RBC Lysate Research Articles

CpG and Rutin Co‐Loaded DNA Tetrahedra for Targeted Therapy of Intracerebral Hemorrhage: Synergistic Hematoma Clearance and Neuroinflammation Inhibition

AbstractIntracerebral hemorrhage (ICH) presents a formidable challenge due to its high mortality and disability rates, primarily attributed to cerebral hematoma formation and ensuing neuroinflammation. Swift hematoma removal is paramount for prognosis, yet existing interventions carry risks and limitations. Notably, elevated CD47 expression on hematoma‐associated RBC triggers a “don't eat me” signal, impeding hematoma clearance, while microglial/macrophage erythrophagocytosis exacerbates oxidative stress and the RBC lysate evokes neuroinflammation. To address this conundrum, a multifunctional nanomedicine (TD‐CFR), employing DNA tetrahedra (TD) as a carrier for ICH treatment is introduced. The investigations reveal that CpG enhances the phagocytosis of CD47‐expressing RBC by microglia/macrophages via lipid metabolism modulation. Integration of CpG into TD preserves its pro‐phagocytic efficacy, while TD's double‐stranded region enables efficient encapsulation of Rutin, a potent anti‐inflammatory and antioxidant flavonoid. Capitalizing on disrupted blood‐brain barrier integrity at the hemorrhage site, TD‐CFR achieves robust enrichment within cerebral hematoma post‐intravenous administration, augmented by folate receptor‐mediated targeting of microglia/macrophages. Efficacy assessments in mouse and rabbit ICH models confirm TD‐CFR's therapeutic benefits, including hematoma clearance, neuroinflammation suppression, and brain function restoration. Leveraging TD's high biosafety profile and dual active ingredient loading capacity, the study unveils a promising drug treatment paradigm for ICH.

Sickle Cell Hemoglobin Induces Autophagy in Human Macrophages

BACKGROUND: Autophagy plays an important role in multiple cell processes including elimination of misfolded proteins and damaged organelles, clearance of intracellular microbes and regulation of innate immunity. Autophagic activity is significantly up regulated in diseases characterized by proteins aggregation such as Alzheimer's, Huntington's, and Parkinson's diseases. Sickle cell anemia (SCA) is associated with E6V mutation in beta-globin gene that induces hemoglobin (Hb) polymerization under low oxygen or acidic conditions. SCA is characterized by chronic presence of low-level plasma Hb (3-10 µM). Hb is cleared from circulation by macrophage's endocytosis, resulting in degradation of Hb in lysosomes. Acidic lysosome environment may induce Hb polymerization, lysosome dysregulation and autophagy activation.HYPOTHESIS: We hypothesize that endocytosis of HbS induces autophagy in human macrophages.METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from three SCA patients and healthy individuals. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 µM, Sigma-Aldrich). Total RNA was depleted of cytoplasmic and mitochondrial ribosomal RNA, and strand-specific libraries were constructed using the Illumina ® TruSeq Stranded Total RNA Gold kit. Libraries were sequenced on an Illumina ® NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped to human reference genome using Dragen RNA v.3.8.4 software (Illumina). Comparisons were conducted using Dragen differential expression software v. 3.6.3 software (Illumina). Transmission electron microscopy (TEM) imaging was performed at Talos 200X transmission electron microscope (Thermo Fisher Scientific). CYTO-ID Autophagy detection kit (Enzo Life Science) was used for detection of autophagy in live cells by fluorescence microscopy, and fluorescent microplate assay.RESULTS: In THP-1-derived macrophages treated with HbS compared to HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 1.5-fold difference, 187 down and 135 up) at 5% FDR. In THP-1-differentiated macrophages treated with SCD RBC lysate compared to control RBC lysate, 30840 genes were detected including 256 differentially expressed genes (0.83%, 1.5-fold difference, 117 down and 139 up) at 5% FDR.We focused our analysis on 33 autophagy-related genes. Among these 33 genes, 30 genes were up-regulated (1.2-1.7-fold) and only 3 genes were down-regulated in macrophages treated with HbS compared to HbA (Fig.1). These changes were statistically significant for 20 genes (p<0.01). No significant differences were found in expression of autophagy-related genes in macrophages treated either with HbS or SCD RBC lysate.Autophagy activation was validated by CYTO-ID Autophagy detection kit, demonstrating significant autophagy activation in HbS treated macrophages compared to HbA treatment (1.2-fold, p=0.0019). Transmission electron microscopy demonstrated accumulation of autophagosomes in the cells treated with HbS compared to non-treated cells and cells treated with HbA.CONCLUSION: Treatment of macrophages with sickle cell hemoglobin or lysate of red blood cells collected from SCD patients up-regulates expression of autophagy-related genes and increased autophagosome formation.ACKNOWLEDGMENTS: We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging; and students Kavita Pandya, Gretchen Whitney High School, Cerritos, CA, and Tharindi Jayatilake, Montgomery Blair High School, Silver Spring, MD, for help in analysis of autophagy-related gene expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years

