Reverse Transcriptase-polymer Chain Reaction Research Articles

Malignant Small Round Cell Tumor: A Rare Aggressive Malignancy-Case Report

Background: Malignant Small round Cell Tumor (MSRCT) is very rare group of tumors. Malignant cells have similar histologic appearance making it difficult to distinguish one from another, and poses a diagnostic challenge with needs for a multimodal approach. Immunohistochemistry and immunophenotyping by flow cytometry, reverse transcriptase polymer chain reaction (RT-PCR) and fluorescence in situ hybridization are needed to confirm. The appropriate and best treatment in early stages of MSRCT is early surgical excision with chemoradiotherapy based on patient specification. Inspite of all these, tumors have poor prognosis and chance of recurrence are high. Method: Case study of a 27-year-old male who presented with a non-productive cough for 4 weeks, with feverish feeling with weight loss and shortness of breath and a new onset precordial chest pain non radiating and worse on exertion. On examination his vitals were: BP 121/91 mm of hg, pulse rate 114bpm, temperature 36.3 degrees Celsius, respiratory rate 24/per min, SPO2 96%.On examination, Elevated Jugular Venous Pressure, hyperactive precordium, displaced apex beat to 6th intercostal space anterior axillary line, muffled heart sounds and bilateral lower limb edema. Respiratory exam was unremarkable. Sputum gene expert was negative for mycobacterium tuberculosis and covid 19 rapid test was negative. Chest X Ray showed cardiomegaly and a transthoracic echocardiography revealed moderate-large pericardial effusion, with mild collapse of right atrium in diastole. Pericardiocentesis done showed hemorrhagic effusion with lymphocytes cell component, patient was initiated on treatment for TB pericarditis empirically. Three months later a chest CT scan done revealed extensive mediastinal/pericardial mass with lung nodules. An ultrasound guided mediastinal/Pericardial biopsy was done, revealed a poorly differentiated small round cell carcinoma. Results: Patient expired 14 weeks from the initial onset of symptoms while awaiting immunohistochemistry studies for specific diagnosis of the tumor and oncology review. Discussion: This demonstrates the complexity of establishing differential diagnosis of MSRCT in a resource limited setting with subsequent delay in treatment initiation, heralds poor prognosis. This also confirms difficulties in establishing differential diagnosis in poorly differentiated tumor.

Padua Score and Coagulopathy Parameters on Survival of COVID-19 Patients at Prof Dr. R. D. Kandou General Hospital Manado

BACKGROUND: Coagulopathy in coronavirus disease 2019 (COVID-19) patients causes a prothrombotic state that increases the risk of thromboembolism. The Padua score and coagulopathy parameter including D-dimer values, fibrinogen, prothrombin time (PT), activated partial thromboplastin time (aPTT), and platelet counts are very important parameters to determine risk of thrombosis and mortality in hospitalized COVID-19 patients. AIM: This study aimed to assess the prognosis of COVID-19 patients with Padua score and coagulopathy parameters. METHODS: This retrospective cohort study was conducted in tertiary university hospital, Prof. Dr. R. D. Kandou Hospital Manado, Indonesia from October 2020 to July 2021. Patients admitted with final diagnosis of COVID-19 confirmed with positive reverse transcriptase-polymer chain reaction test were included in the study. The data were refined by excluding the patients under 18 years old and the patients with no blood test results, D-dimer values, fibrinogen values, PT values, aPTT values, and platelet counts. The association of Padua score and coagulopathy parameters with survival of COVID-19 patients was analyzed by multivariate cox regression and Kaplan–Meier analysis. RESULTS: The probability of survival on day 14 in patients with Padua score <4, D-dimer <0.5 mg/dl, PT ≤16 s, aPTT ≤39 s, and platelets >150,000/μL was 100%, 100%, 84.6%, 81.5%, and 81.4%, respectively. COVID-19 survival was influenced by Padua score ≥4 (heart rate [HR] = 4.199; CI 95% 2.221–7.936), D-dimer ≥0.5 mg/L (HR = 4.772; CI 95% 2.244–10.147), PT >16 s (HR = 2.124; CI 95% 1.608–2.805), aPTT >39 s (HR = 1.449; CI 95% 1.080–1.943), and platelet count <150000/μL (HR = 2.056; CI 95% 1.489–2.840). Padua score has the highest probability of mortality compared to the other coagulopathy parameters (p < 0.001; HR = 3.655; CI 95% 1.927–6.932). CONCLUSION: There was an association of Padua score, D-dimer value, PT value, aPTT value, and platelet count on survival of COVID-19 patients. Padua score being the most influential variable on survivals.

