed with a PeCy5.5-labeled mouse IgG1 anti-human CD45 antibody from Invitrogen  and separation of leukocyte fractions by simultaneously using the following antibodies: PB-labeled mouse IgG1 anti-human CD3, Alx700-labeled mouse IgG1 anti-human CD19 and APC-labeled mouse IgG1 anti-human NKp46 from BD Biosciences , APC-Alx750-labeled mouse IgG2a anti-human CD14 from Invitrogen, FITC-labeled mouse IgG 1 anti-human CD15 from Miltenyi Biotec , and corresponding isotype controls. Flow cytometry was performed on a LSRII Flow Cytometer (BD) using the proper controls to set gates and analyzed with FlowJo8.7.1 software. Dead cells were excluded using propidium iodide labeling and doublets by gating on single cells (FSC-H to FSC-W channel).Umbilical arterial blood specimens for measurement of natural killer (NK) cell number were collected from a separate cohort of preterm infants  .Briefly, TaqMan quantitative real-time (RT)-PCR was performed for 10 human genes deriving from the microarray results  and three housekeeping genes glucose-6-phosphate dehydrogenase (G6PD), succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA), and phosphoglycerate kinase 1 (PGK1) as internal controls for normalization. To test whether the microarray results could be replicated, the gene expression of the same 10 genes was investigated in a validation cohort consisting of 43 new preterm infants by RT-PCR as described in Supplemental Materials and Methods. The patient characteristics of the validation cohort  are given in Supplemental Table Thirty very preterm infants were prospectively enrolled in this study. Twenty-four of 30 samples met the high RNA quality criteria and were further processed for microarray analyses; of these, 16 were retrospectively allocated to the EOI cohort based on the presence of clinical parameters for EOI in the first 72 h of life. Eight infants without EOI were assigned to the control group (non-EOI). The characteristics of the patient cohorts are shown in Table Using a rank-based statistics, comparison of gene expression of infants with and without EOI revealed 292 differentially regulated genes FDR ≤ 0.1). Of these, 219 genes had significantly higher gene expression levels (upregulated genes) in infants with EOI, while 73 genes had significantly lower expression levels (downregulated genes)  = 5.5), chemotaxis (ES 1.9), and leucocyte activation (ES 1.8) as well as in catabolic processes   identified by Rank Products analysis separated the cohort of preterm infants into clusters 1 and 2  and non-EOI (P = 1e-05). Hence, the subclass EOI** activated genes involved in neutrophil activation, T cell proliferation, hypoxia-induced signaling, and carbohydrate metabolism on a higher level than subclass EOI*. For group B genes, no significant differences occurred in the expression level between the subclasses EOI* and EOI** . But both EOI* and EOI** showed a significantly lower DLI for group B genes compared to non-EOI  indicating decreased expression of genes involved in NK cell activation. Clinical variables characterizing EOI* and EOI** subclasses are given in Table The two subclasses EOI* and EOI** were distinguished by the expression of two groups of genes, namely group A and B genes Fig. : Group Aned Fig. : EOI** hNeutrophils play a pivotal role in the innate immune response, as they migrate to the site of infection and help limit microbial infections. Increased activity of neutrophils in infants with EOI, especially in subclass EOI**, is indicated by overexpression of group A genes involved in phagocytotic activity, granula secretion, and respiratory burst of neutrophils as depicted in the interaction network in Fig. NK cells constitute a component of the innate immune system in combating intracellular pathogens and activating and modulating the adaptive immune response. Activation of NK cells is regulated by the expression of a variety of receptors.In EOI, the NK cell activating killer cell lectin-like receptors (KLRs) such as KLR subfamily B member 1 (KLRB1), KLR subfamily C member 2 (KLRC2), and KLR subfamily D member 1 (KLRD1) were downregulated Tables  and 4. GTwo transcription factors, GATA-binding protein 3 (GATA3) and CREB-binding protein (CREBBP), which are known to regulate the expression of KLRs were downregulated. GATA3 is a transcription factor preferentially expressed in NK and T cells that plays an important role in the early phase of NK cell development. Its activity is crucial for the diversification of the NK cell receptor repertoire and interferon γ (IFNG) production and thus pivotal for an effective NK cell response to viruses and bacteria. The downregulation of GATA3 and CREBBP in infants with EOI could explain the reduced gene expression of NK cell receptors in these patients as depicted in the interaction network in Fig. The measurement of NK cell counts in a cohort of preterm infants with and without EOI showed no significant difference in NK cell number between the two groups  each). This result suggested that the downregulation of NK cell activating genes as seen in EOI was not related to the NK cell count.R2 = 0.88) . The overall correspondence between mRNA levels measured by microarrays and by RT-PCR analyses was high  of the gene expression between the exploration and the validation cohort of preterm infants indicating reproducibility of the results  in an independent cohort. Overall, these findings suggest that a qualitative NK cell impairment, measured by gene expression profiling, may be inherent to EOI and provide a superior diagnostic tool for the early detection of EOI.We identified two subclasses within the EOI cohort (EOI* and EOI**) characterized by increased neutrophil activation, T cell proliferation, hypoxia-induced signaling, and carbohydrate metabolism in EOI** preterm infants. Clinically, the group of infants classified as EOI* showed more infection-associated complications when compared with EOI**. In the light of impaired NK cell activation in both EOI subclasses, increased activation of neutrophils, T cell proliferation, hypoxia-induced signaling, and carbohydrate metabolism might indicate protective mechanisms helping to prevent complications of EOI in preterm infants when classified as EOI**.To conclude, the results of this study indicate that (1) transcriptome patterns derived from umbilical arterial blood enable the discrimination of preterm infants with and without EOI and that (2) the group of preterm infants with EOI can be divided in two subclasses showing a common phenomenon of dysregulated NK cell activation but differing in the activation of neutrophils, T cell proliferation, hypoxia-induced signaling, and carbohydrate metabolism.We propose that the addition of NK cell activity into the standard diagnostic repertoire for critically ill (preterm) neonates could be a useful complement to current laboratory diagnostics in order to improve early diagnosis of EOI.ESM 1(PDF 464 kb)


Osteoarthritis is a degenerative joint disease and a world-wide healthcare burden. Characterized by cartilage degradation, subchondral bone thickening and osteophyte formation, osteoarthritis inflicts much pain and suffering, for which there are currently no disease-modifying treatments available. Mouse models of osteoarthritis are proving critical in advancing our understanding of the underpinning molecular mechanisms. The STR/ort mouse is a well-recognized model which develops a natural form of osteoarthritis very similar to the human disease. In this Review we discuss the use of the STR/ort mouse in understanding this multifactorial disease with an emphasis on recent advances in its genetics and its bone, endochondral and immune phenotypes. Animal models are a vital tool for the study of osteoarthritis (OA). In particular they provide scope to examine the early aetiological processes where equivalent human samples are difficult to obtain, and they remain necessary in developing and testing new treatmentset al., 2011 and Mason et al., 2001Numerous mouse strains develop OA with advancing age, offering proof that genetic predisposition or susceptibility is an important factor. They, nonetheless, develop OA with differing incidencesThe STR/1N strain was first isolated by Strong (1951) during an extensive selective-breeding programme designed to identify traits for resistance to tumour induction at the site of injected carcinogensSTR/ort OA susceptibility genetics are uncertain and their phenotype is better characterised. STR/ort mice develop OA in knee, ankle, elbow and temporomandibular jointsOnly a few studies have explored OA-associated pain in this model. Increased basal and evoked prostaglandin E2 release has been observed in knee preparations from 18-week-old STR/1N mice, which may enhance nociceptor sensitivity and chronic OA painet al. (2009) to propose this as the cause of STR/ort OAet al., reported an incidence of patella dislocation in only 22% of STR/ort mice, reinforcing this with radiological studies showing not all mice with OA had displaced patellae, demonstrating that this cannot be a primary eventSeveral lines of evidence suggest that OA development in STR/ort mice involves a mechanical contribution. Indeed, Walton (1977) showed a close relationship between AC lesions and medial patella dislocation, medial collateral ligament calcification/ossification and lateral subluxation