Using immunofluorescence techniques to Identify T cells in the foreskin tissue after medical male circumcision

Background: Medical Male Circumcision (MMC) plays an important role in reducing the risk of acquiring sexually transmitted infections (STIs) such as Human papilloma virus (HPV), Herpes simplex type 2 (HSV-2) and HIV-1. The foreskin tissue (FS) is a site abundant in Langerhans cells (LCs), macrophages and T helper cells that express CD4 and CCR5 that are target markers for HIV1 binding and viral infection. The foreskin tissue may also contribute chemokines and cytokines including those that promote inflammation such as IL-17, IL-1β, IL-8, MCP-1 and MIG. The inner foreskin has been shown to contain higher levels of CD4+CCR5+ cells and thus more susceptible to HIV infection compared to the outer foreskin. It was demonstrated that the majority of chemokines measured were highly expressed in the inner foreskin compared to the outer foreskin including CCL27 which was approximately 7-fold higher in the inner foreskin compared to the outer foreskin, in congruent with the higher density of CD4+CCR5+ observed in the epithelium of the inner foreskin. In this study, we hypothesized that CCL27 upregulation in the inner foreskin triggers the recruitment of CD4+ T cells to the epithelium of the foreskin tissue. This could subsequently lead to increased susceptibility to infections in the inner foreskin tissue. The aims of this dissertation were: 1) to measure the impact of CCL27 on the recruitment of CD4+ T cells to the epithelium of the foreskin tissue using immunofluorescence imaging. 2) to compare manual counting and semi-automated method for counting dually positive cells. 3) to use multiparameter flow cytometry to characterize the cells recruited under the influence of CCL27. Methodology: Inner foreskin tissue (n=11) and outer foreskin tissue (n=4) explants were treated with either TNFα or CCL27 and evaluated using immunofluorescence imaging to quantify the levels of CD3 and CD4 expressing cells. Dually positive CD3+CD4+ cells were counted manually using softworx software on the Deltavision microscope and with semi-automated counting using PIPSQUEAK on ImageJ. TNFα and CCL27 treated inner and outer FS cells were immunophenotyped using polychromatic flow cytometry to measure and compare the densities of Th17 and Th22 cells under the influence of the chemokines. Results: Exogenous exposure of inner foreskin tissue explants to TNFα showed a significant increase in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin (p=0.035) from 78.90 cells/mm2 (IQR: 33.02-127.50) in the unstimulated inner foreskin explants to 134.80 cells/mm2 (IQR: 109.30-206.60). Similarly, the addition of exogenous CCL27 resulted in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin to increase from the unstimulated inner foreskin (value above) to 164.80 cells/mm2 (IQR: 140.30-184.90, p=0.008). No significant difference was observed in the median density of CD3+CD4+ T cells in the outer foreskin tissue explants after exposure to TNFα and CCL27 (36.50 cells/mm2 , IQR: 18.29-96.65 in the unstimulated tissues compared to 65.12 cells/mm2 , IQR: 7.30-202.80 in the TNFα stimulated tissues; p>0.999 and 24 cells/mm2 , IQR: 11.35-149.40 for the CCL27 stimulated tissues; p=0.686). The median density of CD3+CD4+ T cells in the epithelium of unstimulated inner foreskin tissue showed a trend of an increase from the unstimulated outer foreskin tissue but was not statistically different (127.50 cells/mm2 , IQR: 89.22-219.50 in the inner foreskin compared to 36.52 cells/mm2 , IQR:18.29-96.65 in the outer foreskin explants; p=0.057). When comparing the cell counting methods: manual counting vs semi-automated counting, we observed that the manual counting method estimated higher numbers of dually positive cells compared to the semiautomated method in samples measuring 200 cells/mm2 . Despite these differences, there was strong correlation (R=0.782, p0.999) in CCL27 treated explants. The median frequency of Th22 cells in the inner foreskin in the unstimulated tissue explants was 8.80% (IQR: 1.68-12.60%) vs 5.30% (IQR: 0.96-7.67%, p=0.250) in TNFα treated explants and 4.90% (IQR:0.75-7.39%, P=0.125) in CCL27 treated explants. Meanwhile, the median frequency of Th17 cells in the outer foreskin in the unstimulated tissue explants was 21.60% (IQR: 15.40-37.33%) vs 28.20% (IQR: 14.60-39.40%, P=0.750) in TNFα treated explants and 22.90% (IQR:22.90-29.50%, p>0.999) in CCL27 treated explants. The median frequency of Th22 cells in the outer foreskin in the unstimulated tissues was 4.67% (IQR: 2.30-12.90%) vs 5.37% (IQR: 5.34- 7.58%, P=0.750) in TNFα treated tissues and 4.45% (IQR:3.64-5.98%, p>0.999) in CCL27 treated tissues. Furthermore, FS cells isolated using Dispase had significantly lower median frequencies of cells expressing CCR6 (18.35%, IQR:1.33-28.30%) compared to whole tissue controls (41.90%, IQR: 22.46-67%, p=0.031). This impacted the characterization of CD4+ T cell subsets in FS cells and limited our ability to adequately phenotype and measure the impact of TNFα and CCL27 on FS-derived cells using flow cytometry. Conclusion: This study demonstrates that exogenous exposure of FS to TNFα and CCL27 increased the density of CD3+CD4+ T cells in the epithelium of the inner but not the outer foreskin tissue. It was noteworthy that the density of CD3+CD4+ in the epithelium of the inner foreskin was higher than the outer in the unstimulated tissues, suggesting that the proinflammatory environment in the inner FS potentially leads to higher density of T cells in the inner FS even without exogenous stimulation. These results suggest a possible mechanism for recruiting HIV target cells in the inner foreskin tissue associated with higher levels of CCL27 that recruits HIV-1 target T cells during inflammatory responses. A limitation to this conclusion is the small sample size in the outer foreskin. The study also shows potential bias depending on the method used to quantify dually positive cells, whereby semi-automated counting underestimated the densities of CD3+CD4+ T cells compared to manual counting and therefore careful consideration is required when selecting the quantification method. Furthermore, there were no significant difference in the frequencies of Th17 and Th22 cells after exposure to TNFα and CCL27 using flow cytometry. The effects of Dispase on cell surface marker expression and the low cell yield across the experiments impacted the characterization of Th17 and Th22 using flow cytometry and thus limiting capacity to determine how CCL27 influences these T cell subsets