Managing acute acromioclavicular joint dislocation during COVID 19 pandemic by minimally invasive technique with suture anchor and miniplate: A pilot study

BackgroundAcromioclavicular (AC) joint dislocation is common in sportsmen and physically active population. Its management depends upon the grade of injury and functional demands of the patient. A variety of surgical procedures have been described with different limitations and advantages. The present study has assessed the clinical and radiological outcome of acute AC joint dislocation managed with a 3.5 mm Titanium suture anchor and 2 mm miniplate construct which requires lesser dissection, surgical time and thus contact with the patient as mandated by COVID-19 pandemic. MethodsWe enrolled 10 patients of Rockwood type-III, IV and V acute AC joint injury (<3 weeks old) reporting at this hospital from Feb 2020 to May 2021. All were tested for COVID-19 using reverse transcriptase polymer chain reaction test (RTPCR) and managed by closed/open reduction and fixation with a 3.5 mm Titanium Suture Anchor and a 2 mm Titanium miniplate construct. Follow-up was done at 3, 6 and 9 month post-operatively. ResultsThe average age of patients was 31 yrs. RTPCR test for COVID-19 was negative in all patients. Median surgical time was 25 min (Interquartile Range[IQR] = 16–34 min) and median follow-up duration was 36 weeks (IQR = 33–39 weeks). Median visual analogue scale score and IQR at pre-operative, 3 month, 6 month and 9 month follow-up was 7(IQR = 6–8), 3.5(IQR = 2.5–4.5), 2(IQR = 0) and 1(IQR = 0), respectively. Median constant score at pre-operative, 3 month, 6 month and 9 month follow-up were 34(IQR = 25–43), 65.5(IQR = 60.5–70.5), 82.5(IQR = 77.5–87.5) and 88(IQR = 81–95). There was significant improvement in clinical status (non parametric-Friedman test p < 0.001). Radiographs showed no loss of reduction, fracture or implant failure till last follow-up. ConclusionsMinimally invasive technique with a 3.5 mm Ti-suture anchor and 2 mm plate is an easy, fast and reliable construct for the management of acute AC dislocation in physically active population.

Molecular detection and characterization of Potato spindle tuber viroid (PSTVd) infecting Tomato (Solanum lycopersicum L.) in Karnataka State of India.

Potato Spindle Tuber Viroid (PSTVd) is a non-coding, infectious, small, circular RNA known to cause disease in agricultural and horticultural plants. In the present work, an investigation was conducted in the southern districts of Karnataka state to assess the possible pospiviroid infections on tomato plants that are considered natural hosts for viroids. A total of 83 tomato samples showing disease symptoms (virus or viroid-like) along with healthy ones were collected and subjected to viroid detection by conventional Reverse Transcriptase Polymer Chain Reaction (RT-PCR) using universal (Pospi1-RE/Pospi1-FW) and a specific set of primers (3H1/2H1). The study confirmed the presence of PSTVd in one of the samples of tomato collected from Banghatta village of Mandy District, with an expected amplicon of ~ 361bp. The bioassay conducted on tomato plants (cv. Rutgers) proved the association of PSTVd, which was further confirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative sequences were deposited in the NCBI GenBank. The sequence alignment and secondary structure analysis of the isolated viroid with other reference sequences revealed the variations in the pathogenicity, central conserved region, and Terminal right domains. The variations observed between the isolated PSTVd with that of other Indian isolates support that viroid may have been transmitted among the crop plants, possibly through seed or mechanical means.

Detection of three pandemic causing coronaviruses from non-respiratory samples: systematic review and meta-analysis

