scholarly journals Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

2021 ◽  
Vol 10 (10) ◽  
pp. e165101018811
Author(s):  
Fabio Marcelo de Queiróz ◽  
Márcia Aparecida Andreazzi ◽  
Fábio Luiz Bim Cavalieri ◽  
Isabele Picada Emanuelli ◽  
Marcelo Marcondes Seneda ◽  
...  
Keyword(s):  
Animal Origin ◽  
World Population ◽  
Livestock Farming ◽  
Oocyte Donor ◽  
The Mean ◽  
Group 2 ◽  
Group 1 ◽  
Donor Cows

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.

P5392Epicardium derived cells promote sympathetic ganglionic outgrowth towards myocardium in vitro

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
Y Ge ◽  
A M Smits ◽  
J C Van Munsteren ◽  
T Van Herwaarden ◽  
A M D Vegh ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Autonomic Innervation ◽  
Cardiac Damage ◽  
Group 4 ◽  
Group 3 ◽  
Cervical Ganglia ◽  
Group 2 ◽  
Group 1

Abstract Background The autonomic nerve system is essential to maintain homeostasis in the body. In the heart, autonomic innervation is important for adjusting the physiology to the continuously changing demands such as stress responses. After cardiac damage, excessive neurite outgrowth, referred to as autonomic hyperinnervation, can occur which is related to ventricular arrhythmias and sudden cardiac death. The cellular basis for this hyperinnervation is as yet unresolved. Here we hypothesize a role for epicardium derived cells (EPDCs) in stimulating sympathetic neurite outgrowth. Purpose To investigate the potential role of adult EPDCs in promoting sympathetic ganglionic outgrowth towards adult myocardium. Method Fetal murine superior cervical ganglia were dissected and co-cultured with activated adult mesenchymal epicardium-derived cells (EPDCs) or/and adult myocardium in a 3D collagen gel culture system. Four experiment groups were included: Group 1: Vehicle cultures (ganglia cultured without EPDC/myocardium) (n=48); Group 2: ganglia co-cultured with EPDCs (n=38); Group 3: ganglia co-cultured with myocardium (n=95); and group 4: ganglia co-cultured with both EPDCs and myocardium (n=96). The occurrence of neurite outgrowth was assessed in each group. The density of neurites that showed directional sprouting (i.e. sprouting towards myocardium) was assessed as well with a semi-automatic quantification method. Finally, sub-analyses were made by taking gender into account. Results Cervical ganglia cultured with EPDCs alone (group 2) showed increased neurite outgrowth compared to vehicle cultures (group 1), however the neurites did not show directional sprouting towards EPDCs. When co-cultured with myocardium (group 3), directional neurite outgrowth towards myocardium was observed. Compared to the ganglia-myocardium co-cultures, directional outgrowth was significantly increased in co-cultures combining myocardium and EPDCs (group 4), and the neurite density was also significantly augmented. Comparison between males and female ganglia demonstrated that more neurite outgrowth occurred in female-derived ganglia than in male-derived ganglia under the same co-culture conditions. Conclusion Activated adult EPDCs promote sympathetic ganglionic outgrowth in vitro. Sex differences exist in the response of ganglia to EPDCs, and female-derived ganglia appear more sensitive to EPDC-signalling. Results support a role of EPDCs in cardiac autonomic innervation and open avenues for exploring of their role in ventricular hyperinnervation after cardiac damage.