BackgroundThe use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. ObjectivesWe aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. MethodsWe prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at −70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC–tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland–Altman plot, Deming regression). ResultsAt baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. ConclusionsErythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at −70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Comparative Study of Malondialdehyde, Superoxide Dismutase and Glutathione Peroxidase in Urolithiasis Patients: A Case Control Study

Background: Urolithiasis i.e. stone firming disease in the urinary passages is one of the frequently encountered diseases in man. Perhaps the disease is as ancient as the man himself as has been revealed by the archaeological excavation done in different parts of the world such as in Egypt.&#x0D; Aim: Comparative Study of Malondialdehyde, Superoxide Dismutase and Glutathione Peroxidase in Urolithiasis Patients: A Case Control Study&#x0D; Materials and Methods: The study was carried out in the Department of Biochemistry, Datta Meghe Medical College Nagpur in collaboration with Department of Surgery, Division of Urology and Department of Pharmacology.&#x0D; Results: The urolithiasis patients have shown a marked increase in plasma MDA levels. There was a significant increase in the values of superoxide dismutase in patients suffering from urolithiasis (6.26 ± 0.86 µmol/l RBC lysate) as compared to the normal control values (3.40 ± 1.09 µmol/l RBC lysate) in human volunteers (p&lt;0.01).A significantly decreased value of glutathione peroxidase has been observed in patients suffering from urolithiasis as compared to the control group.&#x0D; Conclusion: Enhanced SOD can reduce the formation of Calcium Oxalate crystals and reduce the damage of renal tubular epithelial cell.

Nitric oxide, lipid peroxidation products, and antioxidants in primary fibromyalgia and correlation with disease severity.

Fibromyalgia syndrome (FMS) is characterized by altered pain perception with chronic, widespread musculoskeletal pain. The relationship between nitric oxide, oxidative stress and the severity of FMS has not been studied. This study evaluated NO levels in plasma, LPO products and antioxidants in Red Cell lysate in patients of FMS and correlated it with disease severity. 105 FMS patients who fulfilled 1990 ACR Criteria and 105 age- and sex-matched healthy controls were recruited over two years from 2013 to 2015. Antioxidative enzyme activity was assessed by the estimation of catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) and superoxide dismutase (SOD). Nitric oxide in plasma, MDA marker of lipid per - oxidation (LPO) in the lysate was donen for estimating oxidative stress. FIQR was used to assess the severity of fibromyalgia. The catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase levels were significantly low in patients than controls (p<0.001). Plasma NO levels and LPO were also significantly high (p<0.05). NO and LPO levels showed a significant positive correlation with FIQR (r: 0.57, 0.8 and p: <0.001) whereas a negative correlation was observed between antioxidants (Cat, GR and GPx, but not SOD) and FIQR. Low antioxidants and raised LPO in RBC lysate in patients with FM together with high plasma NO correlated with the severity of FMS.

LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences

Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs.HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016).Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. DisclosuresVieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

Oxidative stress and anti-oxidants in pre and post operative cases of breast carcinoma

To investigate the existence of oxidative stress in the sera of patients with breast cancer and its effects on the consequent breast cancer. This study included 50 control volunteers, 50 patients with breast cancer, and 50 patients with post-operative breast cancer. Patients with pre-operative cancer were clinically and histopathologically diagnosed for breast carcinoma with stage 0, not having therapeutic history. The control 50 healthy female volunteers had the same socio-economic status, and no history of any cancer. After obtaining consent, venous blood was collected from the volunteers by vein puncture using a 10 mL sterile disposable syringe and needle. About 8 mL of blood was collected, 4 mL of which was poured into a heparinized bulb and 4 mL was allowed to clot. The levels of MDA, NO, GSH, and activities of RBC-SOD (in RBC lysate), NOS, copper and zinc GPx, and CAT, and vitamins A, C, and E metabolites were measured in the sera of each group. The activities of RBC-SOD and the levels of MDA, NO, as well as the NOS were significantly higher in the sera of all patients with breast cancer as compared with the controls. However, the levels of GSH and vitamins A, C, and E, as well as the activities of copper and zinc GPx and CAT were decreased in patients with breast cancer when compared with the controls. The study provides further evidence for the presence of oxidative stress in the serum of patients with breast carcinoma. Patients with higher levels of MDA showed deficiencies of antioxidants and trace elements in the serum. A poor dietary antioxidant status and high oxidant levels are associated with the risk of breast cancer, thus suggesting that patients with breast cancer should take nutritive supplements to balance the antioxidant and oxidant levels for better outcomes.

&lt;em&gt;In vivo&lt;/em&gt; correlation of olive leaves extract on some oxidative stress markers in streptozotocin-induced diabetes mellitus in rats

Diabetes mellitus type two (T2DM) is one of the most extensive diseases in the world. Herbal therapy remains a possible adjunct therapy to sustain better glycemic control and reduce complications arising from diabetes. In order to evaluate the curative impacts of olive leaf extract (OLE) on streptozotocin (STZ)-induced diabetic rats, twenty-four Wistar male adult rats were divided into four equal groups; control, diabetic control (45 mg/kg STZ), normal rats treated with OLE (17.8 mg/kg b.wt.), and diabetic rats treated with OLE (45 mg/kg STZ + 17.8 mg/kg b.wt.). The OLE extract was investigated for in vitro antioxidant activity using the DPPH• assay. The phenolic, tannin, and flavonoid contents were determined. The activity of GPX, SOD, and GSH in RBC lysate, CAT in plasma and MDA in serum were measured. The OLE prevented the decrease in GSH and kept MDA around the normal range in the treated diabetic rats. The current study suggests that OLE might be used safely to ameliorate T2DM and its accompanying oxidative stress.

An Atypical Splenic B Cell Progenitor Population Supports Antibody Production during Plasmodium Infection in Mice.

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions and in response to inflammation due to infection or stress. Whereas the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occur in the spleen during acute Plasmodium infection, a critical step in the host immune response. In this study, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a lineage(-)Sca-1(+)c-Kit(-) (LSK(-)) phenotype that proliferates in response to infection with nonlethal Plasmodium yoelii 17X. Infection-derived LSK(-) cells upon transfer into naive congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK(-) cells gave rise to B cells capable of entering into a germinal center reaction, and they developed into memory B cells and Ab-secreting cells that were capable of producing parasite-specific Abs. Differentiation of LSK(-) cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK(-) cells expand and differentiate in direct response to the parasite. However, the ability of LSK(-) cells to differentiate into B cells was not dependent on MyD88, as myd88(-/-) LSK(-) cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B lymphocytes in the spleen and are capable of contributing to the ongoing humoral immune response against Plasmodium infection.

Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50% while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64% respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85% increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose.

Clarithromycin enhances the antimalarial efficacy of mefloquine via its increased bioavailability and disrupting P. falciparum apicoplast.