SARS-CoV-2 has posed an unprecedented challenge to the world. Pandemics have been caused previously by viruses of this family like Middle East Respiratory Corona Virus (MERS CoV), Severe Acute Respiratory Syndrome Corona Virus (SARS CoV). Although these viruses are primarily respiratory viruses, but they have been isolated from non-respiratory samples as well. Presently, the detection rate of SARS‐CoV‐2 RNA from different clinical specimens using Real Time Reverse Transcriptase Polymerized Chain Reaction (qRT‐PCR) after onset of symptoms is not yet well established. Therefore, the aim of this systematic review was to establish the profile of detecting SARS‐CoV‐2, MERS CoV, SARS CoV from different types of clinical specimens other than the respiratory using a standard diagnostic test (qRT‐PCR). A total of 3429 non-respiratory specimens were recorded: SARS CoV (total sample—802), MERS CoV (total sample—155), SARS CoV-2 (total sample—2347). Out of all the samples studied high positive rate was seen for saliva with 96.7% (14/14; 95% CI 87.6–100.0%) for SARS CoV and 57.5% (58/250; 95% CI − 1.2 to 116.2%) for SARS CoV-2, while low detection rate in urine samples for SARS CoV-2 with 2.2% (8/318; 95% CI 0.6–3.7%) and 9.6% (12/61; 95% CI − 0.9 to 20.1%) for SARS CoV but there was relatively higher positivity in urine samples for MERS CoV with detection rate of 32.4% (2/38; 95% CI − 37.3 to 102.1%). In Stool sample positivity was 54.9% (396/779; 95% CI 41.0–68.8%), 45.2% (180/430; 95% CI 28.1–62.3%) and 34.7% (4/38; 95% CI − 29.5 to 98.9%) for SARS CoV-2, MERS CoV, and SARS CoV, respectively. In blood sample the positivity was 33.3% (7/21; 95% CI 13.2–53.5%), 23.7% (42/277; 95% CI 10.5–36.9%) and 2.5% (2/81; 95% CI 0.00–5.8%) for MERS CoV, SARS CoV-2 and SARS CoV respectively. SARS‐CoV‐2 along with previous two pandemic causing viruses from this family, were highly detected stool and saliva. A low positive rate was recorded in blood samples. Viruses were also detected in fluids along with unusual samples like semen and vaginal secretions thus highlighting unique pathogenic potential of SARS‐CoV‐2.

Changes in obstetric practices in the first 3 months of the COVID-19 pandemic in a private tertiary hospital: A descriptive cross-sectional study

BACKGROUND: With the increasing number of COVID positive cases still being reported in the country, coupled with the possible vulnerability of pregnant patients to the disease, aggressive measures must be taken by all institutions to ensure safety of the patients.OBJECTIVE: This study aimed to the determine the clinical profile and pregnancy outcome of patients who were admitted at a tertiary private hospital in Metro Manila from March 16, 2020 to June 15, 2020 in relation to the protocols that were implemented in response coronavirus disease 2019 pandemic.METHODS: The protocols and guidelines implemented by the Department of Obstetrics and Gynecology of a private tertiary hospital in Metro Manila from March 16, 2020 to June 15, 2020 were retrieved. The case record of patients who were admitted and delivered during this period were retrieved and reviewed for pertinent data, which were recorded in a patient data collection form.RESULTS: During the 3-month study period, COVID positive pregnant patients were effectively separated from COVID negative patients from admission to discharge based on a health declaration form and universal reverse transcriptase polymerized chain reaction (RT-PCR) testing. Discharge was facilitated after 12–48 h for uncomplicated deliveries and post partum follow-up was done via telemedicine.CONCLUSION: The separation of COVID positive from COVID negative patients based on symptoms and RT-PCR results were effective in ensuring the safety of patients.

Care of the pregnant woman with coronavirus disease 2019 in labor and delivery: anesthesia, emergency cesarean delivery, differential diagnosis in the acutely ill parturient, care of the newborn, and protection of the healthcare personnel

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2, has been declared a pandemic by the World Health Organization. As the pandemic evolves rapidly, there are data emerging to suggest that pregnant women diagnosed as having coronavirus disease 2019 can have severe morbidities (up to 9%). This is in contrast to earlier data that showed good maternal and neonatal outcomes. Clinical manifestations of coronavirus disease 2019 include features of acute respiratory illnesses. Typical radiologic findings consists of patchy infiltrates on chest radiograph and ground glass opacities on computed tomography scan of the chest. Patients who are pregnant may present with atypical features such as the absence of fever as well as leukocytosis. Confirmation of coronavirus disease 2019 is by reverse transcriptase-polymerized chain reaction from upper airway swabs. When the reverse transcriptase-polymerized chain reaction test result is negative in suspect cases, chest imaging should be considered. A pregnant woman with coronavirus disease 2019 is at the greatest risk when she is in labor, especially if she is acutely ill. We present an algorithm of care for the acutely ill parturient and guidelines for the protection of the healthcare team who is caring for the patient. Key decisions are made based on the presence of maternal and/or fetal compromise, adequacy of maternal oxygenation (SpO2 >93%) and stability of maternal blood pressure. Although vertical transmission is unlikely, there must be measures in place to prevent neonatal infections. Routine birth processes such as delayed cord clamping and skin-to-skin bonding between mother and newborn need to be revised. Considerations can be made to allow the use of screened donated breast milk from mothers who are free of coronavirus disease 2019. We present management strategies derived from best available evidence to provide guidance in caring for the high-risk and acutely ill parturient. These include protection of the healthcare workers caring for the coronavirus disease 2019 gravida, establishing a diagnosis in symptomatic cases, deciding between reverse transcriptase-polymerized chain reaction and chest imaging, and management of the unwell parturient.

