Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome.

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 µM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.

Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression.

In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.

Induced senescence of healthy nucleus pulposus cells is mediated by paracrine signaling from TNF-α-activated cells.

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-α (TNF-α)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-α to induce senescence which was confirmed by demonstrating upregulation of senescence-associated ß-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-α-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-α.

Transforming Growth Factor ß Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro.

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor ß (TGFß) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFß1 or TGFß3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFß1 nor TGFß3 were able to restore the notochordal phenotype. Although TGFß1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020.

Suppression of SPRY4 Promotes Osteogenic Differentiation and Bone Formation of Mesenchymal Stem Cell.

The directed differentiation of human adipose-derived stem cells (hASCs) into different cell types has shown great therapeutic potential in treating various diseases. To maximize the therapeutic potentials, researchers have tried manipulating master transcriptional genes that promote efficient differentiation of mesenchymal stem cells (MSCs) such as the MAPK/ERK signaling pathway. Sprouty (SPRY) is a family of proteins that are known to inhibit the MAPK/ERK signaling pathway. Although the role of some SPRY isoforms in MSC differentiation is known, the function of SPRY4 isoform has not been fully elucidated. In the present study, the role of SPRY4 in the multilineage differentiation of hASCs has been elucidated. To investigate the role of SPRY4 in hASC differentiation and tissue regeneration, we performed a transient knockdown of SPRY expression via a small interfering RNA (siSPRY4). Western blot and quantitative polymerase chain reaction results revealed that the treatment of siSPRY4 before induction of differentiation had no significant effect on adipogenic, but reduced chondrogenic, differentiation of hASCs. Interestingly, SPRY4 transient knockdown had a significant effect on the osteogenic differentiation as indicated by the increased messenger RNA (mRNA) and protein expression of osteogenic markers such as alkaline phosphatase (ALP; 2.3-fold) and osteopontin (OPN; 3.5-fold) and increased calcium deposition measured via Alizarin red staining (3.3-fold). Moreover, in vivo tissue regeneration of siSPRY4-treated hASCs in ectopic bone formation and calvarial defect mouse models showed higher bone volume (5.24-fold) and trabecular number (4.59-fold) assessed via histological and microcomputed tomography analyses. We also determined that the enhanced osteogenic differentiation in SPRY4-treated hASCs was due to the induction of ERK1/2 phosphorylation. Taken together, our results suggest that the regulation of SPRY4 through MAPK signaling is a potentially critical aspect on the osteogenic differentiation of hASCs and for bone tissue regeneration, and thus, may be utilized as a potent technique in the development of effective bone therapeutics. Impact Statement This study tried to expand our current understanding of the osteogenic differentiation of mesenchymal stem cells. The transient downregulation of the SPRY4 expression via small interfering RNA (siRNA) showed significant enhancement of the osteogenic differentiation of adipose-derived stem cells via the induction of ERK 1/2 phosphorylation. This suggests the possible mechanism to maximize the potential of stem cell as therapeutics and has a great potential in treating various bone-related diseases.

Dengue seroprevalence and its socioeconomic determinants in Faisalabad, Pakistan: a cross-sectional study.

INTRODUCTION: Socioeconomic disparities in the community make some groups more vulnerable to dengue infection. METHODS: Fourteen dengue cases (IgM positive) served as index cases for the positive geographic cluster investigations. RESULTS: Of 292 individuals, the overall dengue seroprevalence was 22.9% (IgM positive 4.8%; IgG positive 18.1%). The highest (45%) seroprevalence was reported in the most socioeconomically vulnerable lower class, followed by the middle class (39%). Orthogonal comparisons showed that socioeconomic factors play a significant role in the prevalence of dengue. CONCLUSIONS: An integrated approach is required to control the menace through vector control strategies and improvement of socioeconomic conditions.

Dengue seroprevalence and its socioeconomic determinants in Faisalabad, Pakistan: a cross-sectional study

Abstract INTRODUCTION: Socioeconomic disparities in the community make some groups more vulnerable to dengue infection. METHODS: Fourteen dengue cases (IgM positive) served as index cases for the positive geographic cluster investigations. RESULTS: Of 292 individuals, the overall dengue seroprevalence was 22.9% (IgM positive 4.8%; IgG positive 18.1%). The highest (45%) seroprevalence was reported in the most socioeconomically vulnerable lower class, followed by the middle class (39%). Orthogonal comparisons showed that socioeconomic factors play a significant role in the prevalence of dengue. CONCLUSIONS: An integrated approach is required to control the menace through vector control strategies and improvement of socioeconomic conditions.

Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration.

Advances in mesenchymal stem cells (MSCs) and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain) regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs) into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs), and type II collagen (COL2). RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2) with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y)-box 9 (SOX9) and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPß). Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

Economic burden of dengue in four major cities of Pakistan during 2011.

OBJECTIVE: To assess the economic burden of dengue infection by calculating cost per patient and disability adjusted life years lost. METHODS: The cross-sectional study was conducted in Islamabad, Lahore, Faisalabad and Karachi from July 2012 to March 2013. Residential addresses and telephonic numbers of dengue patients were taken from the records of Pakistan Institute of Medical Sciences, Islamabad, Mayo and Ganga Ram Hospital, Lahore, Civil Hospital, Karachi, and Allied Hospital, Faisalabad. A total of 250 dengue confirmed cases - 50 from each hospital - were randomly selected. Information regarding duration of illness and out-of-pocket expenses were collected to estimate the direct cost, while indirect cost (number of work days missed by the patient) was calculated from disability adjusted life years using Murray's formula. RESULTS: Overall, there were 162(65%) men and 88(35%) with a mean age of 30.4±13.5years. More than half 138(55%) were below 30 years of age. Socio-economically, 145(58%) belonged to low, 70(28%) middle and 35(14%) to high socioeconomic groups. Of the total, 210(84%) cases had dengue fever followed by 32(12.8%) dengue haemorrhagic fever and 8(3.2%) dengue shock syndrome cases. Average duration of illness was 32±7.1 days. Overall direct cost per patient was Rs.35,823 (US$358) and average pre-hospitalisation, hospitalisation and post-hospitalisation was Rs.6154, Rs.21,242 and Rs.8,427 respectively. The overall disability adjusted life years per million population was 133.76. CONCLUSIONS: Although the government had provided free treatment for dengue in public-sector hospitals, still patients had to pay Rs.21,242 during hospital stay, resulting in substantial burden which needs to be addressed.

Demographic and clinico-epidemiological features of dengue fever in Faisalabad, Pakistan.

This cross-sectional study was carried out to explore the epidemiological and clinical features of dengue fever in Faisalabad, Pakistan during 2011 and 2012. During the study period, anti-dengue IgM positive cases were reported in the post-monsoon period during the months of August-December. Certain hotspots for the dengue infection were identified in the city that coincide with the clusters of densely populated urban regions of the city. Out of total 299 IgM positive patients (male 218 and female 81); there were 239 dengue fever (DF) and 60 dengue hemorrhagic fever (DHF) patients. There was decrease in the median age of dengue patients from 31 years in 2011 to 21.5 years in 2012 (p<0.001). Abdominal pain was seen in 35% DHF patients followed by nausea in 28.3%, epistaxis in 25% and rash in 20% patients (p<0.05). Patients reported to be suffering from high-grade fever for an average of 8.83 days in DHF as compared to 5.82 days in DF before being hospitalized. Co-morbidities were found to be risk factor for the development of DHF in dengue patients. Clinical and laboratory features of dengue cases studied could be used for the early identification of patients at risk of severe dengue fever.

Evaluation of diagnostic potential of Rv3803c and Rv2626c recombinant antigens in TB endemic country Pakistan.

To overcome and eliminate tuberculosis (TB), definitive, reliable, and rapid diagnosis is mandatory. Presently, the diagnostic potential of acute and latent stage TB specific antigens i.e., Rv3803c and Rv2626c was determined. Immunogenic recombinant genes of Rv3803c and Rv2626c antigens were cloned in bacterial expression vector pET23b and expressed product was purified. The homogeneity and structural integrity was confirmed by Western blot analysis. Diagnostic potential of Rv3803c and Rv2626c antigens was analyzed using the sera of 140 active TB patients (AFB smear positive) by indirect ELISA. Ten patients of leprosy and 94 healthy individuals were taken as disease and normal control respectively. The data was analyzed using R statistical package. The sensitivity and specificity of Rv3803c in active TB patients was of 69.3% and 76.4% respectively with an area under ROC curve of 0.77, whereas sensitivity and specificity of Rv2626c 77.1% and 85.1%, respectively. The area under ROC curve of Rv2626c was 0.89 which was significantly higher than Rv3803c (p < 0.0001). Recombinant antigens Rv3803c and Rv2626c have potential to be used as diagnostic markers for TB and need to evaluate with other antigens for differential diagnosis of TB.