Cross‐entropy method for distribution power systems reconfiguration

Cross-entropy (CE) is a powerful simulation method for the solution of continuous and combinatory optimization problems. The work presented here utilizes the CE method for the optimal topology of distribution power systems (DPSs). The optimal network switches are determined for the reduction of active power loss. The adapted CE method is tested on three case studies, namely, the 33-node, 83-node, and 880-node DPSs. The results are compared with other reconfigura-tion algorithms to demonstrate the superiority of the proposed algorithm. The impact of the distributed generation is also investigated. The effective integration of the photovoltaic panels at midday, when their production is highest and meets the peak demand, is showed. Finally, the real-time reconfiguration strategy based on the switching effort reduction is proposed and enhanced via an adequate selection of the initial switch states

Prevalence of Salmonella Typhi, Staphylococcus aureus and intestinal parasites among male food handlers in Laghouat Province, Algeria

Background: Food-borne diseases are a global public health problem, most especially in developing countries. Food handlers with a low level of  personal hygiene may be colonized or infected by a wide range of enteric pathogenic micro-organisms including intestinal parasites and bacteria.  The aim of this study is to determine the prevalence of Salmonella Typhi, Staphylococcus aureus and intestinal parasites among male food handlers  in Laghouat province, southern Algeria. Methodology: In this cross-sectional study, stool samples and fingernail samples of both hands were collected from 155 randomly selected male  food handlers. Stool specimens were examined by direct wet mount, formalinether concentration, xenic invitro culture and staining methods for  parasitological identification. For bacterial isolation, standard culture media including Hektoen agar, Salmonella-Shigella (SS), Mannitol salt, and  Blood agar plates were used. Conventional biochemical tests were used for identification of S. Typhi and S. aureus. Antimicrobial susceptibility test  (AST) was performed for bacterial isolates by the Kirby-Bauer disk diffusion method. Data analysis was done using Minitab version 19 software, and  Pearson’s Chi-square test was used to determine association between categorical variables. P value < 0.05 was considered statistically significant.   Results: The overall prevalence rate of intestinal parasites among the study subjects was 40% (62/155). Blastocystis spp was the most frequent  parasite isolated (16.8%), followed by Giardia intestinalis (8.4%), Entamoeba histolytica/dispar (7.7%), Entamoeba coli (3.2%), Trichomonas intestinalis  (2.6%) and Endolimax nana (1.3%). Stool cultures revealed 4 (2.6%) positive samples for S. Typhi, and S. aureus was isolated from fingernail contents  of 23 (14.8%) subjects. All S. Typhi isolates were sensitive to imipenem and ciprofloxacin while S. aureus isolates show high sensitivity to  pristinamycin. Hand washing with soap, finger nail status and clinical manifestations were significantly associated with intestinal parasitic infections,  while clinical manifestation was the only factor associated with S. aureus infection. Conclusion: The present study indicates a high prevalence of pathogenic micro-organisms among male food handlers which highlight the important role of food handlers in the spread and transmission of foodborne infections, and thus requires more attention