of the femurDevelopment of ankle OA in STR/ort mice has similarly been linked with calcaneal dislocation, with elevation progressively more pronounced in ageing mice where it eventually became parallel to the distal tibia (unpublished). The cause for this ankle deformity is unknown, but suggests a possible defect in maintaining joint stability with spontaneous subluxation and later severe disruption of navicular and tarsal bones in male STR/ort miceThe anterior cruciate ligaments of STR/ort mice also exhibit lower ultimate strength, increased collagen metabolism and matrix metalloproteinase (MMP) activity compared to CBA mice at 20–30 weeksWe have recently explored the importance of mechanical loading in lesion induction and pathological OA progression in STR/ort mice using a non-invasive knee joint trauma modelet al. (2008) confirmed Mendelian OA inheritance and concluded that its polygenicity means that the allelic subset involved in OA predisposition unlikely reaches significance in any single-Quantitative Trait Loci (QTL) analysisNumerous genetic and microarray analyses have been performed in STR/ort mice. Studies by Jaeger dickkopf 4 (Dkk4), secreted frizzled related protein 1 (Sfrp1) and fibroblast growth factor 1 (Fgfr1), with 23 polymorphic changes in the Sfrp1 gene identified in STR/ort in comparison to C57BL/6 mice34Revisiting chromosome 8 and fine mapping of the OA-QTL revealed Wnt-related genes associated with altered chondrogenesis, including Our recent studies also support an epigenetic contribution to STR/ort OA (unpublished). Careful joint OA scoring in individually-tracked male mice at 26 weeks of age found an important maternal influence, with a significant correlation between OA severity and maternal litter parity, and to a lesser extent with maternal age. Interestingly, no correlations were found with litter size nor with the time between litters, which suggests an important maternal influence during embryo development that underpins OA severity in STR/ort mice.STR/ort mouse AC undergoes structural demise similar to human OA. Morphologically, STR/ort mouse AC is thicker than in CBA, and whilst STR/ort chondrocytes express a normal spectrum of PGs and collagens, there are early changes in AC matrix integrity and chondrocyte phenotype and functionSubtle changes in STR/ort AC matrix composition, and in glycosaminoglycan PG content in particular, is observed in STR/ort male mice in comparison to age-matched CBA miceCol10a1 mRNA significantly increasing in STR/ort AC compared to non-OA ACAlternative approaches to redress the lack of therapeutics in OA are also now emerging. Evidence suggests that the normally ‘stable’ AC chondrocyte adopts a more ‘transient’ phenotype similar to growth plate chondrocytes in OATUNEL-positive chondrocytes are observed around OA lesions in STR/ort AC, indicating apoptosis and chondrocyte transiency, correlating with OA progressionIt appears that STR/ort mouse AC chondrocytes also have an altered metabolic phenotype, with those in OA–prone regions having low lactate and succinate dehydrogenase activities prior to OA onsetDiscovery of the molecular determinants of OA in STR/ort mice will undoubtedly shed light upon OA aetiopathogenesis in other species and as such, have been well investigated. Our transcriptional profiling of STR/ort AC at various ages revealed differential regulation of many signalling pathwaysMajor pathways such as those provoked by the transforming growth factor (TGF) β superfamily have already been investigated. Expression of TGF-β1 was indeed elevated during OA development in STR/ort compared to age-matched CBA miceEffective regulation of the Wnt pathway is proving critical in OA joint pathologyOxidative stress has been shown to contribute to OA progression. In STR/ort mice, oxidative stress  and the collagen type II degradation (CTX-II) biomarker levels are both higher than in CBA mice prior to OA onset, suggesting that oxidative stress is linked to AC type II collagen degradationSubchondral bone thickening (sclerosis) in OA joints, although often considered secondary, is nonetheless one of the earliest detectable changes and we have observed sclerosis in STR/ort joints with OA onset and developmentSTR/ort mice also have a generalised high bone mass phenotype in cortical and trabecular compartments too (vs C57Bl/6), associated with elevated osteoblast numbers and activity, and impaired osteoclast functionClues to these changing osteochondral relationships in STR/ort mice might be evident in the endochondral ossification required for long bone growth. We recently observed accelerated growth dynamics in comparison to CBA mice with STR/ort mice exhibiting (1) an acceleration in body weight gain and tibia length at sexual maturity (2) Col10a1 and MMP13 expression widely dispersed into the growth plate proliferative zone (3) differences in growth plate maturation zone sizes (4) a dramatic acceleration of growth plate closure with bone bridge formation particularly clustered to medial areas where OA later predominateset al. (1997) found elevated IL-1β levels in AC chondrocytes of STR/ort mice at all agesAlthough OA is not primarily a classic inflammatory disorder, it is accepted that cytokines play an important pathogenic roleTogether these studies suggest that STR/ort OA has an important inflammatory component and this is further cemented by observations of spleen and lymph nodes abnormalitiesSTR/ort mice exhibit increased AC expression of beta-defensins 3 and 4, broad-spectrum antimicrobial components of innate immunity. These findings offer a link between host defence mechanisms and inflammation with AC tissue-remodelling processesObesity is now a recognised OA risk factor and it has been reported that the parent STR/1N strain exhibits higher blood cholesterol and phospholipids compared to DBA/2JN and A/LN strainsMore recently, microarrays on STR/ort AC/subchondral bone described upregulation of 331 genes related to development and function of connective tissues, and 290 genes downregulated linked to lipid metabolism, in particular genes that were directly interacting with peroxisome proliferator-activated receptor (PPAR) alpha/PPARgammaThe STR/ort mouse is an excellent model of spontaneous osteoarthritis with disease pathology starting early in life and showing many similar characteristics akin to human primary OA. The phenotype of the STR/ort mouse is well characterised with pathology observed in the knee, elbow, ankle and temporomandibular joints of male mice; this highlights the sexual dimorphism in this strain, whereby females show higher bone mass and protection from reproducible AC degeneration. The highly defined and reproducible disease pathology of the STR/ort mouse has, to date, offered the unique opportunity to identify the pathological role that key determinants of the AC and subchondral bone phenotypes play in spontaneous OA development, highlighting the attractiveness of this murine model in exploring the aetiopathogenesis of spontaneous OA. Whether research in the OA field should focus upon pre-clinical studies or on clinical studies in man is still a matter of debate, and has been elegantly debated in a recent editorial by Hunter and LittleAll authors were fully involved in the preparation of this manuscript and approved the final version.The authors have no potential conflicts of interest to disclose.Arthritis Research UK for funding .The authors wish to acknowledge


The performances of the models were validated on an independent validation set. The mean of the area under the receiver operating characteristic (ROC) curve for the classifiers was 0.973 with a sensitivity of 0.999 and specificity of 0.959. Finally, we identified CF specific clones that correlate highly with sweat chloride test, BMI, and FEV1% predicted values. For the first time, we show that CF specific serological biomarkers can be identified through immunocreenings of a T7 phage display library with high accuracy, which may have utility in development of molecular therapy.Cystic fibrosis (CF) is an autosomal recessive disorder affecting the cystic fibrosis transmembrane conductance regulator (CFTR). CF is characterized by repeated lung infections leading to respiratory failure. Using a high-throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This library was biopanned to obtain 1070 potential antigens. A microarray platform was constructed and immunoscreened with sera from healthy (n = 49), lung cancer (LC) (n = 31) and CF (n = 31) subjects. We built 1,000 naïve Bayes models on the training sets. We selected the top 20 frequently significant clones ranked with student  There is a tremendous need for developing reliable serum based biomarkers in various diseases including proliferative disorder such as cancer, inflammatory diseases and infections as well as genetic disorders such as cystic fibrosis (CF).1. Currently, there are more than 1300 various mutations in CFTR gene that is known to cause the CF phenotype. The CF phenotype is characterized by chronic bacterial airway infections, neutrophilic inflammation with mucus in airways, progressive bronchiectasis and advanced cystic fibrosis lung disease. Mutations in the CFTR gene affect the epithelial innate immune function in the lungs, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pulmonary pathogens2. Bacterial