Lipoprotein apheresis downregulates IL-1α, IL-6 and TNF-α mRNA expression in severe dyslipidaemia

Background and aimsDyslipidaemias are associated with cardiovascular mortality and morbidity, driven by unstable atherosclerotic plaques with inflammatory infiltrates. Levels of messenger RNA (mRNA) for pro-inflammatory cytokines have been positively correlated with atherosclerotic disease progression. Therapeutic lipoprotein apheresis (LA) reduces plasma lipid levels and reduces inflammation. We evaluated the effects of LA on expression of mRNA coding for key pro-inflammatory cytokines in patients with dyslipidaemia, homo-/hetero-zygous familial hypercholesterolaemia (HoFH, HeFH) or hyperlipoprotein(a)aemia [hyperLp(a)] and associated coronary artery disease (CAD). ApproachTen patients (five males and five females, mean age 47 ± 9.2 years) were enrolled, all with HyperLp(a) or confirmed genetic diagnoses of dyslipidaemia, HoFH, or HeFH; all had associated CAD. mRNA determinations were via reverse transcriptase polymer chain reaction (RT-qPCR). ResultsLA was associated with downregulation of mRNA expression for IL-1α, IL-6 and TNF-α, starting after the first LA session. The observed reduction was progressively enhanced during the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session (IL-1α: −49%, p < 0.001; IL-6: −35%, p < 0.001; TNF-α: −56%, p < 0.001). ConclusionsLA suppresses the expression of IL-1α, IL-6 and TNF-α mRNA in patients with dyslipidaemias. This may contribute to the arterial anti-inflammatory effect of LA.

Toll-like receptor 2 and 4 expression in the pregnant and non-pregnant human uterine cervix

Pelvic infections and sexually transmitted diseases place a burden on health resources and may be associated with premature birth. The mechanisms by which the female reproductive tract (FRT) combats these infections remain ill understood, but are likely to involve the pattern recognition Toll-like receptors (TLR). We sought to compare the expression of TLR-2 and -4 by human pregnant and non-pregnant ectocervical epithelium as a prelude to the investigation of the function of these receptors in this tissue during pregnancy. Using the techniques of reverse-transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry, the gene and protein expression of TLR-2 and -4 were studied in the biopsies of ectocervix obtained from non-pregnant premenopausal women (n=21) undergoing hysterectomy, women in the first trimester of pregnancy undergoing non-medically indicated suction pregnancy termination (n=6), and women at term undergoing elective caesarean section (n=11). The expression of TLR2 and TLR4 genes and proteins were upregulated in early and late pregnant ectocervical epithelium, compared with non-pregnant tissue. These findings suggest that the upregulation of TLR2 and TLR4 in the lower FRT may play a key role in the modulation of the innate immune and inflammatory mechanisms of the ectocervix during pregnancy, interacting with other neuroendocrine factors.

Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling

IntroductionBone marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed as an ideal autologous stem cell source for cell-based therapy for myocardial infarction (MI). However, decreased viability and impaired function of aged MSCs hampered the therapeutic efficacy of engrafted MSCs, and the underlying mechanisms remain unclarified. Here, we investigated the role of inositol phosphates 6 kinase (IP6Ks) inhibition on the therapeutic efficacy of BM-MSCs and its underlying mechanism.MethodsBM-MSCs isolated from young (8-week-old) or aged (18-month-old) donor male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury with or without administration of inositol phosphates 6 kinase (IP6Ks) inhibitor TNP (10 μM). MSC apoptosis induced by H/SD was determined by flow cytometry and TUNEL assays. Protein expressions were evaluated by Western blot assay. Furthermore, the paracrine effects of MSCs were measured by reverse transcriptase–polymerized chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses.ResultsAged BM-MSCs exhibited more Inositol pyrophosphate 7 (IP7) production, compared with young BM-MSCs. Meanwhile, the expression of phospho-Akt (Thr308) was significantly decreased in the aged MSCs, resulting in enhanced Bad activation and decreased Bax/Bcl-2 ratio. Moreover, the apoptosis in aged BM-MSCs was increased, compared with young BM-MSCs. Furthermore, TNP administration significantly inhibited IP7 production and increased the phosphorylation of Akt under both normoxic and hypoxic conditions. Meanwhile, IP6Ks inhibition reduced apoptotic index of aged MSCs, associated with decreased expressions of pro-apoptotic proteins Bax and Bad and increased anti-apoptotic protein Bcl-2. The expressions of angiogenic factors, including VEGF, bFGF, IGF-1 and HGF, were decreased in MSCs from aged mice. In addition, TNP administration enhanced the paracrine efficiency of aged BM-MSCs under normoxic and hypoxic conditions.ConclusionsThis study demonstrates for the first time that IP6Ks and IP7 play critical role in the aging related vulnerability to hypoxic injury and impaired paracrine efficiency of BM-MSCs, which is associated with impaired Akt activation.