Renal effect of vitamin D metabolites: evidence for the essential role of the 25(OH) group

1983 ◽  
Vol 244 (6) ◽  
pp. F674-F678 ◽  
Author(s):  
M. M. Friedlaender ◽  
Z. Kornberg ◽  
H. Wald ◽  
M. M. Popovtzer
Keyword(s):  
Vitamin D ◽  
Vitamin D3 ◽  
Essential Role ◽  
Fractional Excretion ◽  
Vitamin D Metabolites ◽  
Group 2 ◽  
Fractional Excretion Of Phosphate ◽  
Group 1

The effects of 1 alpha (OH)vitamin D3 [1 alpha (OH)D3] and 24,25(OH)2vitamin D3 [24,25(OH)2D3] on the phosphaturic action of parathyroid hormone (PTH) were studied in two groups of parathyroidectomized (PTX) rats. In group 1, PTX PTH-infused rats received intravenous 1 alpha (OH)D3, and in group 2, PTX PTH-infused rats received intravenous 24,25(OH)2D3. PTX PTH-infused rats served as controls. The effects of both vitamin D metabolites on renal PTH-activated adenylate cyclase (AC) were studied in vitro. In group 1, PTH increased fractional excretion of phosphate (CP/CIn) from 0.045 +/- 0.012 (+/- SE) to 0.263 +/- 0.011 (P less than 0.005). 1 alpha (OH)D3 failed to influence this response. In group 2, PTH increased CP/CIn from 0.055 +/- 0.008 to 0.289 +/- 0.027 (P less than 0.005). 24,25(OH)2D3 reduced the PTH-induced rise in CP/CIn from 0.289 +/- 0.027 to 0.192 +/- 0.021 (P less than 0.01) and decreased the urinary excretion of adenosine 3',5'-cyclic monophosphate. In vitro, 24,25(OH)2D3 blunted the PTH-activated AC, whereas 1 alpha (OH)D3 had no effect. These results show that 24,25(OH)D3, similar to two other 25(OH) metabolites of vitamin D-25(OH)vitamin D3 and 1,25(OH)2vitamin D3-suppresses the phosphaturic action of PTH, whereas 1 alpha(OH)D3, which is devoid of a 25(OH) group, lacks this effect. This suggests that a 25(OH) group is a prerequisite for the antiphosphaturic effect of vitamin D, whereas the 1 alpha (OH) group is not essential for this action.

scholarly journals An Evaluation of Shear Bond Strength of Self-Etch Adhesive on Pre-etched Enamel: An in vitro Study

2013 ◽  
Vol 14 (6) ◽  
pp. 1036-1038
Author(s):  
Abdul Mujeeb ◽  
Bhadra Rao ◽  
Satti Narayana Reddy ◽  
Kanchan Mehta ◽  
G Saritha
Keyword(s):  
Statistical Analysis ◽  
Bond Strength ◽  
Shear Bond Strength ◽  
Occlusal Surfaces ◽  
Group 3 ◽  
The Mean ◽  
Group 2 ◽  
Self Etch ◽  
Group 1

ABSTRACT Aim To determine the shear bond strength of self-etch adhesive G-bond on pre-etched enamel. Materials and methods Thirty caries free human mandibular premolars extracted for orthodontic purpose were used for the study. Occlusal surfaces of all the teeth were flattened with diamond bur and a silicon carbide paper was used for surface smoothening. The thirty samples were randomly grouped into three groups. Three different etch systems were used for the composite build up: group 1 (G-bond self-etch adhesive system), group 2 (G-bond) and group 3 (Adper single bond). Light cured was applied for 10 seconds with a LED unit for composite buildup on the occlusal surface of each tooth with 8 millimeters (mm) in diameter and 3 mm in thickness. The specimens in each group were tested in shear mode using a knife-edge testing apparatus in a universal testing machine across head speed of 1 mm/ minute. Shear bond strength values in Mpa were calculated from the peak load at failure divided by the specimen surface area. The mean shear bond strength of all the groups were calculated and statistical analysis was carried out using one-way Analysis of Variance (ANOVA). Results The mean bond strength of group 1 is 15.5 Mpa, group 2 is 19.5 Mpa and group 3 is 20.1 Mpa. Statistical analysis was carried out between the groups using one-way ANOVA. Group 1 showed statistically significant lower bond strength when compared to groups 2 and 3. No statistical significant difference between groups 2 and 3 (p < 0.05). Conclusion Self-etch adhesive G-bond showed increase in shear bond strength on pre-etched enamel. How to cite this article Rao B, Reddy SN, Mujeeb A, Mehta K, Saritha G. An Evaluation of Shear Bond Strength of Self-Etch Adhesive on Pre-etched Enamel: An In vitro Study. J Contemp Dent Pract 2013;14(6):1036-1038.