Many important drugs like mefloquine are not being used because of the development of resistance and other related issues. In the present study, we aimed to control drug resistance by using combination therapy and tried to understand the mechanism involved. We have explored in vitro interaction of clarithromycin (CLTR), and mefloquine (MQ) against Pf3D7 and PfK1 strains. Bioavailability of MQ in parasitized RBC lysate was checked in the presence/absence of CLTR using HPLC method. Further tufA mRNA/protein expression was investigated to know the effect of both drugs on apicoplast by using qPCR and Western blotting. MQ and CLTR inhibited growth of Pf3D7 and PfK1. CLTR showed its delayed antimalarial effect by its low IC50 values in the second cycle which indicates its effect on apicoplast. Downregulation of tufA expression on both mRNA and protein level supports this hypothesis. MQ and CLTR showed synergism/additiveness (mean ∑FICs = 0.89 and 1.26) against Pf3D7 and PfK1 respectively. It is evidenced from HPLC data that CLTR might have reduced metabolism of MQ in Plasmodium falciparum, leading to increased levels of MQ to produce enhanced antimalarial activity. The metabolism of CLTR is also reduced may be due to competitive metabolism of MQ via CYP3A4. The present study reveals that broad spectrum biological activities (i.e. antimalarial and antiviral) of MQ can be saved by using suitable partner drug like CLTR. This study also shows that CLTR increases the concentration of MQ and disrupts the apicoplast.

Biochemical assessment of red blood cells during storage by (1)H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress.

Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra. The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.

Effect of non surgical periodontal therapy on salivary and RBC lysate superoxide dismutase levels in periodontitis: A clinico biochemical study

To compute superoxide dismutase (SOD) levels in saliva and plasma of chronic periodontitis patients as compared to healthy controls, to find the association, if any, with clinical parameters and to verify the role of non surgical periodontal therapy (NSPT) in amending SOD activity. Forty two subjects were divided into two groups based on periodontal parameters; Group A: 20 chronic periodontitis patients (CP) and Group B: 22 periodontally healthy controls (PH).Clinical parameters, saliva samples (modified draining method) and plasma samples (by venipuncture) were evaluated at baseline, at 3 months and 6 months after NSPT for changes in clinical parameters and SOD activity. SOD levels were determined by Mc Cord and Fridovich method and absorbance was read by spectrophotometer at 560 nm. Significantly lower levels of salivary and plasma SOD (p<0.05) were detected in CP patients with a negative co-relation with clinical parameters at baseline in group-A. Post therapy SOD levels increased significantly (p<0.05) and plasma SOD levels rose even above that of control group.

In Vivo Alteration of Phase II Transformation Enzyme (GST) Levels by Stainless Steel Orthodontic Appliances

Orthodontic appliances includes bands, brackets and wires which are made up of stainless steel with the composition of roughly 8-12% nickel and 17-22% chromium and other trace metals. Corrosion of orthodontic appliances can have a toxic effect on surrounding oral tissues. The main aim of the study is to analyze the Glutathione s transferase activity in saliva and blood samples of patients undergoing Orthodontic treatment. The study group included 40 participants and Samples were taken in different time intervals: Group 1- Collection of samples before the appliances fixed, Group 2- Collection of sample after one week of fixed appliances, Group 3- Collection of sample after three months of fixed appliances each from the Orthodontic Department of A.B Shetty Memorial institute of dental sciences. Glutathione S transferase was estimated using 1-chloro, 2, 4-dinitrobenzene (CDNB) method. In case of Saliva group 3 (211.17±1.84) significantly increased compare to group 1 (87.05±3.86) and group 2 (118.74±2.8). In case of RBC lysate group 2(532.33±3.2) and group 3(512.40±1.16) were increased when compare to group 1(107.12±2.54). The Glutathione s transferase levels of patients increased over a period of time after treatment with orthodontic appliances but other factors like age of the patient, diet rich in antioxidants, light and strenuous physical activity, medication, amalgam fillings and genetic constitution of an individual also influenced the results.