Comparative performance of the probable case definitions of dengue by WHO (2009) and the WHO-SEAR expert group (2011)

BackgroundThe primary objective of this study was to find the performance of the 2009 probable case definition of dengue and compare it with the definition given by the WHO-SEAR expert group in 2011.MethodsA cross-sectional study was conducted in Thiruvananthapuram district of Kerala, which is hyperendemic for dengue. A consecutive series of 851 participants defined by the selection criteria were recruited from the primary, secondary, and tertiary health care settings. Sensitivity, specificity, predictive values, and likelihood ratios of the clinical case definitions were calculated using reverse transcriptase-polymerized chain reaction (RT-PCR) as gold standard in case of fever less than or equal to 5 days and serology (IgM positivity) for fever >5 days. Diagnostic odds ratio (DOR) was also calculated as a single indicator of performance of the case definition.ResultsThe 2009 World Health Organization (WHO) case definition had a sensitivity of 76·4% (69·6–82·1) and negative predictive value of 87·5%. The 2011 WHO-SEAR expert group case definition had a higher sensitivity of 87·9% (82·2–91·9) but lower negative predictive value of 86·6%. The three independent criteria which were significantly associated with dengue were thrombocytopenia less than 150 000 (OR 2·80), leukopenia (OR 2·28), and absence of backache (OR 2·68). The performance of 2009 case definition was better (DOR 2·4) than the 2011 WHO-SEAR expert group case definition. This was further enhanced when thrombocytopenia was specified as platelet count less than 150 000 (DOR2·7). When ‘no backahe’ was added as an additional criteria, the performance of both definitions improved.ConclusionsThe 2009 WHO case definition has better discriminatory power than the 2011 WHO-SEAR expert group case definition. The performance of 2009 WHO case definition is enhanced by specifying thrombocytopenia as platelet count less than 150 000. The inclusion of ‘no backache’ further improves the discriminatory power. This may be more useful in primary care settings, to rule out dengue.

Inhibitory effects of curcuminoids from Curcuma longa on matrix metalloproteinase-1 expression in keratinocytes and fibroblasts

Because aging and inflammation mechanisms share common signaling pathways and mediators, natural products with anti-inflammatory effects are also good candidates as anti-aging agents. Bismethoxycurcumin, demethoxycurcumin, and curcumin are three curcuminoids that can be isolated from Curcuma longa and are well known for their anti-inflammatory and anti-cancer activities. In this study, we examined the anti-wrinkle effects of three curcuminoids from Curcuma longa in keratinocytes and fibroblasts to determine whether an anti-inflammatory agent could also be developed as an anti-aging treatment. The inhibitory effects of UV-B-induced upregulation of nuclear factor-kappaB (NF-κB) activity were detected by a reporter gene assay. Tumor necrosis factor (TNF-α) release was detected by reverse transcriptase-polymer chain reaction (RT-PCR), and matrix metalloprotease-1 (MMP-1) expression was determined by western blot and enzyme-linked immunosorbent assay (ELISA). UV-B induced TNF-α release through NF-κB activation in keratinocytes. Three curcuminoids inhibited the UV-B-induced TNF-α mRNA expression. Specifically, demethoxycurcumin and bisdemethoxycurcunim inhibited UV-B-induced NF-κB activation in HaCaT keratinocytes and reduced the expression of MMP-1 in both keratinocytes and fibroblasts. We found that curcuminoids have anti-wrinkle activity through the inhibition of UV-B induction in keratinocytes and TNF-α induction in fibroblasts. Our results suggest that wrinkle formation and inflammation have common signaling pathways.