THE ROLE OF NASOGASTRIC TUBE AFTER LAPAROSCOPIC REPAIR OF SMALL PERFORATION OF DUODENAL ULCERS IN LOW RISK PATIENTS

2019 ◽  
pp. 66-70
Author(s):  
Huu Tri Nguyen
Keyword(s):  
Nasogastric Tube ◽  
Laparoscopic Repair ◽  
Postoperative Hospital Stay ◽  
Low Risk ◽  
Duodenal Ulcers ◽  
Risk Patients ◽  
The Mean ◽  
Group 2 ◽  
Group 1

Background: The aim of this study was to evaluate the role of nasogastric tube after laparoscopic repair of small peforation of duodenal ulcers in low risk patients. Methods: A retrospective study on 69 consecutive perforated duodenal ulcer patients with size of perforation of less than 5 mm, ASA score of less than 4, Boey score of less than 2, treated with laparoscopic repair at Hue University of Medicine and Pharmacy Hospital from January 2012 to June 2018. Patients were divided into two groups: group 1 with postoperative nasogastric tube and group 2 without postoperative nasogastric tube because patients were uncooperative and removed the nasogastric tube themselves. Results: The mean age was 47.8 ± 14.7 years. Male/female ratio was 22. The mean of duration from symptom onset until surgery was 7.5 ± 5.5 hours. 60 patients (87.0%) had a Boey score of 0 and nine patients (13.0%) had a Boey score of 1. The mean of size of perforation was 3.5 ± 1.0 mm. All of perforations were on the anterior duodenal wall. The patients in the group 2 had a significantly shorter interval between surgery and passage of first flatus than in group 1 (1.8 ± 0.5 days vs 2.6 ± 0.7 days (p = 0.042)), had a significantly shorter postoperative hospital stay than in group 1 (4.5 ± 0.6 days vs 5.8 ± 0.8 days (p = 0.026)). There was no significant difference between group 1 and group 2 in the duration of analgesic use (2.3 ± 0.5 days vs 2.8 ± 0.8 days, p = 0.097). There was no morbidity or mortality in two groups. Conclusions: The patients without postoperative nasogastric tube had a significantly shorter interval between surgery and passage of first flatus and postoperative hospital stay. The use of postoperative nasogastric tube in small perforations of duodenal ulcers in low risk patients seems to be unnecessary. Key words: Perforated duodenal ulcer, laparoscopic repair, laparoscopic surgery, nasogastric tube

scholarly journals Fracture Resistance of Composite Veneers with Different Preparation Designs

2016 ◽  
Vol 20 (2) ◽  
pp. 99-103
Author(s):  
Katerina Zlatanovska ◽  
Ljuben Guguvcevski ◽  
Risto Popovski ◽  
Cena Dimova ◽  
Ana Minovska ◽  
...  
Keyword(s):  
Fracture Resistance ◽  
In Vitro Study ◽  
Fracture Load ◽  
Group 3 ◽  
The Mean ◽  
Group 2 ◽  
Preparation Design ◽  
Frequent Localization ◽  
Group 1

Summary Background: The aim of this in vitro study was to examine the fracture load of composite veneers using three different preparation designs. Material and methods: Fifteen extracted, intact, human maxillary central incisors were selected. Teeth were divided into three groups with different preparation design: 1) feather preparation, 2) bevel preparation, and 3) incisal overlap- palatal chamfer. Teeth were restored with composite veneers, and the specimens were loaded to failure. The localization of the fracture was recorded as incisal, gingival or combined. Results: Composite veneers with incisal overlap - palatal chamfer showed higher fracture resistance compared to feather preparation and bevel preparation. The mean (SD) fracture loads were: Group 1: 100.6±8.0 N, Group 2: 107.4±6.8 N, and Group 3: 122.0±8.8 N. The most common mode of failure was debonding for veneers with feather preparation and fracture when incisal edge is reduced. The most frequent localization of fracture was incisal. Conclusion: The type of preparation has a significant effect on fracture load for composite veneers. This study indicates that using an incisal overlap- palatal chamfer preparation design significantly increases the fracture resistance compared to feather and bevel preparation designs.

