Hamid Saeed

Osteoblastic cells: differentiation and trans-differentiation.

The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation.

Telomerase-deficient mice exhibit bone loss owing to defects in osteoblasts and increased osteoclastogenesis by inflammatory microenvironment.

Telomere shortening owing to telomerase deficiency leads to accelerated senescence of human skeletal (mesenchymal) stem cells (MSCs) in vitro, whereas overexpression leads to telomere elongation, extended life span, and enhanced bone formation. To study the role of telomere shortening in vivo, we studied the phenotype of telomerase‐deficient mice (Terc−/−). Terc−/− mice exhibited accelerated age‐related bone loss starting at 3 months of age and during 12 months of follow‐up revealed by dual‐energy X‐ray absorptiometric (DXA) scanning and by micro–computed tomography (µCT). Bone histomorphometry revealed decreased mineralized surface and bone‐formation rate as well as increased osteoclast number and size in Terc−/− mice. Also, serum total deoxypyridinoline (tDPD) was increased in Terc−/− mice. MSCs and osteoprogenitors isolated from Terc−/− mice exhibited intrinsic defects with reduced proliferating cell number and impaired osteogenic differentiation capacity. In addition, the Terc−/−‐MSC cultures accumulated a larger proportion of senescent β‐galactosidase+ cells and cells exhibiting DNA damage. Microarray analysis of Terc−/− bone revealed significant overexpression of a large number of proinflammatory genes involved in osteoclast (OC) differentiation. Consistently, serum obtained from Terc−/− mice enhanced OC formation of wild‐type bone marrow cultures. Our data demonstrate two mechanisms for age‐related bone loss caused by telomerase deficiency: intrinsic osteoblastic defects and creation of a proinflammatory osteoclast‐activating microenvironment. Thus telomerization of MSCs may provide a novel approach for abolishing age‐related bone loss.

Mesenchymal stem cells (MSCs) as skeletal therapeutics–an update

Mesenchymal stem cells hold the promise to treat not only several congenital and acquired bone degenerative diseases but also to repair and regenerate morbid bone tissues. Utilizing MSCs, several lines of evidences advocate promising clinical outcomes in skeletal diseases and skeletal tissue repair/regeneration. In this context, both, autologous and allogeneic cell transfer options have been utilized. Studies suggest that MSCs are transplanted either alone by mixing with autogenous plasma/serum or by loading onto repair/induction supportive resorb-able scaffolds. Thus, this review is aimed at highlighting a wide range of pertinent clinical therapeutic options of MSCs in the treatment of skeletal diseases and skeletal tissue regeneration. Additionally, in skeletal disease and regenerative sections, only the early and more recent preclinical evidences are discussed followed by all the pertinent clinical studies. Moreover, germane post transplant therapeutic mechanisms afforded by MSCs have also been conversed. Nonetheless, assertive use of MSCs in the clinic for skeletal disorders and repair is far from a mature therapeutic option, therefore, posed challenges and future directions are also discussed. Importantly, for uniformity at all instances, term MSCs is used throughout the review.

Telomere dysfunction reduces microglial numbers without fully inducing an aging phenotype.

The susceptibility of the aging brain to neurodegenerative disease may in part be attributed to cellular aging of the microglial cells that survey it. We investigated the effect of cellular aging induced by telomere shortening on microglia by the use of mice lacking the telomerase RNA component (TERC) and design-based stereology. TERC knockout (KO) mice had a significantly reduced number of CD11b(+) microglia in the dentate gyrus. Because of an even greater reduction in dentate gyrus volume, microglial density was, however, increased. Microglia in TERC KO mice maintained a homogenous distribution and normal expression of CD45 and CD68 and the aging marker, ferritin, but were morphologically distinct from microglia in both adult and old wild-type mice. TERC KO mice also showed increased cellular apoptosis and impaired spatial learning. Our results suggest that individual microglia are relatively resistant to telomerase deficiency during steady state conditions, despite an overall reduction in microglial numbers. Furthermore, telomerase deficiency and aging may provide disparate cues leading to distinct changes in microglial morphology and phenotype.

Telomerase activity promotes osteoblast differentiation by modulating IGF-signaling pathway

The contribution of deficient telomerase activity to age-related decline in osteoblast functions and bone formation is poorly studied. We have previously demonstrated that telomerase over-expression led to enhanced osteoblast differentiation of human bone marrow skeletal (stromal) stem cells (hMSC) in vitro and in vivo. Here, we investigated the signaling pathways underlying the regulatory functions of telomerase in osteoblastic cells. Comparative microarray analysis and Western blot analysis of telomerase-over expressing hMSC (hMSC-TERT) versus primary hMSC revealed significant up-regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin. In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc−/−). The low bone mass exhibited by Terc−/− mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced skeletal mRNA expression of Igf1, Igf2, Igf2r,Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc−/− MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase deficient osteoblastic cells.

Antibiotic Self-Prescribing Trends, Experiences and Attitudes in Upper Respiratory Tract Infection among Pharmacy and Non-Pharmacy Students: A Study from Lahore.