Maternal Undernutrition Programs Offspring Adrenal Expression of Steroidogenic Enzymes

The aim of this study was to determine the influence of maternal undernutrition (MUN) on maternal and offspring adrenal steroidogenic enzymes. Pregnant Sprague-Dawley rats were 50% food-restricted from day 10 of gestation until delivery. Control animals received ad libitum food. Offspring were killed on day 1 of life (P1) and at 9 months. We determined the messenger RNA (mRNA) expression of steroidogenic enzymes by real-time reverse transcriptase polymerized chain reaction (RT-PCR). Maternal undernutrition inhibited maternal adrenal expression of P450 cholesterol side-chain cleavage enzyme (CYP11A1), 11 beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), and adrenocorticotropic hormone (ACTH) receptor (ACTH-R; MC2 gene) compared with control offspring. There was a marked downregulation in the expression of CYP11B1, CYP11B2, 11 β-hydroxysteroid dehydrogenase type 1 and 2 (HSD1 and HSD2), CYP11A1, ACTH receptor, steroidogenic acute regulatory protein (STAR), and mineralocorticoid receptor (MCR; NR3C2 gene) mRNA in P1 MUN offspring (both genders), with no changes in glucocorticoid receptor (GCR). Quantitative immunohistochemical analysis confirmed the PCR data for GCR and MCR in P1 offspring and demonstrated lower expression of leptin receptor protein (Ob-Ra/Ob-Rb) and mRNA in P1 MUN offspring. In 9-month adult male MUN offspring, the expression of HSD1, CYP11A1, CYP11B2, Ob-Ra/Ob-Rb, and GCR mRNA were significantly upregulated with a trend toward an increase in ACTH-R and a decrease in 17 alpha-hydroxylase (CYP17A1) expression. In adult female MUN offspring, similar to males, the expression of CYP11A1, ACTH-R, and Ob-Rb mRNA were increased, whereas GCR and CYP17A1 mRNA were decreased. These results indicate that the adrenal gland is a target of nutritional programming. In utero undernutrition has a global suppressive effect on maternal and P1 offspring adrenal steroidogenic enzymes in association with reduced circulating corticosterone levels in P1 offspring, which may be secondary to a negative feedback from elevated maternal GC levels and or leptin levels in MUN dams. Gender-specific differences in steroidogenic enzyme expression were found in adult MUN offspring. The common finding of increased ACTH receptor expression in MUN adults of both genders suggests an increased sensitivity of these offspring to stress.

Effect of endothelial cells on bone regeneration using poly(L‐lactide‐co‐1,5‐dioxepan‐2‐one) scaffolds

Our recent in vitro study demonstrated that endothelial cells (ECs) might influence the differentiation of bone marrow stromal cells (BMSCs). Therefore, the aim of this study was to describe this effect in vivo, using a rat calvarial bone defect model. BMSCs were isolated from femurs of two-donor Lewis rats and expanded in α-minimum essential medium containing 10% fetal bovine serum. One fifth of BMSCs were induced and differentiated into ECs in an Endothelial Cell Growth Medium-2 and then characterized by a flow cytometry. The remaining BMSCs were cultured in freshly prepared osteogenic stimulatory medium, containing dexamethasone, ascorbic acid and β-glycerophosphate. Either BMSCs alone (BMSC-group) or co-cultured ECs/BMSCs (CO-group) were seeded into poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds, cultured in spinner flasks, and then implanted into symmetrical calvarial defects prepared in recipient rats. The animals were sacrificed after 2 months. The formation of new bone was evaluated by radiography and histology and by the expression of osteogenic markers using reverse transcriptase-polymerized chain reaction (RT-PCR). To investigate vessel formation, histological staining was performed with EC's markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group. However, the expression of EC's marker was similar on both groups by histological analysis after 2 months postoperatively. Furthermore, the CO-group exhibited greater expression of osteogenic markers as demonstrated by RT-PCR. The results are consistent with the previous in vitro findings that poly(LLA-co-DXO) scaffold might be suitable candidate for bone tissue engineering. In vivo, bone regeneration was enhanced by a construct of the polymer scaffold loaded with co-cultured cells.