Characterisation of Leukaemic Stem Cell Populations in Childhood AML Indicate Distinct Disease Subtypes with Diverse Clinical Features

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1500-1500
Author(s):  
Victoria J Weston ◽  
Tracey A Perry ◽  
Katie Brown ◽  
Shaun R Wilson ◽  
Pamela R Kearns
Keyword(s):  
Stem Cell ◽  
Cd38 Expression ◽  
Group 4 ◽  
Childhood Aml ◽  
Group 3 ◽  
The Mean ◽  
Group 2 ◽  
Stem Cell Populations ◽  
Group 1

Abstract Abstract 1500 Five year survival rates for childhood AML in children are currently 55–65%. AML is an extremely heterogeneous disease, and while prognostic significance of some karyotypic abnormalities has become evident, the biology of the disease remains largely unknown. Full characterisation of leukaemia initiating cells which may be responsible for relapse has not yet been undertaken. We investigated the leukaemic stem cell populations from 10 childhood primary AML samples by comparing expression of CD34, CD38 and CD45RA, a marker of a committed granulocyte-macrophage progenitor (GMP)-like population frequent in adult AML, in vitro daunorubicin sensitivity and engraftment in immuno-compromised NOD/Shi-scid/IL-2Rgnull (NOG) mice. Consequently, we were able to classify AML samples into 4 subgroups. These comprised Group 1, CD34+CD38- AML (n=1); Group 2, CD34-CD38+CD45RA- (<10% CD34+ blasts) AML (n=4); Group 3, CD34+CD38+CD45RA- (>10% CD34+ blasts) AML (n=4); and Group 4, CD34-CD38+CD45RA+ (<10% CD34+ and >10% CD45RA+ blasts) (n=1). There was no apparent enrichment for high risk prognostic karyotypes in any of the groups. The Group 1 AML presented at 3y with t(16;21); In Group 2 AMLs, the mean presentation age was 11y, 2 carried good prognostic t(8;21), while 1 had MLL involvement and 1 had FLT3-ITD with chromosome 13 isodisomy, both higher risk indicators; The Group 3 AMLs presented with a mean age of 11.9y and 2 carried good prognostic inv(16) whereas 2 had FLT3-ITD one with additional chromosome 13 isodisomy, t(5;11) and TP53 loss. Finally, the Group 4 AML presented at 1.5y with a normal karyotype. When we compared the 2 most frequent subgroups, Group 2 had a much shorter mean EFS of 122d (n=2) compared with a 275d (n=4) for Group 3 (the mean follow up was 282d and 1013.5d, respectively). We next sorted four cell subpopulations based on CD34 and CD38 expression (CD34+CD38-, CD34+CD38+, CD34-CD38- and CD34-CD38+ blasts) and compared in vitro sensitivity to daunorubicin. In Group 1, CD34- and CD34+ cells were equally sensitive at nanomolar IC50 doses. In 2 of the Group 2 AMLs,CD34-CD38- cells were the most resistant to daunorubicin at micromolar IC50 doses (2.5-10mM) whereas the CD34-CD38+ cells (also the dominant subpopulation in this group) were the most sensitive cells exhibiting nanomolar IC50 doses (750–800nM). In contrast, the Group 3 AMLs were overall more sensitive to daunorubicin exhibiting lower nanomolar IC50 doses. Again in this group, the CD34-CD38- cells were typically the most resistant (this time being the dominant subpopulation) whereas the CD34+CD38+ were the most sensitive cells. Finally, in the Group 4 AML, while CD45RA+ cells rapidly underwent spontaneous apoptosis, CD45RA- cells exhibited extreme resistance to daunorubicin (IC50 >10mM) and CD38 expression had no impact on sensitivity. The reduced sensitivity of Group 2 AMLs to daunorubicin compared with Group 3 could, therefore, be an underlying factor contributing to shorter EFS. Finally, we initiated comparison of the stem cell qualities of the different subpopulations from representative samples from each of the two major subgroups, first by assessment of differentiation potential in vitro, and second by engraftment in vivo using NOG mice. In on-going experiments, the time to leukaemia will be compared between mice injected with unsorted and sorted cells and, at terminal cull, cells harvested from organs will be characterised by karyotype and immunophenotype and tested for clonogenic potential via subsequent serial transplantations. Peripheral blood sampling currently suggests higher human CD45+ engraftment in mice injected with sorted versus unsorted cells, and these are CD34+CD33+CD3- recapitulating the AML phenotype. We anticipate that particular subpopulations will be enriched for AML stem cells with the ability to repopulate the leukaemia. Overall, we have shown that childhood AML is diverse with respect to stem cell characteristics. AMLs with low CD34 (Groups 2 and 4) exhibit the greatest overall resistance to daunorubicin as well as shorter EFS. Furthermore, in the majority of AMLs, CD34-CD38- blasts exhibit the least sensitivity to daunorubicin. Novel therapies which can target these resistant subpopulations with leukaemia initiating activity could significantly improve the treatment responses in this clinically challenging disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of routine upper endoscopy before bariatric surgery in the Middle East population: a review of 1278 patients

