Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Production,Fate and Pathogenicity of Plasma Microparticles in Murine Cerebral Malaria

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.     

Bioactive Molecules Derived from Snake Venoms with Therapeutic Potential for the Treatment of Thrombo-Cardiovascular Disorders Associated with COVID-19

The Protein Journal - As expected, several new variants of Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) emerged and have been detected around the world throughout this Coronavirus...     

Structural changes of vegetation and its association with microclimate in a successional gradient of low thorn forest in northeastern Mexico

Understanding how microclimate and vegetation are associated during secondary succession is of primary importance for plant conservation in the face of the increasing land cover modification. However, these patterns are still unstudied for many plant communities. This study aimed to evaluate the structure (species richness, Shannon's diversity index, Simpson´s dominance index, abundance of each species, average height of species, species cover (%), species composition, and indicator values) of a low thorn forest fragment and to analyze its relation with microclimate along a successional gradient. Four stages of succession were delimited by the analysis of Landsat images, in the state of Tamaulipas, northeast Mexico. Statistical models incorporated species richness, diversity indices, abundance, height, and cover, as variables for searching differences between stages, or to evaluate microclimate associations. A total of 70 species, 54 genera, and 27 families were determined. Height of tree layer was the most important variable for discrimination of the successional stages. Conserved areas differed floristically from other stages, associated mainly with the lowest values of wind speed originated by tree layer characteristics. A significant association between species and microclimate was found, being wind speed and relative humidity the most important variables. Some species, due to their high importance values and their patterns of association with microclimate, may be considered as key taxa for low thorn forest, which is a threatened semitropical community in northeast Mexico. Conserved and late successional areas account for climatic regulation of this plant community, and the importance of these forest patches may be considered when establishing biodiversity protection areas.

     

Predictors of HVTN 503 MRK-AD5 HIV-1 gag/pol/nef Vaccine Induced Immune Responses

Background

Phambili, the Merck (MRK)-Adenovirus Type 5 (Ad5) HIV-1 gag/pol/nef subtype B vaccine study, conducted in South Africa, suspended enrollment and vaccination when companion study, Step, was found non-efficacious. Although the vaccine did not prevent HIV-1 infection or lower viral-load setpoint, immune responses recognized clades B and C HIV-1 subtypes. We investigated predictors of the vaccine-induced antigen-specific immune responses.

Methods

Vaccine-induced immunogenicity was ascertained by interferon-γ ELISpot assays on the first 186 enrolled participants receiving two vaccinations. Analyses, stratified by study arm/sex, were performed on baseline demographics [sex, age, Body Mass Index (BMI), site, Adenovirus Type-5 (Ad5) titer, Herpes Simplex Virus Type-2 (HSV2) status, heavy drinking]. Multivariate logistic regression determined predictors.

Results

Of the 186 participants, 53.7% (n = 100) were female, median BMI was 22.5 [IQR: 20.4–27.0], 85.5% (n = 159) were Ad5 seropositive, and 18.8% (n = 35) drank heavily. All vaccine recipients responded to both clade B (n = 87; 47%) and/or C (n = 74; 40%), p = 0.17. In multivariate analysis, female sex [Adjusted Odds Ratio (AOR): 6.478; p = 0.0159], overweight/obese BMI (AOR: 0.186; p = 0.0452), and heavy drinking (AOR: 0.270; p = 0.048) significantly predicted immune response to clade C for any antigens. A marginally significant predictor of clade C-pol antigen was female sex (AOR: 3.182; p = 0.0500).

Conclusions

Sex, BMI, and heavy drinking affected vaccine-induced HIV-1 specific immune responses to clade C antigens. The role of female sex and overweight/obese BMI boosting and suppressing vaccine-induced HIV-1 specific immune responses, respectively, requires elucidation, including any effect on HIV vaccine efficacy, especially in the era of colliding epidemics (HIV and obesity).     

Adverse Obstetric and Perinatal Outcomes following Treatment of Adolescent and Young Adult Cancer: A Population-Based Cohort Study

Objective

To investigate obstetric and perinatal outcomes among female survivors of adolescent and young adult (AYA) cancers and their offspring.

Methods

Using multivariate analysis of statewide linked data, outcomes of all first completed pregnancies (n = 1894) in female survivors of AYA cancer diagnosed in Western Australia during the period 1982–2007 were compared with those among females with no cancer history. Comparison pregnancies were matched by maternal age-group, parity and year of delivery.

Results

Compared with the non-cancer group, female survivors of AYA cancer had an increased risk of threatened abortion (adjusted relative risk 2.09, 95% confidence interval 1.51–2.74), gestational diabetes (2.65, 2.08–3.57), pre-eclampsia (1.32, 1.04–1.87), post-partum hemorrhage (2.83, 1.92–4.67), cesarean delivery (2.62, 2.22–3.04), and maternal postpartum hospitalization>5 days (3.01, 1.72–5.58), but no excess risk of threatened preterm delivery, antepartum hemorrhage, premature rupture of membranes, failure of labor to progress or retained placenta. Their offspring had an increased risk of premature birth (<37 weeks: 1.68, 1.21–2.08), low birth weight (<2500 g: 1.51, 1.23–2.12), fetal growth restriction (3.27, 2.45–4.56), and neonatal distress indicated by low Apgar score (<7) at 1 minute (2.83, 2.28–3.56), need for resuscitation (1.66, 1.27–2.19) or special care nursery admission (1.44, 1.13–1.78). Congenital abnormalities and perinatal deaths (intrauterine or ≤7 days of birth) were not increased among offspring of survivors.

Conclusion

Female survivors of AYA cancer have moderate excess risks of adverse obstetric and perinatal outcomes arising from subsequent pregnancies that may require additional surveillance or intervention.     

Isolation and characterization of a biosurfactant‐producing Fusarium sp. BS‐8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m^?1) with a critical micelle concentration of ≥ 1.2 mg mL^?1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L^?1 pure biosurfactant having significant emulsifying index (E₂₄, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1065–1075, 2014