Antitumor activity of the water-soluble polysaccharide from Hyriopsis cumingii in vitro

As a freshwater pearl mussel, Hyriopsis cumingii is widely cultured in China to produce pearls. This study was made to explore the antitumor activity of water-soluble polysaccharide (WSP) from mantles of H. cumingii. Cell viability of human hepatocarcinoma HepG2 cells was estimated by MTT method. Cell cycle analysis was determined by flow cytometry. Apoptosis was observed under fluorescence microscopy and confirmed by DNA fragment assay. Reverse transcriptase-polymerized chain reaction (RT-PCR) and immunocytochemistry were carried to evaluate c-myc, bcl-2 and cyclinD1 gene expressions in HepG2 cells. Furthermore, intracellular reactive oxygen species (ROS) production was assessed by flow cytometry. After WSP treatment, the growth of HepG2 cells was inhibited and most of cells arrested in G(0)/G(1) phage (p < .01); apoptotic changes in nucleus and cytoplasm were also observed in WSP-treated cells; percentage of apoptotic rate significantly increased in a dose-dependent manner (p < 0.001); DNA fragmentation was detected in treated HepG2 cells; The mRNA level and protein level of c-myc, bcl-2 and cyclinD1 were decreased in the treated HepG2 cells. ROS was significantly increased in a dose- and time-dependent manner as well. The results suggested that WSP has potent antitumor activity.

Effect of Hormones on Matrix Metalloproteinases Gene Regulation in Human Aortic Smooth Muscle Cells

Postmenopausal women receiving hormone replacement therapy have more adverse outcomes after vascular reconstructions. Estrogen-binding receptors have been identified on vascular smooth muscle cells (VSMCs), indicating that vascular function may be under direct hormonal control. A key group of enzymes involved in vascular remodeling are matrix metalloproteinases (MMPs). Here we studied the effect of estrogen (Est) and progesterone (Prog) on MMP gene expression in human VSMCs. VSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est+Prog combination (Est/Prog), and interleukin-1beta (100 U/mL; IL-1beta). Gene array analysis indicated Est+IL-1beta increased the expression of MMP-3. Reverse transcriptase-polymer chain reaction (RT-PCR) analyses revealed MMP-3 mRNA levels were significantly increased by Est/Prog+IL-1beta treatment. However, Western blot and further RT-PCR analyses indicated no change in MMP-3 in response to hormones alone. RT-PCR analyses revealed membrane type 1 (MT1)-MMP mRNA levels, not MMP-2 or tissue inhibitor of MMP (TIMP), were significantly increased by Est/Prog+IL-1beta, and Western blot analyses confirmed a significant increase in MT1-MMP protein in response to Est alone. Estrogen and progesterone affect the MMP pathway of VSMCs via isoform specific mechanisms and may lead to unbalanced MMP regulation. Estrogen up-regulates MT1-MMP without a corresponding increase in TIMP-2, known activator and inhibitor of MMP-2, respectively. Additionally, estrogen up-regulates MMP-3 only in the presence of IL-1beta. This differential regulation, combined with case-specific variations in degree of inflammatory response, may explain why some women receiving exogenous hormone therapy at the time of vascular interventions are more susceptible to complications.

Therapeutic Potential of Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Ischemic Myocardium

Background and Objectives: We designed this study to determine the therapeutic potentials of umbilical cord blood (UCB)-mesenchymal stem cells (MSCs) , as compared with bone marrow (BM)-MSCs. Materials and Methods: MSCs were isolated from UCB and BM. For the in vivo study, myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD) in rats for 30 min, and this was followed by release; the MSCs were then injected into a designated point around the infarcted area. Echocardiographs were performed two weeks after surgery. For the in vitro study, a cDNA microarray and cytokine array were performed to compare the MSCs from UCB and from BM. Cell migration was assessed by a wound scratch assay, and the level of cardiac ankyrin repeat protein (CARP) was determined by reverse transcriptase-polymer chain reaction (RT-PCR) or Western blot analysis. Results: For the echocardiograph findings, the fractional shortening (FS) was 43.9% in the UCB-MSCs group and it was 38.6% in the BM-MSC group. The ejection fraction (EF) was 79.8% in the UCB-MSC group and it was 72.4% in the BM-MSC group (control FS: 26.2% and the control EF: 56.6%). CARP was one of the highly expressed genes in the UCB-MSCs on the cDNA microarray. The mRNA and the expressed level of CARP protein in the UCB-MSCs were higher than those in the BM-MSCs. The cell migration of the CARP small interfering ribonucleic acid (siRNA) transfected UCB-MSCs was delayed compared to that of the normal UCB-MSCs (p<0.05). Conclusion: Our study directly compared the two types of MSCs from UCB and BM, and we suggest that the CARP molecule might be responsible for the motility of UCB-MSCs. (Korean Circ J 2008;38:446-454)

Effect of recombinant human TNF–binding protein-1 and GnRH antagonist on mRNA expression of inflammatory cytokines and adhesion and growth factors in endometrium and endometriosis tissues in baboons