infections in CF are characterized by organisms that have substantial genetic flexibility to evade phagocytic clearance and develop resistance to multiple antibiotics2. Repeated or chronic microbial infections are thought to be the major contributor to excessive inflammation leading to CF lung damage. In addition to chronic lung infections, CF subjects may exhibit exocrine pancreatic insufficiency, diabetes mellitus, and sexual organ dysfunction.Cystic fibrosis is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR)5. Various proteins and protein degradation products have been explored as candidate biomarkers for clinical outcome, such as neutrophil elastase, IL-86, and degradation products of lung surfactant protein SP-A10. A variety of proteomic approaches exploited antigenic biomarkers that could provide candidates for the diagnosis of infection, prognostic indicators or vaccine development. Pedersen et. al. used antibodies from CF patients to probe a protein array of body fluids prepared by two-dimensional gel electrophoresis for antigenic biomarker detection in Pseudomonas aeruginosa5. Others identified the outer membrane protein OprL as a seromarker for the initial diagnosis of Pseudomonas aeruginosa infection in CF patients11.Circulating autoantibodies and autoantigens in CF sera have been widely reported, yet their significance is unknown13. Because the CSL represents a segment of the human lung microbiome, we hypothesize that it contains potential antigens relevant to CF. To test this, we immunoscreened our microarray platform with sera form healthy controls, CF and lung cancer patients using the power of antibody recognition present in human sera to discover potential serological biomarkers in CF.Recently, we developed a heterologous cDNA library derived from bronchoalveolar cells (BAL) and total white blood cells (WBC) from sarcoidosis patients and combined it with cultured human monocytes and embryonic lung fibroblasts cDNA libraries to build a complex sarcoidosis library (CSL)12. A microarray platform was constructed and immunoscreened with 111 sera  of the lungs. The demographics of the study subjects are shown in Table t-test on 1,000 training sets (FDR < 0.01) between CF vs. healthy controls samples. A total of 599 clones appeared significant at least once. We calculated the frequency of each significant clone and ranked the top 20 clones according to their significance and frequency. Furthermore, we performed an unsupervised PCA for all 1070 clones with data from 111 study subject sera. As shown in Fig. A panel of potential antigens was randomly selected from two highly enriched pools of T7 phage cDNA libraries through biopanning of the CSL libraryNext, we performed unsupervised hierarchical clustering with all 1070 clones on 111 samples. We observed that the magenta cluster has a mix of samples and lacks specific sub-clusters of CF samples Fig. . In contNext, we applied the classifier model and calculated the AUC values on accumulating numbers of clones (see method section) on test and validation sets. Figure 12. Protein blast using algorithms of the BLAST program were applied to identify the highest homology to identified peptides. Additionally, we compared these results with corresponding nucleotide sequences using nucleotide blast and determined the predicted amino acid in frame with phage T7 10 B gene capsid proteins. Among 20 clones four CF reactive antigens comprise relatively large peptides, while 16 CF antigens are coded by the inserted gene fragments leading to out-of-frame-peptides, hereby meeting the definition of mimotopes14 (Table 15. Full length of peptides and genes of the top 20 CF clones are shown in Supplementary Table 16. We found highest spearman correlation (r = −0.54) between sweat chloride values and the clone p51_BP3_113 (GEM_5047)  between BMI and the P51_BP3_47 clone (dnaJ homolog) . After obtaining the sequences of clones, Expasy program was used to translate the cDNA sequences to protein sequences47) Fig. . By comb47) Fig. . BMI is og) Fig. . By combog) Fig. . Additioog) Fig. . The corog) Fig. . Table 318. Most of these methods led to the discovery of a series of markers or expression signatures but failed to be useful in clinical practice18. In view of this background, we applied a novel high throughput technology to overcome the current gap by constructing phage-protein microarrays in which peptides were derived from a unique sarcoidosis cDNA library and expressed as a phage fusion protein. Through immunoscreening and rigorous statistical analysis, we identified 20 highly significant CF clones as biomarkers that are able to discriminate between CF and healthy controls as well as lung cancer sera. One important issue in biomarker discovery is the validation of biomarkers and sample selection. To overcome this issue, we randomly assigned samples into 1000 training sets instead of using one training set. Then, we compared the healthy controls and CF samples for each pair of such random sets. The ranking of the top 20 clones was based on the significance and frequency of each clone (how many times each clone appears significant at FDR < 0.01).CF is characterized by a self-perpetuating cycle of airway obstruction, chronic bacterial infection, and vigorous inflammation that results in bronchiectasis, progressive obstructive lung disease, and marked shortening of life expectancy. Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with F508del homozygous CF demonstrate significant variability in severity of pulmonary disease and infection. Non-invasive serological biomarkers that can aid to monitor disease progression or evaluate response to therapy would be extremely valuable. Several groups attempted to identify specific biomarkers to predict inflammation in CF using various biofluids such as sputum, BAL and serum20. Additionally, subjects with smoking related chronic obstructive lung disease (COPD) can develop a similar clinical phenotype with recurrent respiratory infections, mucus inspissation and airway obstruction that is attributed to acquired CFTR deficiency21. In unsupervised HC, we have seen few false positive classification that might have been due to the selection of control groups , who had a significant smoking history. Interestingly, these subjects had more than 50 pack years of smoking history. Because it is known that cigarette smoking affects bacterial clearance22, we speculate that long-term cigarette smoking in these subjects might have led to a similar immunoreactivity to our microarray platform as CF individuals. Therefore, it is probable that if we choose a younger non-smoker group as control subjects, we would have no false positive classification.Environmental stresses including cigarette smoking, hypoxia, and chronic inflammation have also been implicated in reduced CFTR function23. Another epitope was DnaJ homologue (Hdj)-1/heat shock protein (Hsp) 40, a protein chaperon, which along with its co-chaperone Hsp70 regulates protein folding and trafficking in the endoplasmatic reticulum (ER) and facilitates degradation of misfolded proteins24. It has been shown that Hsp40 and Hsp70 facilitate CFTR assembly25. We found DnaJ homologue was increased in sera of CF patients and had a negative correlation with BMI of CF subjects. Another epitope (Thioredoxin like protein) was decreased in CF patients. Studies have shown that excessive neutrophil elastase activity in the airways of pediatric and adult CF patients resulted in lung damage28. Disruption of neutrophil elastase activity by adding exogenous thioredoxin or dihydrolipoic acid in the sputum of CF patients reduced the neutrophil elastase activity29. Another in-frame epitope with relevance to FEV1% predicted was Thymosin β-4 (TMSB4X). In vitro addition of Thymosin β-4 in the sputum of CF patients decreases the sputum cohesivity by depolymerizing actin30.Furthermore, we sequenced the top 20 discriminating antigens for CF and identified homologies in a public database. The range length of identified peptides for CF antigens was between 8–213 amino acids (AA). Among the 20 CF specific phage peptides, five out-of-frame peptides and one epitope were increased in sera of CF patients. One epitope (HLA-DR) was three times randomly selected , suggesting the importance of HLA-DR in pathology of CF. Recently, studies have demonstrated that the transcript levels of HLA-DR and HLA-DQ are reduced in CF patients14. Although the significance of mimotopes is not clear, it has been shown that some out-of-frame peptides can be immunogenic and can activate MHC class I molecules31. Due to smaller peptide sequences of mimotopes, they may have homology with diverse proteins. Prior studies using similar techniques have identified out-of-frame peptides32. We identified two sequenced peptides (narX and barA_4) with similarity to histidine kinases that belong to a large family of membrane-spanning proteins found in many prokaryotes and some eukaryotes. This gene controls the bacterial virulence, growth and biofilm formation in CF patients33. Similarly, IgG response to Burkholderia capacia 80-kDa outer membrane protein has been shown to be significantly higher in patients with CF34. Interestingly, when we explored the correlation of biomarkers with sweat chloride values, we found a good correlation with the outer membrane porin35. Another