Pharmacists are the custodians of drugs; hence their education, training, behaviors and experiences would affect the future use of drugs at community and hospital pharmacies. Therefore, we aimed at evaluating the self-prescribing antibiotic trends, knowledge and attitudes among pharmacy and non-pharmacy students. We found that pharmacy students had higher risks of experiencing URIs related symptoms such as cough (RR; 1.7, p = 0.002), allergy (RR; 2.07, p = 0.03) and running nose (RR; 3.17, p<0.005), compared to non-pharmacy students -resulting in higher probabilities of selecting cough syrups (OR; 2.3, p<0.005), anti-histamines (OR; 1.8, p = 0.036) and anti-inflammatory/anti-pyretic (OR; 2.4, p<0.005) drugs. Likewise, bachelor’s degree pupils (OR; 2, p = 0.045), urban area residents (OR; 2.44; p = 0.002) and pharmacy students (OR; 2.9, p<0.005) exhibited higher propensities of antibiotic self-use–notable classes include, b-lactams (45.9%) followed by macrolides (26.5%) and augmentin (28.94%), respectively. Surprisingly, pharmacy and non-pharmacy students had higher odds of using antibiotics in common cold (OR; 3.2, p<0.005) and pain (OR; 2.37, p = 0.015), respectively. Unlike non-pharmacy students, pharmacy students were likely to select alternative therapy, such as Joshanda (OR; 2.22, p = 0.011) and were well acquainted with antibiotic hazards, with 77% reduction in risk of antibiotics re-use. In conclusion, university students exhibited antibiotic self-prescribing trends in conditions that does not warrant their use, thus are irrational users. The pharmacy education confers very little benefit to rational self-prescribing practices among students, while non-pharmacy students are more vulnerable to repeated antibiotic usage. Thus, the educational and training modules should be designed for university students to disseminate targeted information regarding the potential hazards of antibiotic self-use and importance of consultation with qualified and registered medical doctor/pharmacist before starting with antibiotics.

Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)

BackgroundTelomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.ResultsTo examine these effects, Terc-/- BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr – known transcription regulators of osteogenesis. Similarly, in Terc-/- BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.ConclusionTaken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.

Polymeric nanocapsules embedded with ultra-small silver nanoclusters for synergistic pharmacology and improved oral delivery of Docetaxel

Despite of the remarkable cytotoxic and imaging potential of ultra-small metal nanoclusters, their toxicity-free and targeted delivery to cancerous cells remains a substantial challenge that hinders their clinical applications. In this study, a polymeric scaffold was first synthesized by grafting folic acid and thiol groups to chitosan (CS) for cancer cell targeting and improved gastric permeation. Furthermore, silver nanocluster (Ag NCs) were synthesized in situ, within CS scaffold by microwave irradiation and core-shell nanocapsules (NCPs) were prepared with hydrophobic docetaxel (DTX) in the core and Ag NCs embedded CS in the shell. A significant cytotoxicity synergism (~300 folds) was observed for DTX with co-delivery of Ag NCs against breast cancer MDA-MB-231 cells. Following oral administration, the DTX-Ag-NCPs increased bioavailability due to enhanced drug transport across gut (9 times), circulation half-life (~6.8 times) and mean residence time (~6.7 times), as compared to the control DTX suspension. Moreover, 14 days acute oral toxicity of the DTX-Ag-NCPs was performed in mice and evaluated for changes in blood biochemistry parameters, organ to body weight index and histopathology of liver and kidney tissues that revealed no significant evidence of toxicity suggesting the safety and efficiency of the DTX-Ag-NCPs as hybrid nanocarrier for biocompatible delivery of metal nanoclusters.

Genderwise clinical response of antipsychotics among schizophrenic patients: a prospective observational study from Lahore, Pakistan

Abstract Objective: The study was aimed to evaluate the gender specific response to adherence and occurrence of side effects among schizophrenic patients in Lahore, Pakistan. Methods: A prospective study was performed for a period of 1 year among 180 newly diagnosed schizophrenics, aged 20–60 years to observe the symptoms, medication adherence and side effects. Morisky–Green–Levine Scale was used to evaluate medication adherence, LUNSER for side effects and PANSS to measure positive and negative symptoms. Data were analyzed using SPSS. Results: Positive symptoms (Male: Baseline 36.14 vs. endpoint 23.58, Female: 35.29 vs. 23.74) and negative symptoms (Males 27.9 vs. 20.05, Females 28.41 vs. 20.2) of schizophrenia were equally reduced after a follow up of 1 year in both the genders. Male population suffered more accumulative side effects (11.4 in males vs. 6.40 in females), extrapyramidal symptoms such as tardive dyskinesia and tremors (1.21 in males vs. 0.57 in females) and other side effects as compared to women (p ≤ .005). Males were found poorly adherent to antipsychotic treatment than females (93.3% in males vs. 6.7% in females (p ≤ .005). Conclusions: Prescribing practices should not overlook sex specific factors like hormonal changes, altered brain morphology and socioeconomic factors that may be responsible for the difference in the response to the course of schizophrenia.

Exploring the knowledge and attitude of medical and pharmacy students about generic medicine in Lahore, Pakistan:

Background The age of out-of-pocket health-care expenditures demands the practice of generic medicine. Our objective was to evaluate the knowledge and attitude about generic medicine among medical and pharmacy students of Lahore, Pakistan. Methods A cross-sectional study was performed among calculated sample size (via online Raosoft calculator) of 295 students including 185 pharmacy and 110 medical students from third, fourth, and final year of studies using convenient sampling approach. A pre-validated questionnaire was used to assess the knowledge of students regarding generic medicine. Data were analyzed using SPSS version 20. Results We found that a majority of both pharmacy and medical students were aware of the meaning of brand and generic medicine where 86 (29.7%) students believed that generic medicine is bioequivalent to brand name product and 108 (36.5%) students agreed that cost burden will be reduced with the use of generics. There were concerns about quality (P ≤ 0.05) and side effects (P ≤ 0.005) of generic medicine as compared to brands. Significant associations were found between the knowledge of medical and pharmacy students (P ≤ 0.005). Conclusion Educational interventions and policies regarding generic medicine practices are strongly needed to overcome the knowledge deficits among pharmacy and medical students.