2018 ◽  
Vol 06 (10) ◽  
pp. E1171-E1176 ◽  
Author(s):  
Bassem Abou Hussein ◽  
Ali Khammas ◽  
Mariam Shokr ◽  
Maiyasa Majid ◽  
Mariam Sandal ◽  
...  
Keyword(s):  
Bariatric Surgery ◽  
Middle East ◽  
Meta Analysis ◽  
Preoperative Assessment ◽  
Upper Endoscopy ◽  
Bariatric Patients ◽  
The Mean ◽  
Group 2 ◽  
Group 1

Abstract Introduction The routine use of preoperative endoscopy for patients undergoing bariatric surgery is controversial. Although many surgeons find it unnecessary, others still consider it a mandatory tool in preoperative assessment. Patients and methods We reviewed the medical records of all patients who had undergone preoperative endoscopy before bariatric surgery in Rashid Hospital between January 2013 and June 2016. The patients were divided into three groups: Group 0 included patients with normal endoscopy; Group 1 included patients with abnormalities that did not affect the timing or type of procedure; Group 2 included patients with abnormalities that had a direct impact on the procedure. Results The files of 1473 patients were reviewed. Endoscopy results were not present in 195 files, so those patients were excluded, and the remaining 1278 files were included. The mean age of patients was 41.3 ± 12.7 years, 61 % were female and 39 % were male. The mean body mass index (BMI) was 43.7 ± 8 kg/m2. Endoscopy was normal in 10.6 % of patients and abnormal in 89.4 %. The most common abnormalities were gastritis, positive Campylobacter-like organism test (CLO test), gastroesophageal reflux disease (GERD) with esophagitis and hiatal hernia. Group 0 included 10.6 % of patients (n = 135), Group 1 included 25.6 % of patients (n = 327), while Group 2 included 63.8 % of patients (n = 816). Conclusion Routine endoscopy seems to play an important part in the preoperative preparation of patients planned for bariatric surgery in the Middle East population. Further studies or meta-analysis could help in building up clear solid evidence and guidelines that could be approved by international bariatric associations with regard to indications for preoperative upper endoscopy in bariatric patients.

scholarly journals The Utility of Capsule Endoscopy in Patients under 50 Years of Age with Recurrent Iron Deficiency Anaemia: Is the Juice Worth the Squeeze?