The Growth Inhibition Effect on the Causative Bacteria of Bacterial Vaginosis by Bacterial Strains Isolated from the Vagina of a Healthy Woman

Two Gram-positive rod strains isolated from the healthy vagina of a woman were tested for the possibility as probiotics. One strain was identified as Steroidobacter denitrificans (YH1) and the other as Lactobacillus crispatus (YH2) by 16S rRNA partial sequencing. The Casman agar and Man-Rogosa-Sharpe (MRS) agar were mixed in same quantity, supplemented with 5% human rbc lysate (CMS agar). The Wilkins-Chalgren agar and MRS agar were mixed in same quantity (WCM agar). Gardnerella vaginalis was cultured in Casman broth, supplemented with 5% human rbc lysate and 1,000 x-diluted with normal saline. Bacteroides fragilis, Mobiluncus mulieris and Peptostreptococcus asaccharolyticus were cultured in Wilkins-Chalgren anaerobe broth and 2,000x-diluted. S. denitrificans YH1 and L. crispatus YH2 were cultured in MRS broth anaerobically and 100x-diluted. The diluted suspensions of B. fragilis, M mulieris and P. asaccharolyticus were inoculated on WCM agar and G. vaginalis on CMS agar by cotton swabs. Ten ㎕ aliquots of YH1 and YH2 were inoculated on the center of WCM agar and CMS agar. The growth inhibition zone diameters of B. fragilis, G. vaginalis, M. mulieris and P. asaccharolyticus by YH1 were 35 ㎜, 35 ㎜, 25 ㎜ and 60 ㎜. The inhibition diameters by YH2 were 25 ㎜, 30 ㎜, 20 ㎜ and 40 ㎜, respectively. These results implicate that S. denitrificans YH1 can be the stronger probiotics for the treatment of bacterial vaginosis than L. crispatus, compared inhibition zone diameters by YH1 and YH2.

An Essential Role for C5aR Signaling in the Optimal Induction of a Malaria-Specific CD4+ T Cell Response by a Whole-Killed Blood-Stage Vaccine

The protective immunity induced by the whole-killed parasite vaccine against malarial blood-stage infection is dependent on the CD4(+) T cell response. However, the mechanism underlying this robust CD4(+) T cell response elicited by the whole-killed parasite vaccine is still largely unknown. In this study, we observe that immunization with Plasmodium yoelii-parasitized RBC lysate activates complement C5 and generates C5a. However, the protective efficacy against P. yoelii 17XL challenge is considerably reduced, and the malaria-specific CD4(+) T cell activation and memory T cell differentiation are largely suppressed in the C5aR-deficient (C5aR(-/-)) mice. An adoptive transfer assay demonstrates that the reduced protection of C5aR(-/-) mice is closely associated with the severely impaired CD4(+) T cell response. This is further confirmed by the fact that administration of C5aR antagonist significantly reduces the protective efficacy of the immunized B cell-deficient mice. Further study indicates that the defective CD4(+) T cell response in C5aR(-/-) mice is unlikely involved in the expansion of CD4(+)CD25(+)Foxp3(+) T cells, but strongly linked to a defect in dendritic cell (DC) maturation and the ability to allostimulate CD4(+) T cells. These results demonstrate that C5aR signaling is essential for the optimal induction of the malaria-specific CD4(+) T cell response by the whole-killed parasite vaccine through modulation of DCs function, which provides us with new clues to design an effective blood-stage subunit vaccine and helps us to understand the mechanism by which the T cell response is regulated by the complement system.

Alterations in erythrocyte membrane fluidity and Na+/K+-ATPase activity in chronic alcoholics

