Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells.

The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.

Effect of local probiotic (Lactobacillus reuteri) vs systemic antibiotic therapy as an adjunct to non-surgical periodontal treatment in chronic periodontitis.

AIM: The aim of the present study was to assess and compare the clinical efficacy of local probiotic Lactobacillus reuteri (L. reuteri) and systemic antibiotics as adjunct to scaling and root planing (SRP) in the treatment of chronic periodontitis (CP). METHODS: Thirty systemically-healthy participants (15 probiotic and 15 antibiotic), clinically diagnosed with CP, were enrolled. All patients underwent SRP. Adjunctive probiotics were administered twice daily for 3 months, whereas a combination of amoxicillin and metronidazole were given three times daily for 7 days. RESULTS: Intragroup analysis showed statistically-significant improvement in all clinical parameters: plaque index, bleeding on probing, periodontal pocket depth, and clinical attachment level gain at each follow-up visit. However, intergroup comparison of clinical periodontal parameters did not show statistical significance. CONCLUSION: The adjunctive use of L. reuteri and systemic antibiotics along with SRP showed similar improvement in all clinical periodontal parameters. This indicates that both adjunctive therapeutic agents showed similar efficacy in resolving inflammation and improving periodontal outcomes.

Effects of variation in umbilical artery resistive index on placental morphology and birth weight in pregnancy induced hypertension.

BACKGROUND: Pregnancy induced hypertension results from defective trophoblast invasion and increased umbilical artery resistance which in turn results in decreased blood supply to the placenta and hence to the foetus. This arterial resistance varies in different cases of PIH thus causing variable effects on placenta and foetus. The objective of this study was to study the morpho-metric changes in placenta and alterations in birth weight with differences in umbilical artery resistive index in hypertensive pregnancies. METHODS: Ninety pregnant women with pregnancy induced hypertension were selected with gestational age greater than 35 weeks. Doppler ultrasound examinations were carried out to record umbilical artery resistive index (UARI). 2 groups were made on the basis of median values of UARI. Plain ultrasound examination was then carried out to record presentation, site of placentation, grade of maturity, insertion of the cord, cord thickness, placental thickness, vacuolation and amniotic fluid index (AFI). After delivery, foetal birth weight was noted and placentae examined for placental weight, infarcts, number of cotyledons, umbilical cord insertion, cord thickness and placental thickness were noted. Foetoplacental weight ratio was also calculated. RESULTS: Significantly higher UARI was seen in the high-resistance group. Significantly lower values of placental thickness, AFI, birth weight, placental weight and placental thickness, whereas greater number of grade-III maturity, infarcts and marginal cord insertion were noted in the high-resistance group. CONCLUSION: Increased UARI leads to a spectrum of changes in the placenta and also decreased birth weight. Marginal cord insertion causes greater risk of increased UARI.

Unraveling the role of metal ions and low catalytic activity of cytochrome C oxidase in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by high levels of aluminum and certain other metal ions in the brain: The disease is also characterized by low activity of brain cytochrome c oxidase (COX) but whether the elevated metal ions and the low COX activity are linked is not known. Moreover, COX is known to exhibit two catalytic rates (V (max)) and two substrate binding constants (K (m)) but it is not known which of these is affected in AD. In this study, we employed the Klatzo AD rabbit model to evaluate the impact of elevated metal ions on brain COX activity. New Zealand white rabbits were injected intra-cerebrally with 1.4% solutions of either AlCl(3), FeCl(3), CaCl(2), or MgCl(2); and 10 days, later the brain mitochondria were isolated. Polarographic assay revealed that compared to the controls, all four metals led to decreases in the V (max) of the enzyme's low affinity site. The respective decreases were; 16%, 36%, 18%, and 30%. The results suggest a sequence of events in vivo in which oxygen radical damage to mitochondria and COX leads to low ATP production and excess heme establishing conditions thought to be ideal for neurodegeneration.

Low activity and poor membrane tethering for rabbit brain cytochrome C oxidase in cholesterol-copper Alzheimer's model.

Alzheimer's disease (AD) is a major cause of dementia and death in the elderly, but its etiology is poorly understood. In recent years, Sparks and Schreurs (2003) have developed a rabbit model which displays 12 AD characteristics; however, the activity of brain cytochrome c oxidase (COX), which is usually low in AD, has not yet been assessed. In this study, we assessed activity of brain COX for Sparks' model. New Zealand white rabbits were maintained on the specified cholesterol-copper diet for a 10-week period following which brain mitochondria were isolated. The activity of COX within the mitochondria was assessed by polarographic assay; we also evaluated the spectral properties of the mitochondria and employed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to investigate their protein composition. Finally, we attempted to isolate COX and studied its subunit composition by SDS-PAGE. Polarographic assay revealed that compared to the controls, 44% of the rabbits on the cholesterol-copper diet had significantly decreased brain COX activity; on average, the V (max) of the high affinity site of mitochondria from the cholesterol-copper-fed rabbits was 26% lower than that of the controls. In addition, the difference spectra of brain mitochondria obtained from 33% of the rabbits raised on the cholesterol-copper diet, showed 35-40% diminished absorbance in the 430 nm region suggesting decreased concentration or reducibility of COX or another heme protein. SDS-PAGE analysis revealed that, for the rabbits raised on the cholesterol-copper diet, a number of COX subunits (VI-VII) were loosely held and easily lost during attempts to isolate the enzyme.

Increased BAEP latency and reduced brain cytochrome c oxidase activity observed in the Suzuki model of Alzheimer's disease [abstract]

Cytochrome c oxidase (COX), the terminal respiratory enzyme, is reported to be deficient and to exhibit reduced activity in a number of neurodegenerative disorders. The Alzheimer's rabbit model is based on the intracerebral injection of Holt's adjuvant, an aluminum salt solution. This model was used to compare the brain activity and the activity of brain COX in treated and controlled rabbits. Of a total of 26 New Zealand white rabbits, thirteen were injected intracerebrally with Holt's adjuvant solution, five with physiologically saline solution and the remainder were kept as controls. The auditory responses (BAEP) of the rabbits were monitored for 14 days before injections and 14 days thereafter. The animals were then sacrificed and brain mitochondrial extracts were used for SDS-PAGE analysis, difference spectra spectrophotometry and polarographic assays. The results of SDS-PAGE showed that there were no differences in the protein composition of the brain mitochondria of the three groups. Similarly, difference spectra (reduced minus oxidized, 400-630 nm) from both treated groups were identical to that of the control with characteristic maxima centered around 434, 550 and 604 nm. Polarographic assays, however, showed that while the enzyme from both treated groups displayed the characteristic biphasic kinetics, there was reduced activity in the enzymes from the brain of rabbits receiving the adjuvant but not the saline solution. This result is significant in light of the fact that several researchers have reported reduced activity of COX from Alzheimer's disease (AD) patients. Of particular significance, BAEP results reveal an increase in the interpeak latency between peaks III and V of the Holt's injected but not the saline injected or control rabbits. This latter result suggests that monitoring the BAEP might provide a simple non-invasive method for confirming AD. (AU)