significant biomarker detected is beta-lactamase. Several studies have shown association between the development of resistance to beta-lactam antibiotics and high beta-lactamase production in CF patients36.Among the 20 sequenced CF specific phage peptides we identified 16 antigens with relatively short out-of-frame peptides meeting the criteria as mimotopes (mimetic sequence of a true epitope)38. One in-vitro study showed that Pseudomonas aeruginosa toxin regulates TetR family transcriptional regulator and hence regulates CFTR expression through transcriptional repression37. Interestingly, TetR is involved in the regulation of antibiotic resistance and controls the expression of membrane-associated proteins that are involved in antibiotic resistance39. Through immunoscreening, we identified decreased NADPH dehydrogenase subunit I. Similarly, studies have shown that mitochondrial complex I activity is reduced in cells with impaired cystic fibrosis transmembrane conductance regulator40. CFTR chloride channels belong to the superfamily of ABC transporter ATPases41. Interestingly, we identified reduced ABC transporter substrate binding protein expression in CF patients. The ABC transporters are widespread in prokaryotes and eukaryotes containing nucleotide-binding domains (NBD) and two transmembrane domains (TMDs). ATP hydrolysis on the NBD drives conformational changes in the TMD, resulting in alternating access from inside and outside of the cell for unidirectional transport across the lipid bilayer42.Among 16 mimotopes, we found eight with decreased expression in CF patients Table . Interes13. The phage display technology and immunoscreening has utilities not only in identifying of diagnostic biomarkers, but also may enable us to develop a novel targeted therapy utilizing the peptide sequences (mimotopes) as vehicles to deliver specific drugs. For instance, among highly significant clones, we found a sequence peptide homologous to histidine kinase (narX) with high specificity and sensitivity. Bacterial histidine kinases are promising targets for the development of antibacterial therapy. Currently efforts have been made to identify specific compounds targeting the inhibition of histidine kinase as antibacterial therapy43. Additionally, this technology might enable us to discover unknown epitopes targeting specific bacterial antigens leading to immunogenicity and antibody production in CF subjects, as well as providing us with a better understanding of host immune defenses in CF subjects. Furthermore, this microarray platform can be hybridized to detect IgA in sera or saliva of CF patients that may have clinical values.To our knowledge no previous study used phage display technology to detect CF serum biomarkers. We detected novel antigens for CF using a heterologous library derived from sarcoidosis subjects. Lungs are highly exposed to numerous bacteria and our library is predominantly derived from sarcoidosis BAL cells and WBCs containing diverse immune cells, including macrophages that were exposed to various pathogens. Hence, we postulate that the CSL represents a segment of the lung microbiome containing diverse antigens including CF specific antigens, sarcoidosis and TB specific antigens44. The present study describes a novel approach to identify CF biomarkers. Further studies with a larger cohort group of patients and/or longitudinal studies are needed to investigate the role of these antigens in CF, their mechanism of action and their utilities in drug design and monitoring of therapy.In summary, we have developed a novel T7 phage display library derived from BALs and leukocytes of patients with sarcoidosis that displays a significant segment of the potential antigens that can recognize IgG antibodies in CF sera with high accuracy. Furthermore, we have identified a set of CF clones that highly correlate with clinical measures such as, sweat chloride values, BMI and FEV1. Microarray and immunoscreening has a value in clinical practice in antibody detection as it is non-invasive and requiring a minimal amount of blood. The identified sequences can be used to develop peptide/protein-coated magnetic nonoparticles for clinical testing or for applications in drug deliveryAll chemicals were purchased from Sigma-Aldrich  unless specified otherwise. LeukoLOCK filters and RNAlater were purchased from Life Technologies . The RNeasy Midi kit was obtained from Qiagen, . The T7 mouse monoclonal antibody was purchased from Novagen . Alexa Fluor 647 goat anti-human IgG and Alex Fluor goat anti-mouse IgG antibodies were purchased from Life Technologies .This study was approved by the institutional review board at Wayne State University, the Detroit Medical Center and Cystic Fibrosis Center. Sera collected from 3 groups: 1) healthy volunteers; 2) confirmed CF subjects, and 3) sera from subjects with adenocarcinoma of the lungs. All study subjects signed a written informed consent. All methods were performed in accordance with the human investigation guidelines and regulations by the IRB (protocol Number = 055208MP4E) at Wayne State University.45. All spirometric studies were performed using a calibrated pneumotachograph and lung volumes were measured in a whole-body plethysmograph . All CF subjects were ambulatory patients. Sweat chloride test values were obtained from the medical records.Pulmonary function tests were performed following ATS guidelines in a licensed laboratory in all patients unless contraindicated12.Using standardized phlebotomy procedures blood samples were collected and stored at −80 °C12. Differential biopanning for negative selection was performed using sera from healthy controls to remove the non-specific IgG, and sarcoidosis sera for positive enrichment12.T7 phage display libraries from BAL, WBC, EL-1 and MRC5 were made to generate a complex sarcoid library (CSL)12.Informative phage clones were randomly picked and amplified after four rounds of biopannings and their lysates were arrayed in quintuplicates onto nitrocellulose FAST slides  using the ProSys 5510TL robot . The nitrocellulose slides were hybridized with sera and processed as described previouslyIndividual phage clones were PCR amplified using T7 phage forward primer 5′ GTTCTATCCGCAACGTTATGG 3′ and reverse primer 5′ GGAGGAAAGTCGTTTTTTGGGG 3′ and sequenced by Genwiz , using T7 phage sequence primer TGCTAAGGACAACGTTATCGG.12. Using the ImaGene 6.0 (Biodiscovery) image analysis software, the binding intensity of each peptide with IgGs in sera was expressed as log2 (red/green) fluorescent intensities. These data were pre-processed using the limma package in the R language environment47 and normexp method was applied to correct the background48. Within array normalization was performed using the LOESS method49. The scale method was applied to normalize between arrays49. Intensity ratio of a clone in CF samples divided by the same clone intensity ratio from healthy control samples were calculated to determine the fold change of a clone.Following the immunoreaction, the microarrays were scanned in an Axon Laboratories 4100 scanner  using 532 and 647 nm lasers to produce a red (Alexa Fluor 647) and green (Alexa Fluor 532) composite image. Cy5 (red dye) labeled antihuman antibody was used to detect IgGs in human serum that were reactive to peptide clones, and a Cy3 (green dye) labeled antibody was used to detect the phage capsid proteint-test. To evaluate the significant CF antigens identified with t-test, we applied principal component analysis (PCA), agglomerative hierarchal clustering (HC), heatmap, and naïve Bayes classifier. To avoid the problem of over-fitting the classifiers, we randomly split the CF and healthy controls samples into: i) training, ii) test, and iii) validation sets. Out of the 31 CF samples, 21 samples were randomly assigned into training (10 samples) and test (11 samples) sets. We repeated 1000 times random processing to generate 1000 training and test sets. The remaining 10 CF samples were used as an independent validation set. The 1000 training and testing sets for the healthy controls were randomly selected from 33 out of 49 samples (16 training and 17 test set). Therefore, the number of samples for the validation set for healthy controls was 16. While 31 LC samples were randomly split into test (15 samples) and validation (16 samples) sets. For CF clones specific selection, we applied a t-test between the 1000 CF training sets vs. 1000 healthy control-training sets. To correct for multiple comparisons, we applied the false discovery rate (FDR) algorithm with a threshold of 0.01 FDR50. The frequency of each significant clone (FDR < 0.01) across all 1000 runs was calculated and sorted based on their frequency of occurrence. The top 20 clones were considered highly significant CF clones. We built a naïve Bayes classifier on each of the 1000 training sets and tested the classifier model on the 1000 testing sets. Finally, the classifier model was validated on a complete independent validation set. The range of clones starts with the most frequent clone followed by adding one clone at a time. We constructed the models on training sets and applied the model on testing sets, as well as validation set. Finally, we determined correlation of biomarkers with body mass index (BMI) and % predicted forced expiratory volume (FEV1) of CF patients. We calculated combinations of 5 clones from the top set of markers. For each combination, the aggregated vector was calculated from the mean of 5 clones and Pearson correlation between the aggregated vector and BMI and FEV1% predicted was determined.To detect frequently differentially expressed antigens for CF we applied a two-tailed Supplementary Dataset