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Prabhjot Singh Sidhu ◽  
Mark E. McAlindon ◽  
Kaye Drew ◽  
Reena Sidhu
Keyword(s):  
Iron Deficiency ◽  
Capsule Endoscopy ◽  
Iron Deficiency Anaemia ◽  
Significant Proportion ◽  
Deficiency Anaemia ◽  
Significant Pathology ◽  
The Mean ◽  
Group 2 ◽  
Group 1

Background and Aims. The role of capsule endoscopy (CE) in the <50 years of age patients with iron deficiency anaemia (IDA) remains unclear. We aim to assess its utility in this cohort.Methods. All patients referred for CE for recurrent IDA were included retrospectively. Patients were divided into Group 1 (<50 years) and Group 2 (≥50 years).Results. There were 971 patients with recurrent IDA and 28% belonged to Group 1. The mean age was 40 years in this group with a DY of 28% (n=76). Significant diagnoses included erosions and ulcers (26%;n=71), small bowel (SB) angioectasia (AE) (10%;n=27), SB tumours (3%;n=7), Crohn’s disease (3%;n=7), SB bowel strictures (1%;n=3), and SB varices (1%;n=2). On logistic regression, the presence of diabetes (P=0.02) and the use of warfarin (P=0.049) was associated with increased DY. The DY in Group 2 was 38% which was significantly higher than in Group 1 (P=0.02). While SB tumours were equally common in both groups, AE was commoner in Group 2 (P<0.001).Conclusion. A significant proportion of patients <50 years are referred for CE. Although the DY is lower compared to those ≥50 years, significant pathology is found in this age group. CE is advisable in patients <50 years old with recurrent IDA and negative bidirectional endoscopies.

scholarly journals Comparison of the Accuracy of Fixture-Level Implant Impression Making with Different Splinting Techniques

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Mehrdad Nateghi ◽  
Ramin Negahdari ◽  
Sahar Molaei ◽  
Ali Barzegar ◽  
Sepideh Bohlouli
Keyword(s):  
Setting Time ◽  
Acrylic Resin ◽  
Study Groups ◽  
Significant Difference ◽  
Group 3 ◽  
The Mean ◽  
Group 2 ◽  
Main Model ◽  
Group 1

Objectives. The impression-taking technique is one of the most critical factors that not only prevents the shrinkage caused by polymerization but also enhances the accuracy of implant impressions. Also, choosing the right time of taking impressions after splinting implants is one of the important criteria that affects the impression-taking technique. Accordingly, the present study aimed to evaluate the accuracy of different splint methods for implant impressions made at different times. Methods. In this in vitro study, a two-piece metallic index was prepared, and the patient’s jaw was simulated by placing self-cured acrylic resin in the lower part of the index. Then, two holes were made in the acrylic resin at a specific distance from each other, and the analogs were placed in these holes. Splinting of impression copings was carried out with autopolymerized acrylic resin (GC Pattern resin LS, GC America Inc., USA), and an open tray impression approach was performed. Thirty-six casts in three groups (n = 12) were fabricated from the acrylic model. After scanning the casts, the impression accuracy was compared between the three study groups by measuring the distance between the outer portions of the scan bodies screw-retained on implant analogs inside the cast using the Exocad software (2015.07 version). Group 1: splinting impression copings with autopolymerized acrylic resin and impression making immediately after the setting time (4 minutes); group 2: splinting and impression procedure after 17 minutes with splint sectioning and reconnection; group 3: splinting and impression procedure after 24 hours with splint sectioning and reconnection. The data were analyzed using SPSS 17 using the Kruskal–Wallis test. Results. The mean distance measured in group 1 was 19.14 ± 0.029 mm, which was significantly lower than the main model. The distances were 19.15 ± 0.039 and 19.159 ± 0.33 mm in groups 2 and 3, respectively. These two groups were not significantly different from the main model. Moreover, the mean distance measured in the three impression techniques was similar. Conclusions. There was no significant difference in the measurements between group 2, group 3, and the main model. Therefore, dentists can make an impression after 17 minutes to reduce chair time.