Ethanol disorders biological membranes causing perturbations in the bilayer and also by altering the physicochemical properties of membrane lipids. But, chronic alcohol consumption also increases nitric oxide (NO) production. There was no systemic study was done related to alcohol-induced production of NO and consequent formation of peroxynitrite mediated changes in biophysical and biochemical properties, structure, composition, integrity and function of erythrocyte membranes in chronic alcoholics. Hence, keeping all these conditions in mind the present study was undertaken to investigate the role of over produced nitric oxide on red cell membrane physicochemical properties in chronic alcoholics. Human male volunteers aged 44 +/- 6 years with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics (~125 g of alcohol at least five times per week for the past 10-12 years). Elevated nitrite and nitrate levels in plasma and lysate, changes in erythrocyte membrane individual phospholipid composition, increased lipid peroxidation, protein carbonyls, cholesterol and phospholipids ratio (C/P ratio) and anisotropic value (gamma) with decreased sulfhydryl groups and Na(+)/K(+)-ATPase activity in alcoholics was evident from this study. RBC lysate NO was positively correlated with C/P ratio (r = 0.547) and anisotropic (gamma) value (r = 0.428), Na(+)/K(+)-ATPase activity was negatively correlated with RBC lysate NO (r = -0.372) and anisotropic (gamma) value (r = -0.624) in alcoholics. Alcohol-induced overproduction of nitric oxide reacts with superoxide radicals to produce peroxynitrite, which appears to be responsible for changes in erythrocyte membrane lipids and the activity of Na(+)/K(+)-ATPase.

Identification of the Major Arsenic-Binding Protein in Rat Plasma As the Ternary Dimethylarsinous−Hemoglobin−Haptoglobin Complex

Chronic exposure to arsenic causes a wide range of diseases such as hyperkeratosis, cardiovascular diseases, and skin, lung, and bladder cancers, and millions of people are chronically exposed to arsenic worldwide. However, little is known about the mechanisms underlying these toxic actions. The metabolism of arsenic is essential for understanding the toxic actions. Here, we identified the major arsenic-binding protein (As-BP) in the plasma of rats after oral administration of arsenite by the use of two different HPLC columns, gel filtration and anion exchange ones, coupled with an inductively coupled argon plasma mass spectrometer (ICP MS). The molecular mass of the As-BP was estimated to be 90 kDa based on results using the former column, and arsenic bound to this protein only in the form of dimethylarsinous acid (DMA (III)) in the plasma in vivo. In addition, the purified As-BP was shown to consist of two different proteins, haptoglobin (Hp) of 37 kDa (three bands) and the hemoglobin (Hb) alpha chain of 14 kDa (single band), using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), respectively, suggesting that the As-BP was the ternary DMA (III)-Hb-Hp complex. To confirm the present observations, an arsenic-binding assay was carried out in vitro . Although DMA (III) bound directly to fresh rat plasma proteins, they were different from that identified in vivo. However, when a DMA (III)-exposed rat RBC lysate (DMA (III) binds to Hb in rat RBCs) was added to control rat plasma, a new arsenic peak increased at the expense of the arsenic-Hb one. Furthermore, this new arsenic peak was consistent with the As-BP identified in the plasma in vivo, suggesting that arsenic bound to Hb further binds to haptoglobin (Hp), forming the ternary As-Hb-Hp complex.

HPLC assay with UV detection for determination of RBC purine nucleotide concentrations and application for biomarker study in vivo

ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50–100 μL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 μg/mL for ATP and ADP, and 20–400 μg/mL for AMP, GTP, and GDP with coefficients of determination ( r 2) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague–Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.

Hitachi Hemolytic Index correlates with HBOC-201 concentrations: Impact on suppression of analyte results

Objectives To determine the feasibility of laboratories to use an instrument's Hemolytic Index (HI) to determine if a test result would be accurate in the presence of hemoglobin-based oxygen carrier HBOC-201. Design and methods HI values from the Roche Hitachi Modular P800 for samples containing HBOC-201 were determined. HI limits for 24 tests determined by addition of RBC lysate hemoglobin to serum and the instrument manufacturer's HI limits were compared to HI limits for reporting the same tests determined by adding HBOC-201 to plasma. Results There is a linear relationship ( R 2 = 0.99) between the HI value on the Modular P800 and HBOC-201 concentrations. Twenty-one of 24 HI limits for reporting results as determined by the manufacturer or adding RBC lysate to serum were more conservative or equal to the limit determined by HBOC-201 interference testing. HBOC-201 interference testing was more conservative for albumin, creatinine, and uric acid. Conclusion For most analytes, the Modular P800 HI provides an accurate, conservative and automated method to determine whether laboratory results should be reported or suppressed when samples contain HBOC-201.