Bone regeneration is a process that involves several molecular mediators, such as growth factors, which directly affect the proliferation, migration and differentiation of bone-related cells. The osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP(10–14) have been shown to stimulate the proliferation, differentiation, alkaline phosphatase activity and matrix mineralization of osteoblastic lineage cells. However, the exact molecular mechanisms that promote osteoblastic proliferation and differentiation are not completely understood. This review presents the main chemical characteristics of OGP and/or OGP(10–14), and also discusses the potential molecular pathways induced by these growth factors to promote proliferation and differentiation of osteoblasts. Furthermore, since these peptides have been extensively investigated for bone tissue engineering, the clinical applications of these peptides for bone regeneration are discussed. Interest in developing bioactive biomaterials capable of promoting bone regeneration has grown significantly over the past few years. Different approaches and materials have been developed to regenerate bone defects wherein physiological processes to a bone neoformation are insufficient to repair the injury. In addition, some researchers have focused on investigating biological mediators and the signaling pathways that influence bone metabolism, in order to better understand this complex process. In this context, comprehension of the key signaling pathways involved in bone metabolism is extremely important for the development of bioactive biomaterials related to bone repair or regeneration.Bone regeneration is a complex process which involves a combination of many biological factors . Growth 2-ALKRQGRTLYGFGG-OH). This peptide was isolated from blood during osteogenic remodeling of post-ablation of marrow regeneration OGP(10–14) and cyclic analog OGP(10–14) in comparison with OGP(10–14). Ovariectomy-induced bone loss in mice model showed that the OGP reverses the majority of trabecular bone loss and that OGP(10–14) and desamino[Tyr10]OGP(10–14) have an even stronger osteogenic effect than OGP [In vivo studies indicated better bioactivity effects of the peptides desamino[Tyrthan OGP . Moreovethan OGP .5-Gly6 or Thr8-Leu9 bonds.The influence of hormones and local peptidic factors on cell metabolism is often regulated by enzymatic activity of cell surface peptidases such as neprilysin (NEP) . NEP is The osteogenic properties of OGP and OGP(10–14) have been most commonly evaluated in in vitro studies using cellular systems based on osteoblastic cell lines or marrow stromal cells of either murine or human origin . These s−13 M, indicating a considerably higher sensitivity of these cells to OGP peptide [−13 M [−13 up to 10−8 M) of OGP(10–14) were evaluated in primary human osteoblast (hOB) cells [−9 M OGP(10–14) stimulated bone formation and mineralization through osteocalcin synthesis and phosphatase activity. However, the ideal concentration of OGP(10–14) to stimulate the proliferation of hOB cells was 10−12 M. In addition, OGP(10–14) inhibited apoptosis and prevented the decrease in osteoprotegerin secretion induced by exposure of hOB cultures to dexamethasone, with a maximal effect at 10−9 M and 10−12 M OGP(10–14) concentrations, respectively. These results suggest the use of the OGP(10–14) as a possible anabolic treatment for osteoporosis [The ideal concentration of OGP to stimulate the proliferation and activity of osteoblasts was found in the MC3T3-E1 in which cell number was increased in peaks at peptide concentrations of 10 peptide . The alke [−13 M . SimilarB) cells . In hOB oporosis .Furthermore, OGP and OGP(10–14) are also potent regulators of marrow stromal cells, wherein the main OGP activity in marrow systems is a marked stimulation of ALP activity and matrix mineralization ,55,56,57Although the exact molecular mechanisms governing osteoblastic proliferation and differentiation are not completely known, some signaling cascades involved in these processes have been investigated ,56,57.i protein kinase mitogen-activated protein (MAP) by OGP(10–14), an increased expression and activation of MAPKAPK2 phosphorylation and increased transcriptional activity of the cAMP response element-binding (CREB) transcription factor were observed, which resulted in cell proliferation [In MC3T3-E1 osteoblastic cells, OGP(10–14) showed a mitogenic effect involving the rapid phosphorylation of Extracellular signal–regulated kinases 1/2 (ERK1/2), and de novo Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) mRNA and protein synthesis. After phosphorylation of ERK1/2 via activation of Gferation   in multipotent cells, some studies have investigated the mechanisms whereby OGP regulates the differentiation of MSCs into osteoblasts ,56,57. IFei et al.  found thDuring the process of cell differentiation, the membrane expression of the active form of Ras Homolog Gene Family Member (RhoA) in MSCs was increased, leading to investigation of the RhoA/Rho-associated protein kinase (ROCK) signaling. RhoA becomes activated after binding to the GTP protein. Subsequently, ROCK 1 and 2 are the major proteins associated with RhoA kinase D.Increased binding of RhoA to the GTP protein was observed in human MSC cells treated with OGP . MoreoveAdditionally, the molecular mechanism of OGP, as a regulator of osteogenic differentiation of MSCs, has revealed that AK141205  may enhance the expression of C-X-C motif ligand 13 (CXCL13) resulting in osteogenic differentiation  Figure E. The inThe in vivo effect of exogenously administered OGP in bone formation was first described by Bab et al. . In thisSimilarly, using a different model of bone healing, Zhao et al.  evaluateThe use of OGP and OGP(10–14) has also been proposed in osteoporosis treatment ,52. Chenco-glycolic acid) (PLGA) scaffolds in comparison with systemically administered OGP in the healing of segmental long bone defects in rabbits. In this study, 100 µg of OGP were incorporated by adsorption into each PLGA scaffolds. Histomorphometric and radiographic analysis showed that bone formation and union were significantly higher after local application of the OGP than after systemic administration. Moreover, degradation of the scaffold material occurred while new bone tissue was formed. Based on these results, the PLGA porous scaffold associated with OGP could be considered a therapeutic alternative to the use of autologous or allogeneic bone grafts in orthopedic surgery.Based on the positive effects of systemically-administered OGP and OGP(10–14), the peptides were considered good candidates for bone tissue engineering applications. Therefore, studies have been carried out to evaluate the effectiveness of OGP and OGP(10–14) in tissue-engineered scaffolds/membranes/hydrogels . Shuqian−9 M. The BC-HA membranes promoted better bone formation in critical-size mice calvarial defects. However, the analysis of gene expression showed that the BC-HA-OGP/OGP(10–14) membranes promoted an acceleration of osteoblast differentiation/activity in the early periods of analysis. Nevertheless, the incorporation of the peptides at concentrations of 10−9 M did not improve bone regeneration potential in long-term analysis. These results suggest that material modifications are needed to improve the effectiveness of the peptides release in the required concentration for longer periods [In dentistry, OGP has been incorporated into regenerative membranes used in the Guided Tissue Regeneration (GTR) technique. GTR is a therapy that utilizes membranes as biological barriers to prevent subepithelial connective tissue invagination into intrabony defects, preserving the space for bone neoformation . Saska e periods .Stakleff et al.  developeIn order to specifically induce the osteogenic differentiation of human MSCs (hMSCs), a multifunctional injectable vehicle for the co-delivery of hMSCs and osteoinductive peptides was proposed by Maia et al. . HydrogePolicastro et al.  successfThe identification of new approaches to stimulate osteoblast function and bone formation is critical to improve the treatment of fractures and other bone diseases and ultimately improve quality of life for patients. For this purpose, extensive investigations of the signaling pathways involved in the influence of OGP and OGP(10–14) peptides on osteoblast activity are still necessary. This review discussed about some studies that investigated the signaling and interaction between these peptides and other molecules during osteoblast proliferation and differentiation. Therefore, new discoveries in this direction may result in new approaches that involve combining different molecules that interact with these peptides in order to improve their effectiveness in bone repair/regeneration. Therefore, further investigation studies about OGP and OGP(10–14) peptides are necessary to improve and extensively enable the clinical use of these peptides in humans for medicine regenerative.