Influence of themo-mechanical treatments and microstructural state on the fatigue behaviour of a weald seam: case of API X60 steel

The aim of this work is the study of the fatigue behaviour of API X60 steel and the influence of thermal and mechanical treatments. The evaluation of the integrity and safety of welded structures dictates the approach taken in this research. The microstructural observations on the different zones of the weld seam indicates that the variation of heterogeneous structure is a progressive destruction of the strips of lamination which cause a new phase leading to a drop in the mechanical properties requiring treatment after welding. The fatigue cracking rate diverges beyond the threshold of DK, but no deviation of the crack from its propagation axis was noticed, which confirms the correct choice of filler metal over that of the base metal with an overmatching M = 1.1, and the treatments applied to the structure. This fatigue cracking rate transversal to the welding direction initially presents an aspect similar to that of BM but registers a delay as soon as the crack tip enters the second zone (HAZ) then it progresses rapidly. This evolution is characterized by a disturbance due to the repeated change of microstructure

Optimal Fractional-Order PI Control Design for a Variable Speed PMSG-Based Wind Turbine

© 2021 IIETA. . This is an open access article distributed under the Creative Commons Attribution License, to view a copy of the license, see: https://creativecommons.org/licenses/by/4.0/This paper focusses on the design of optimal control strategies for a variable-speed wind energy system based on Permanent Magnet Synchronous Generator (PMSG). The fractional order PI controller, denoted PIλ, is an extension of the classical PI controller, which provides greater flexibility, better performance and robustness, however the tuning of the controller parameters is challenging. In this work, Particle Swarm Optimization (PSO) and Genetic Algorithm (GA) provide approximate solutions to various problems and form a good optimization. In our system, they are used to have the PI regulator parameters and tune the parameters of the proposed controllers. The proposed controllers have been applied as maximum power point (MPPT) controllers for the wind turbine and to regulate the PMGS currents under variable weather conditions and. The results show that, among all these controllers, the fractional order PI controller optimized by the PSO leads to better performance in terms of the transient response characteristics such overshoot, rise time and settling time.Peer reviewedFinal Published versio

Effect of thermal aging above the glass transition temperature on the mechanical properties of rigid PVC tube

Thermal aging of standard samples of rigid PVC (u-PVC) taken from plastic tubes near and above the glass transition temperature (Tg) was used to investigate the effect of elevated temperatures on the mechanical properties of rigid (u-PVC) tubes used in industrial areas. Three aging protocols were used, each lasting 90 days at 80 °C, 100 °C, and 120 °C. Crosslinking and chain scission reactions changed the tensile properties, either increasing them or decreasing them. For all three aging protocols, it was discovered that Young's modulus increased with aging time. Furthermore, the rate of increase of the modulus was observed to increase with aging temperature. The Yield stress showed the same pattern. Their increase in both cases is due to material stiffening caused by a crosslinking process. This last one was revealed by DSC, where Tg increased as a result of the chemical process. The decrease in elongation at break, which occurred progressively in all aging protocols and more precisely at temperatures above Tg, on the other hand, demonstrated the relationship between chain scissions and temperature. The material becomes more brittle as the temperature rises. Brittleness is caused by a high activity of chain scissions on the surface sample, which allows micro-cracks to form. When subjected to tensile forces, cracks propagate from the surface toward the sample's thickness.