To evaluate the mechanism of action of recombinant human tumor necrosis factor (TNF)-binding protein-1 by assessing differential expression of messenger RNA (mRNA) for cytokines, matrix metalloproteinases, and growth and adhesion factors in baboons. Analysis of gene expression in a prospective randomized study. University Fertility Center. In the in vivo study, 14 baboons were randomly and subcutaneously (SC) treated with either phosphate-buffered saline (PBS), GnRH antagonist, or recombinant human TNF-binding protein-1 at the time of induction. In the ex vivo study, 4 baboons were treated by menstrual endometrium that had been incubated randomly with either PBS or recombinant human TNF-binding protein-1 before intrapelvic injection. In the in vivo study, analysis of 11 endometrial and 10 endometriosis biopsies included either PBS (n = 5), GnRH antagonist (n = 8), or recombinant human TNF-binding protein-1 (n = 8). In the ex vivo study, 2 endometrial and 4 endometriosis biopsies were analyzed from 4 baboons. The mRNA expression of TNF-alpha, IL-8, IL-6, transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, intercellular adhesion molecule-1, matrix metalloproteinase-1, and regulated on activation, normal T-cell expressed and secreted were investigated using real-time reverse transcriptase-polymer chain reaction (PCR). TGF-beta mRNA expression was decreased in endometriotic lesions from baboons treated with recombinant human TNF-binding protein-1 when compared with the placebo group. Except TGF-beta, mRNA expression of inflammatory cytokines and adhesion/growth factors is not affected in endometrial and endometriosis biopsies from baboons after induction of endometriosis combined with either systemic injection of recombinant human TNF-binding or GnRH antagonist or ex vivo treatment with recombinant human TNF-binding protein-1. Further studies are needed to elucidate the mode of action on how inhibition of TNF-alpha activity prevents the development of endometriosis.

Synaptic specializations exist between enteric motor nerves and interstitial cells of Cajal in the murine stomach

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC-IM) in the stomach and colon and ICC in the deep muscular plexus (ICC-DMP) of the small intestine. In the absence of ICC-IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC-IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse-associated proteins between nerve terminals and ICC-IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP-25 were detected. Synaptotagmin and SNAP-25-immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC-IM but not smooth muscle. W/W(V) mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC-IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP-25 were detected in W/W(V) tissues. In the absence of ICC-IM, synaptotagmin and SNAP-25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD-93 and PSD-95 in the stomach and expression levels of PSD-93 and PSD-95 were reduced in W/W(V) mutants. These data support the existence of synaptic specializations between enteric nerves and ICC-IM in gastric tissues. In the absence of ICC-IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals.

Amino Acid-Based Peritoneal Dialysis Solution Stimulates Mesothelial Nitric Oxide Production

Ultrafiltration failure is a common problem in continuous ambulatory peritoneal dialysis. Recent work has indicated a role of enhanced expression of nitric oxide synthase (NOS) in ultrafiltration failure. However, the conditions predisposing to increased generation of NO by the peritoneum have not been studied in detail and the cell types potentially involved have not been tested individually. We performed experiments in human peritoneal mesothelial cells (HPMC) in culture. Amino acid-based dialysis solution (Nutrineal; Baxter Deutschland GmbH, München, Germany), L-arginine, and glucose-containing control solutions were used and we observed the effects on the HPMC. We reasoned that amino acid-based dialysis solutions containing L-arginine, the substrate of NOS, might influence mesothelial NO generation. Nitric oxide production was measured in the supernatant using the Griess reaction. We studied the effect of the combined NOS inhibitor L-NMMA and specified the isoform of NOS involved. In serum-free control medium, the cells exhibited baseline generation of nitrite at a rate of 5.4 +/- 0.5 micromol/g protein. Addition of 6 mmol/L L-arginine to the control medium increased nitrite significantly (11.8 +/- 0.66 micromol/g protein, p < 0.002), as did amino acid-based dialysis solution (15.7 +/- 1.3 micromol/g protein, p < 0.002); L-NMMA caused a significant reduction of this nitrite. HPMC expressed eNOS (NOSIII) when grown in L-arginine-supplemented medium, shown by immunocytochemistry and by reverse transcriptase-polymer chain reaction. Biochemical exposure to a calcium ionophore in 1 micromol/L concentration approximately doubled the nitrite production by L-arginine-incubated cells. Peritoneal mesothelial cells generate NO in vitro. Generation of NO increased further in response to L-arginine supplementation of the culture medium and to amino acid-containing dialysis solution. Mesothelial cells express eNOS, which was likely involved in the observed peritoneal NO generation.