P = 0.0313). This study demonstrates that CTC clusters are found in locally advanced patients, and this may be an important prognostic marker. In vivo and in vitro studies are warranted to determine the role of these CTC clusters, in particular, whether leukocyte involvement in CTC clusters has clinical relevance.Distant metastasis (DM) from head and neck cancers (HNC) portends a poor patient prognosis. Despite its important biological role, little is known about the cells which seed these DM. Circulating tumour cells (CTCs) represent a transient cancer cell population, which circulate in HNC patients’ peripheral blood and seed at distant sites. Capture and analysis of CTCs offers insights into tumour metastasis and can facilitate treatment strategies. Whilst the data on singular CTCs have shown clinical significance, the role of CTC clusters in metastasis remains limited. In this pilot study, we assessed 60 treatment naïve HNC patients for CTCs with disease ranging from early to advanced stages, for CTC clusters utilizing spiral CTC enrichment technology. Single CTCs were isolated in 18/60–30% (Ranging from Stage I-IV), CTC clusters in 15/60–25% (exclusively Stage IV) with 3/15–20% of CTC clusters also containing leukocytes. The presence of CTC clusters associated with the development of distant metastatic disease( Despite the fact that primary treatment is usually both intensive and highly morbid, up to 50% of HNC patients still fail locoregionally or systemically. The more advanced the locoregional disease, the greater the risk of presenting with established metastatic disease, or harbouring micrometastatic disease, resulting in systemic failure at a later point. Circulating tumour cells (CTCs), which are shed from primary or secondary tumours and circulate in patients’ blood represent an important window into the mechanisms and characteristics of tumour metastasis4. They may also help direct locoregional and systemic treatment, by stratifying patients’ risk of systemic failure, allowing better selection of treatment individually.Head and neck cancers (HNC) accounts for the fifth most common non-skin cancer globally8. CTCs can be measured non-invasively from the blood and have direct clinical application9. In 2004, the FDA approved the first CTC enumeration platform, CellSearch (Janssen Diagnostics)11. It was demonstrated on this platform that single CTCs, in a number of tumour types, had clinical utility10. The enumeration of CTCs and cut off values of 5 or more CTCs in 7.5 ml of blood for metastatic breast cancer and prostate cancer has been associated with a poor prognosis and predictive of shorter progression free survival (PFS) and overall survival (OS)12. However, due to its inherent pre-selection of epithelial tumour cells expressing EpCAM, this system has shown poor sensitivity in detecting CTCs isolated from HNC patients bloods14.CTCs were first described by Thomas Ashworth in 1869, as ‘cells identical with those of the cancer itself’, and the field has rapidly advanced in the last decade17. In a number of studies, CTC clusters or circulating tumour microemboli (CTM), composed of platelets, stromal and hematopoietic cells, have been reported18. Critically, these tumour cell aggregates, are protected from the shear stressors in the blood, and are better suited to survive the journey through the circulation by cooperation of heterogeneous cell types within the cluster, which can include immune evading cells19. The prevalence and the number of CTC clusters can be underestimated due to their short detection window and lack of appropriate detection methods20. Studies have also documented that CTC clusters have a shorter circulation half-life, with faster entrapment within distant organs where metastatic growth may initiate22. CTC clusters may provide clues to their evolution during the course of cancer treatment and the mechanisms of cluster mediated treatment resistance23. CTC clusters have been associated with decreased metastasis-free survival and a greater metastatic capacity than single CTCs21. Notably, it has been shown that CTC clusters, held by plakoglobin-dependent adhesions, have arisen from oligoclonal expansion of tumour cell groupings, rather than aggregation or proliferation of single CTCs17. Recent studies suggest that CTC clusters have the ability to traverse narrow capillaries in a ‘single-file’ and retain the ability to re-form the intact cluster upon exiting, highlighting the metastatic seeding capacity of these large cellular aggregates that were previously thought to extravasate upon reaching narrow capillaries24.Of recent, there has been a shift, from marker-based CTC assays to marker-independent assays, to capture a greater population of CTCs from circulation, including CTC clusters26.To date, the role of CTC clusters, including CTM, has not been well established. Despite their biological importance, in comparison to single CTCs, the data on CTC clusters remains unclear. In this pilot study, we investigated whether CTC clusters and CTM were found in the circulation of HNC patients. This study evaluated 60 treatment-naive HNC patients, with disease ranging from early to advanced stages, for CTC clusters using spiral microfluidic CTC enrichment technology. This technology enriches for CTCs by a function of cell size and deformability and provides a robust methodology for CTC enrichmentPatients’ HNC disease staging ranged from early to advanced stages of HNC (Stage I-IV). CTCs and CTC clusters were successfully isolated using the spiral microfluidic chip. All patients had no evidence of distant metastatic disease upon presentation, however, at later time points (3–6 months follow up rescan), 7 stage IV patients developed lung and/or liver lesions Fig. . CTC cluSingle CTCs were detected in 20/60 patients (33.3%) ranging from 1–10 CTCs/5 ml  ranging from 1–3 clusters/5 ml (consisting of 3–13 cells)  events were observed in the 10 normal healthy volunteer (NHV) cohort.29. CTC clusters are defined as ≥3 tumour cells, held in close proximity by strong cell-cell adhesions, detected in the blood of cancer patients31. Studies have reported that CTC clusters have a shorter half-life in blood, an increased metastatic capacity compared to single CTCs30, and that disrupting the interactions within clusters may provide a strategy to reduce CTC cluster mediated metastasis16. An example of this would be the knockdown of plakoglobin, a protein which is highly expressed in CTC clusters that may facilitate in the reduction of cluster generation32. The observation of CTC clusters or CTMs has been associated with adverse outcomes33.Single CTCs have been previously reported in other solid tumour types including HNC37. In our cross sectional pilot study, single CTCs were found in 33.3% of patients (Stage I-IV) and CTC clusters in 25% of patients (Stage IV). Whilst the number of single CTCs is comparable to previous HNC studies41, the presence of CTC clusters in 25% of the cohort is of importance43. Furthermore, from 7 stage IV HNC patients in the study, that progressed to developing lung/liver metastasis (3–6 months later), 6 patients presented with CTC clusters (P = 0.0313). In patients with the absence of CTC clusters, the predicitive value of not developing distant disease within 6 months was found to be 95%. Chalmers et al., 2012 has shown that HNC CTC clusters can co-express Vimentin and CD44, epithelial-mesenchymal transition (EMT) and stemness traits which may represent an aggressive phenotype34. It is not fully understood whether the expression of mesenchymal traits on CTC clusters is due to single proliferating CTCs which had undergone EMT or an EMT transformed CTC cluster44. Single CTCs, CTC clusters, or both cell types were found in 25/60 of the sampled HNC patient bloods. Importantly, the presence of CTC clusters did not depend on single CTCs being present and could be potentially used as an independent prognostic marker44.To date, the clinical utility of CTCs in HNC remains limited, and few studies have reported on the presence of these subpopulations of CTC clusters47. To this end, recent studies have highlighted that PD-L1 is frequently expressed on CTCs and may be involved in immune evasion48. Studies have shown that the presence of non tumour cells (e.g. platelets and leukocytes) within CTC clusters promotes metastasis, by protecting the clusters from the shear stressors and immune attacks49.Notably, in 3/21 of the CTC clusters from stage IV HNC patients, white blood cells were found within the cluster. This feature in a number of CTC clusters is of importance as the incorporation of white blood cells (WBCs) in the CTC cluster may provide a mechanism by which these CTC clusters evade the immune systemThis pilot study challenges the notion of only reporting on individual CTCs in HNC studies. Whilst single CTCs were found in the screened population, a comparable population presented with CTC clusters. Whilst the role of CTC clusters, including clusters containing WBCs is not fully understood, studies into this area are warranted to understand cluster mediated immune escape and their role in metastasis.This prospective study was conducted across three major academic hospitals in Brisbane, Austrlia. Ethics approval was obtained from the Metro South and Health Service District Human Research Ethics Committee (HREC/12/QPAH/381 and HREC/11/QPAH/331) in accordance with the National Health and Medical Research Council’s (NHMRC) guidelines to collect blood from the Royal Brisbane and Women’s Hospital (RBWH), Logan Hospital and Princess Alexandra Hospital (PAH). This study also has QUT ethics approval (1400000617 and 1100001420). All participants gave written informed consent and 10 ml blood samples were collected in BD Vacutainer K2E tubes (EDTA) from 60 HNC patients before treatment and 10 normal healthy volunteers (NHV), with CTC assessment made as described below.26. In brief, after the spiral chip had an initial priming run, the sample was loaded onto a syringe and pumped through the spiral chip at 1.7 ml/min. The CTC output were collected and spun down at 300 × g for 5 mins.An initial red blood cell (RBC) lysis  was performed to the 10 ml blood sample to reduce the cellular components passing through the spiral chip. Thereafter, cells were centrifuged and the pellet resuspended in 10 ml of sheath buffer . The spiral device was setup as previously describedCTC enriched cells were cytospun onto glass slides and CTCs/CTM identified using the CellSearch antibody cocktail  (Janssen Diagnostics). Cells were further characterized for surface EGFR using anti-EGFR antibody . Briefly, the glass slides were incubated with an antibody cocktail of CellSearch Reagents  for 1 hour at room temperature, washed 3 times in PBS, coverslipped and imaged on the Olympus IX73 epifluorescence microscope.CTCs were visualized using immunofluorescence post enrichment. Cells were classified as CTCs after meeting the following criteria (i) high nucleus to cytoplasmic ratio (ii) morphologically larger than the background cells with intact nuclei (iii) cytokeratin-8,18,19 positive (iv) EGFR positive (v) CD45 negative. CTC clusters were reported as 3 or more CTCs in close proximity and CTM when CTC clusters included leukocytes (CD45 positive cells). The results were reported as the number of CTCs, CTC clusters, and CTM per 5 ml whole blood.P-values <0.05 considered statistically significant.The development of distant metastatic disease (confirmed by imaging and biopsy where possible) were compared to CTC groups using Fisher’s exact test. All statistical analysis were performed using Graphpad Prism 7.0 software, were two-sided, and All data generated or analysed during this study are included in this published article.