Quick, Cheap, and Easy Method for HIV-1 Human Blood Screening.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2902-2902
Author(s):  
Tomasz Rozmyslowicz ◽  
Krzysztof Wroblewski ◽  
Jaynathan Moodley ◽  
Glen N. Gaulton
Keyword(s):  
31P Nmr ◽  
Uninfected Cell ◽  
Cell Extracts ◽  
Infected Cells ◽  
Group 3 ◽  
The Mean ◽  
Group 2 ◽  
Hiv 1 ◽  
Group 1

Abstract When performed under strict conditions of temperature and pH, Perchloric acid (PCA) extraction is claimed to be a reliable method of providing cytosolic composition without quantitative or qualitative changes. We applied this technique to human lymphocytes, and obtained 31P-NMR spectra of both infected and uninfected cell extracts. Our studies show that the phosphomonoester (PME) to phosphodiester (PDE) ratio in uninfected cell extracts is up to three times higher than in extracts of HIV-1 infected cells. While PME signals include mainly phosphocholine and phosphoethanolamine, the PDE was identified mainly as glycerophosphorylcholine. Changes are clearly visible within 8–12 hours after infection. We conducted our initial studies on cells infected in vitro with HIV-1. We used the cell lines U87, MAGI, SupT1 and CEM, and primary lymphocytes isolated from blood of healthy donors (obtained from Bergen Community Regional Blood Center, Paramus, NJ) infected in vitro with HIV-1 type IIIB (X4-type) or 89.6 (X4/R5-type) at an M.O.I. of 0.01. The infection of these cells was confirmed by PCR analysis. The mean PDE/PME ratio of 30 uninfected lymphocyte samples was 0.482 ± 0.094 while the mean PDE/PME ratio of 30 infected samples was 0.206±0.056. Prior to extraction all cells were isolated from dead ones using Ficoll procedure, then cell pellets containing at least 5×106 cells were placed on ice and extracted with PCA. Subsequently, the KHCO3 neutralized extract was analyzed using phosphorus NMR spectroscopy. The mechanisms that determine the content of phosphodiesters and their modulation by the presence of HIV-1 comprise another target for future investigation, but its analytical value is clearly visible. Lastly, to determine whether the observation of reduced PDE/PME ratio was applicable in the analysis of HIV-1 sero-positive patients, we analyzed, in a blinded fashion, the 31P-NMR spectra of lymphocyte extracts obtained from six HIV-1 infected individuals. HIV-1 infected cells and plasma were isolated from subjects enrolled in the AIDS clinic at the University of Durban, South Africa. These individuals constitute both HIV-1 infected asymptomatic and AIDS patients, and were previously untreated by any regimen for HIV-1 infection. This population was confined to individuals aged 18–50 and consisted of ∼50% male participants, of which 100% were Black. The mean PDE/PME ratio for these samples was 0.203 ± 0.076, and again all six samples fell below the mean PDE/PME ratio of uninfected cells minus Standard Deviation. Statistical analysis was then conducted among all of these groups: uninfected (group 1), in-vitro infected (group 2) and in-vivo infected (group 3), using the Student t-test for unpaired samples. For group 1 vs. group 2 the t = 5.34, probability = 0.000040, for group 2 vs. group 3 the t = 0.142, probability = 0.89, and for group 1 vs. group 3 the t = 4.88, probability = 0.00011. The method developed here is self-calibrating (thus it measures the ratio of two blood extract components), easy to implement, and allows for measurements to be done automatically, using methods which are much more sensitive than NMR spectroscopy- for example HPLC. This technology is also much faster and cheaper than any currently used screening technique.

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