Strikingly, similar to heterozygote expression of SMPD1, either preventative (p-smpd1+/+) or therapeutic (t-smpd1+/+) pharmacological treatment strategies with desipramine – a functional inhibitor of acid sphingomyelinase (FIASMA) – significantly improved liver function and survival. The inhibition of sphingomyelinase exhibited a protective effect on liver function in the acute-phase, and the reduction of HSC activation diminished development of sepsis-associated liver fibrosis in the post-acute phase of sepsis. In summary, targeting sphingomyelinase with FDA-approved drugs is a novel promising strategy to overcome sepsis-induced liver dysfunction.The molecular mechanisms of maladaptive response in liver tissue with respect to the acute and post-acute phase of sepsis are not yet fully understood. Long-term sepsis survivors might develop hepatocellular/hepatobiliary injury and fibrosis. Here, we demonstrate that acid sphingomyelinase, an important regulator of hepatocyte apoptosis and hepatic stellate cell (HSC) activation, is linked to the promotion of liver dysfunction in the acute phase of sepsis as well as to fibrogenesis in the long-term. In both phases, we observed a beneficial effect of partial genetic sphingomyelinase deficiency in heterozygous animals (smpd1 The development of organ dysfunction resulting in multiple organ failure is a hallmark of the disease continuum which remains one major cause for poor outcome of these patients3. In particular, hepatic excretory dysfunction is an early and common consequence of sepsis4. In addition to challenges in managing the acute phase of the overwhelming host response to infection, long-term consequences of sepsis in survivors are still an underestimated issue compromising quality of life and resulting in an increased mortality risk following hospital discharge5. Previous studies have demonstrated that sepsis leads to increased liver-stiffness6 and prolonged sepsis-associated cholestasis with associated mortality8. The latter even with the risk to result in progressive sclerosing cholangitis9. These phenomena are also mimicked in a long-term murine model of sepsis where fibrosis of the liver subsequent to hepatic stellate cell (HSC) activation was shown in survivors10.Sepsis continues to be a serious problem worldwide with mortality rates reaching up to 18% due to consecutive organ failure and maladaptive host response13. Previous data suggest a positive association between an increased concentration as well as activity in plasma and disease severity allowing discrimination of outcome of these patients14. Furthermore, the regulation of this enzyme by a functional inhibitor improved either the survival of mice in an endotoxemia model11 or overcoming from cardiomyopathy due to polymicrobial sepsis15. SMPD1 is responsible for the fast and transient breakdown of membrane-embedded sphingomyelin to ceramide which in turn accumulates in lipid rafts of the outer layer of the cell membrane thus reorganizing protein complexes and supporting downstream signal transduction of inflammatory pathways17. SMPD1 is necessary for numerous signaling pathways controlling proliferation, autophagy, differentiation and apoptosis20. On a molecular level, there is also evidence of a direct regulation of the activation process of HSC into activated liver myofibroblasts (aLMF) by SMPD1 controlling extracellular matrix (ECM) production and deposition. The inhibition of SMPD1 abrogates the transdifferentiation of primary mouse hepatic stellate cells in vitro and reduces fibrogenesis in an in vivo model of bile-duct ligation21. Moreover, numerous studies have also provided a direct impact of SMPD1 activity on hepatocyte damage in vitro as well as in vivo23. In hepatocytes of SMPD1 deficient animals, the total loss of enzyme function protects these cells from Fas- and TNF-α-mediated apoptosis25.Among the plethora of pathophysiological changes in sepsis, the enhanced activity of the stress responsive enzyme sphingomyelin-phosphodiesterase 1 (SMPD1) might function as a promising target for therapeutic interventions+/+ animals (p-smpd1+/+) for seven days prior to the induction of sepsis with desipramine, a well-known functional inhibitor of SMPD1 (FIASMA)26. Furthermore, we established a therapeutic strategy treating smpd1+/+ mice (t-smpd1+/+) with desipramine 6 hours following induction of polymicrobial sepsis, in order to evaluate a potential role of its therapeutic use. Additionally, we compared these animals with septic smpd1+/+ littermates as well as with mice partially deficient for the enzyme (smpd1+/−). At three time points representing different phases of sepsis-associated liver damage and fibrogenesis , we monitored the role of SMPD1 in the maintenance of hepatic (dys)function during the acute-phase and in the prevention of hepatic fibrosis during the post-acute phase of sepsis.In order to characterize the effects of SMPD1 in the setting of acute- and post-acute liver dysfunction following the host response during sepsis, we opted for different inhibition strata: As a model of pharmacological inhibition, we preventively treated smpd127. Measurement of the SMPD1 activity in serum samples of smpd1+/+ littermates revealed an increased activity at 24 hours and 3 days following sepsis induction, whereas at day 28 the SMPD1 activity did not differ from that in sham-treated animals. In contrast, smpd1+/− animals demonstrated significant lower activity levels at all time points as compared to smpd1+/+ mice  levels in liver homogenates at 24 hours following sepsis induction indicating noticeable oxidative stress. The partial genetic inhibition of the enzyme significantly improved oxidative stress levels in the acute phase of sepsis  and cysteine and glycine rich protein 2 (Csrp2), both of which are well established measures of activated liver myofibroblasts, confirmed a significant increase in smpd1+/+ animals, which was not evident in smpd1+/− animals  with subsequent antibiotic rescue. As shown in Fig. +/+ animals in an observation period of 96 hours. In contrast, smpd1+/− animals displayed a significant reduction of mortality following polymicrobial sepsis with similar findings for preventative treatment with desipramine 7 days prior to sepsis induction as well as therapeutic treatment with desipramine 6 hours following sepsis induction.As a previous study has already demonstrated a beneficial role of SMPD1 inhibition for survival in a murine model of sterile endotoxemiaThis study provides first evidence of a detrimental role of the conserved stress responsive enzyme SMPD1 in the development of sepsis-induced liver dysfunction and fibrosis, which both affect the outcome from sepsis and the quality of life in sepsis survivors. We show that reducing SMPD1 activity, either by partial genetic deficiency or mostly important by preventative or therapeutic pharmacological inhibition, is beneficial for liver function in the acute phase of sepsis and for preventing inflammation, HSC activation and hepatic fibrogenesis as a prolonged consequence of sepsis.29. Protective effects due to deficiency could be reversed by treatment with exogenously added ceramide31. In addition, in vitro treatment of rat hepatocytes with human placenta SMPD1 induced oxidative stress and initiated apoptosis32.Altered SMPD1 activity has been linked to the pathogenesis of numerous disease entities such as alcoholic cirrhosis, non-alcoholic steatohepatitis, as well as cachexia and mortality in chronic heart failure11, in a Staphylococcus aureus or polymicrobial sepsis model27. In order to clarify the role of SMPD1 as a stress enzyme in sepsis-induced liver dysfunction, we used mice, which are partially deficient in the enzyme. It is controversially discussed whether it is appropriate to use this strain of genetically deficient mice in order to prove pathophysiological effects and cellular mechanisms based on the fact that mice compensate their total loss of enzyme function33. The complete knock-out of SMPD1 gene results in Niemann-Pick disease which is characterized by rapid central nervous degeneration alongside an impairment of liver function34. However, minimal residual enzymatic activity is sufficient to prevent Niemann-Pick disease and its neurological symptoms35. In line with these studies, we observed increased oxidative stress as well as elevated cytokine responses in knock-out animals36, which encouraged us to investigate mice heterozygous for SMPD1 (smpd1+/−) presenting with decreased but still residual SMPD1 activity levels. In addition to the beneficial effect of partial deficiency of SMPD1 in a bile duct ligation21 as well as in a Fas-mediated apoptosis model25, we infer from our sepsis model that residual activity of SMPD1 in smpd1+/− mice is sufficient to prevent maladaptive effects caused by the overwhelming activity in smpd1+/+ animals.Moreover, an increased activity of secreted SMPD1 in plasma samples of septic patients correlates with disease severity and poor outcome, and its inhibition improved survival rate in a murine model of severe endotoxemia+/− mice had a favorable phenotype resulting in improved liver function and reduced hepatic oxidative stress in the course of sepsis. In the case of sepsis-induced liver dysfunction there is still a broad discussion regarding its etiology and underlying molecular mechanism. One of these concepts denotes that immune cells, such as liver invading monocytes, contribute to the pathogenesis of sepsis-induced liver failure due to the overwhelming production of cytokines37. Consistent with the fact that SMPD1 is a key player in the regulation of macrophage differentiation and cytokine secretion, we demonstrate that activation of these cells and further cytokine expression is a function of SMPD1 activity. Furthermore, reduced hepatic fibrogenesis as a long-term sequela of sepsis, was proven by the expression profile of Acta238 and Csrp239, which are well established markers for activated liver myofibroblasts. Our data revealed that in smpd1+/− mice the number of aLMF was reduced which might be mirrored in a diminished deposition of ECM in the liver. In fact, the expression profile of two components of ECM  was found reduced on both, the molecular and the histological level. Our data are in line with a previous study from Moles et al. identifying SMPD1 as a key player HSC activation and hepatic fibrogenesis21.In this study smpd1i.e. proliferation) or even a phenotypic dissociation of stellate cells affected, one might consider the temporal-spatial conditions of liver tissue undergoing adaptive response, but also the presence of granulomatous inflammation as a response to persistent inflammation/infection10 due to inflammasome activation in stellate cells40. Then, the generation and distribution of tissue abundant abscesses and the status of inflammasome activation might be correlated with a putative phenotypic dissociation of stellate cells.In our study we elaborated causes and consequences of sepsis-induced fibrogenesis and its modulation by approved drugs. For that issue we analyzed samples from tissue homogenates and sections from representative locations. To solve the more specific question, whether there is not only a change in (trans-)activation state of hepatic stellate cells but also a variation in number  all of which regulate the activity of SMPD1 and have been used in daily clinical care for decades in treatment of neurological disorders+/+ mice as well as in smpd1+/− mice.In addition, we show an improved survival rate after polymicrobial sepsis in desipramine-treated smpd1ex-vivo experimentation or animal experiments with distinct (mixed) genotypes might function as a reliable tool to get further insights into the complex regulatory mechanisms of membrane integration of MDR2, its cytoskeletal anchoring (phosphorylation/ dephosphorylation), or ultimately reorganization upon inflammation in membranous subdomains characterized by a specific lipid composition4. Since the lipid mediator ceramide is known to affect and also to control mostly all of these key events, the exploration of the  kinetic) and dynamic of ceramide generation and its accumulation is of great interest, especially with respect to MDR2-function.With the aim of fostering the excretory capacity also the functionality of hepatocytes and cholangiocytes as well as the restitution of excretory function in presence of inhibitors of sphingomyelinase needs enhancing perception for upcoming experimental work. Primary hepatocytes for +/−, p-smpd1+/+ and t-smpd1+/+ mice are even sufficient for regulation of adaptive mechanisms in the liver during host response. Finally, our data highlights the favorable effect of functional SMPD1 inhibition, i.e. by desipramine, in order to protect the liver from hepatocyte damage in vivo which is underlined by obtained data from smpd1+/− animals. Furthermore, the pharmacological inhibitor diminished the activation of HSC and prevented fibrogenesis in survivors as a prolonged consequence of severe infection. We are proposing a concept of a causative therapeutic strategy by evaluation of a novel target. As these SMPD1 inhibiting drugs are FDA-approved drug, which are frequently prescribed for neurological disorders, observational studies in well-defined patients could unravel, whether FIASMA use is associated with reduced liver injury in patients with sepsis.Overall, results from our study support the notion that an increased activity level of SMPD1 displays a detrimental role with respect to regulation of liver function in both the acute and post-acute phase of sepsis. The decreased, but residual SMPD1 activity in smpd149 of human origin, was cultured under standard conditions  media, 5% CO2). Cells were treated 24 hours with different concentrations of desipramine  prior to stimulation with cytokine mix  for 24 hours as described previously51.An immortalized hepatic stellate cell line (LX-2)+/− animals with a partial deficiency in SMPD1 function to smpd1+/+ littermates52. For each experiment, similar proportions of male and female gender were randomly selected at the age of 8–12 weeks (mean B.W. 22.2 g). Animals were housed under controlled/standardized day-night conditions (12 h/12 h) at room temperature , received a standard diet and water ad libitum and were allowed to adapt to laboratory conditions for at least 2 days prior to the start of the experiments.All experiments were performed in accordance with the German legislation on protection of animals and with approval of the local animal welfare committee . We compared smpd1+/+ animals were treated 7 days prior or 6 hours following sepsis induction with desipramine-hydrochloride  every 24 hours subcutaneously which was continued daily up to euthanasia. Sepsis was induced by the standardized and established peritoneal contamination and infection approach53. Briefly, intraperitoneal injection of fecal slurry  was performed (3.0 µl/g B.W.) into the right lower quadrant of the abdomen with a 21-gauge cannula. Without supportive treatment, severity of the insult results in an approximately 100% mortality rate within 48 hours (data not shown). However, administration of antibiotics and volume resuscitation rescued animals and adjusted survival rate to 50% . In the post-acute phase (day 3) treatment was restrained to desipramine-HCl/vehicle administration once daily . Disease progression and outcome of mice during host response was continuously assessed using the clinical severity scores (CSS) (data not shown) as previously described53. Animals were deeply anesthetized before any procedure by isoflurane (2%) which was followed by harvesting tissues and whole blood (right-ventricular heart puncture).Smpd1+/+, smpd1+/−, p-smpd1+/+, t-smpd1+/+) at baseline, 24 h, 3 and 28 days following the insult. SMPD1 activity was determined by the hydrolysis of fluorescently labeled sphingomyelin  as a substrate, chromatographic product separation, and image analysis as described previously55. Serum samples were diluted 1:10 with the incubation buffer  before analysis. Finally, the reaction mixture was composed from 20 μl diluted serum and the extraction was carried out using a SpeedVac concentrator Plus .Serum was collected from each stratum  and 4 volumes of 25% metaphosphoric acid. Following centrifugation , GSH was measured in the supernatants photometrically. For analysis, data were obtained from n = 4 for baseline and n = 4 animals following polymicrobial sepsis from each stratum .Glutathione in its reduced form (GSH) was determined according to Ellman+/+, smpd1+/−, p-smpd1+/+, t-smpd1+/+) at baseline, 24 hours, day 3 and 28 following sepsis induction was used for analysis of clinically used laboratory parameters of hepatobiliary damage and dysfunction  using the clinical chemistry analyzer Fuji Dri-Chem 3500i  according to manufacturer’s instructions.Serum from each stratum 57. The expression profile of selected mRNA was measured using Rotor-Gene Q 2plex system. The following primer sequences were used:Fresh frozen liver tissue samples (15–20 mg) were homogenized in 600 µL lysis buffer . RNA was isolated using RNeasy Mini Kit according to manufacturer’s instructions . RNA concentration and integrity were analyzed using NanodropACTA2fw: tgatcaccatcggaaatgaa, rv: agaggtccttcctgatgtcaaCOL1A1 fw: caagaaccccaaggacaaga, rv: aggaaggtcagctggatggGAPDH fw: ctctgctcctcctgttcgac, rv: caatacgaccaaatccgttgacActa2fw: ccgagatctcaccgactacc, rv: tccagagcgacatagcacagf4/80 fw: tttcctcgcctgcttcttc, rv: ccccgtctctgtattcaaccCcl2/mcp1 fw: aggtgtcccaaagaagctgtag, rv: aatgtatgtctggacccattccTgfb fw: tatagcaacaattcctggcg, rv: tgctgtcacaagagcagtgTnfa fw: gtctactgaacttcggggtgat, rv: atgatctgagtgtgagggtctgIl1b fw: gaagagcccatcctctgtga, rv: ttcatctcggagcctgtagtgCsrp2 fw: gctacggaaagaagtatggacc, rv: ctcagtcagagttgtagactccCol1a1 fw: caaggtccttctggatcaagtg, rv: cctttatgcctctgtcaccttgCol3a1 fw: acgtagatgaattgggatgcag, rv: gggttggggcagtctagtgMrp2 fw: aacttgggttgctccatga, rv: caggaccaggattttggatttActb fw: gctcttttccagccttcctt, rv: cggatgtcaacgtcacactt+/+, smpd1+/−, p-smpd1+/+, t-smpd1+/+ were fixed in neutral buffered formaldehyde 4% for at least 24 hours directly following harvesting. Samples were dehydrated in rising alcohol concentration and then embedded in paraffin. Livers were sliced into 3 µm sections and deparaffinized through xylene and reversed graded alcohol series. At least seven organs of each stratum were stained in Sirius Red, which was used to detect and to visualize extracellular matrix deposition. Two experienced researchers independently scored collagen deposition using a semi-quantitative scoring system in 20 randomly sampled high-power fields per animal semi-quantitatively in a blinded fashion as 0 (absent), 1 (mild), 2 (moderate), 3 (strong). Scores were accumulated for each animal.Representative liver samples from smpd1For survival analysis, log rank test was used. Unpaired Mann-Whitney-U test (MWU) was performed to determine statistical differences between groups and over time. A level of p ≤ 0.05 was considered to be statistically significant. With respect to analyses of expression profile values log2 fold variations at least ± 1 were considered to be of biological significance.