Development of a high-throughput image cytometric screening method as a research tool for immunophenotypic characterization of patient samples from clinical studies.

Immunophenotyping has been the primary assay for characterization of immune cells from patients undergoing therapeutic treatments in clinical research, which is critical for understanding disease progression and treatment efficacy. Currently, flow cytometry has been the dominant methodology for characterizing surface marker expression for immunological research. Flow cytometry has been proven to be an effective and efficient method for immunophenotyping, however, it requires highly trained users and a large time commitment. Recently, a novel image cytometry system (Cellaca® PLX Image Cytometer, Revvity Health Sciences, Inc., Lawrence, MA) has been developed as a complementary method to flow cytometry for performing rapid and high-throughput immunophenotyping. In this work, we demonstrated an image cytometric screening method to characterize immune cell populations, streamlining the analysis of routine surface marker panels. The T cell, B cell, NK cell, and monocyte populations of 46 primary PBMC samples from subjects enrolled in autoimmune and oncological disease study cohorts were analyzed with two optimized immunophenotyping staining kits: Panel 1 (CD3, CD56, CD14) and Panel 2 (CD3, CD56, CD19). We validated the proposed image cytometry method by comparing the Cellaca® PLX and the AuroraTM flow cytometer (Cytek Biosciences, Fremont, CA). The image cytometry system was employed to generate bright field and fluorescent images, as well as scatter plots for multiple patient PBMC samples. In addition, the image cytometry method can directly determine cell concentrations for downstream assays. The results demonstrated comparable CD3, CD14, CD19, and CD56 cell populations from the primary PBMC samples, which showed an average of 5% differences between flow and image cytometry. The proposed image cytometry method provides a novel research tool to potentially streamline immunophenotyping workflow for characterizing patient samples in clinical studies.

Interleukin 17 and Growth Differentiation Factor 9 Influence in a Sample of Iraqi Infertile Males.

Infertility is defined as the inability of couples to conceive after 1 year of regular unprotected intercourse, which affects 10-15% of couples. The present study aimed to investigate the influence of Interleukin-17 (IL-17) and growth differentiation factor 9 (GDF9) on three groups of infertile males, including control, azoospermia, and oligozoospermia. In total, this study was performed on 93 participants, consisting of 18, 65, and 10 subjects in the Azoospermia, oligozoospermia, and control groups, respectively. The mean plasma levels of IL-17 in the azoospermia and oligozoospermia groups were 21.317±3.605 and 15.101±2.416 ng/l, respectively, which were significantly higher than that in the control group (5.392±1.731 ng/l). Furthermore, the mean plasma levels of GDF9 in the azoospermia and oligozoospermia groups were 3.299±1.051 and 6.2603±2.621 ng/l, respectively, which was significantly higher than that in the control group (12.807±2.170 ng/l). One-way analysis of variance and least significant difference post-hoc test were performed to assess significant differences among means. R-squared measures how well the linear regression model fits the data. It can be interpreted as the proportion of variance of the outcome Y explained by the linear regression model. R-squared is a number between 0 and 1. In non-obstructive forms of severe oligozoospermia and azoospermia, like the case in the current study, intracytoplasmic sperm injection is suggested by using testicular biopsy for spermatozoa extraction, if viable spermatozoa are present.

Development of a T-ARMS-PCR Assay for Detecting Genetic Polymorphism in the Catalase (rs7943316) Gene in the Iraqi Population with Breast Cancer.

Numerous investigations have demonstrated that oxidative stress is markedly increased in breast cancer patients compared to their healthy counterparts. Catalase (CAT), a crucial antioxidant enzyme, plays a pivotal role in safeguarding cells against oxidative damage initiated by reactive oxygen species (ROS). The CAT (rs7943316) gene encodes catalase, and certain genetic variations in this gene have been observed to modify catalase activity and levels. Such changes can lead to an altered response to oxidative stress, potentially increasing the risk of breast cancer. In light of this, a novel tetra-primer amplification-refractory mutation system (T-ARMS)-PCR assay was developed to investigate the possible correlation between the CAT (rs7943316) gene polymorphism and the development of breast cancer in patients. This method employs a one-step PCR, which is faster, more cost-effective, and more precise than existing techniques. Sanger sequencing was performed to validate the accuracy of our findings. The T-ARMS-PCR assay revealed a significant association between the A/T allele of the CAT (rs7943316) gene and breast cancer. Specifically, individuals with the TT genotype had a higher risk of developing breast cancer than those with the AA genotype. The T allele frequency was greater among breast cancer patients than in the control group, and genotype frequencies were consistent with the principles of the Hardy-Weinberg Equilibrium. This study is the first to showcase a rapid, cost-effective, and high-throughput method for detecting the SNP in the CAT (rs7943316) gene. This method has the potential to be employed in large-scale clinical trials.

Association of the Manganese Superoxide Dismutase (Mn-SOD) Gene C47T Polymorphism with Lung Cancer: A Case-Control Study.

OBJECTIVE: The objective of this study was to determine the association between manganese superoxide dismutase (Mn-SOD) gene C47 T polymorphism and the risk of malignant lung cancer in Iraqi smokers. METHODS: Blood samples were obtained from 260 lung cancer patients (88 females and 172 males) and 295 healthy individuals (91 females and 204 males). DNA was extracted from blood samples and the SOD2 gene was amplified using specific primers. The nucleotide sequences of the SOD2 gene were analyzed by using BLAST server at National Center for Biotechnology Information (NCBI) and the Raptorx app. RESULTS: TT, CT, and CC genotypes concentrations were 48.1%, 33.2%, and 18.7%, respectively, in the control group. The concentrations of TT, CT, and CC genotypes were 43.5%, 31.5%, and 25%, respectively, in the case group. There were no statistical differences between cases and controls in terms of genotype frequency of SOD2C47T polymorphism. We observed that SOD2C47T polymorphism CT genotype did not increase the risk of lung cancer development compared to those with TT genotype (OR= 0.951, 95% CI = 0.648-1.396; P = 0.798). In addition, it was observed that CC genotype did not increase the risk of lung cancer development in comparison with TT genotype ( OR=0.673, 95% Cl=0.435-1.041: P=0.075). CONCLUSION: These results indicated that there was no association between SOD2C47T polymorphism and the risk of lung cancer development in Iraqi smokers.

A Rare Case of Choroid Plexus Papilloma of the Third Ventricle in an Adult.

Colloid cysts are the commonest masses of the third ventricle. Third ventricle neoplasms are uncommon. They include tumors arising from the choroid plexus (papillomas, carcinomas), tumors arising from other than the choroid plexus (ependymomas, meningiomas), metastases, and lymphoma. Choroid plexus tumors usually occur in the lateral ventricle in children and fourth ventricle in adults, and often present with hydrocephalus. We herein describe the extremely rare occurrence of third ventricle choroid plexus papilloma in a 35-year-old man who presented to the emergency department with a long history of intermittent headaches, occasionally associated with photophobia. CT and MR imaging revealed a lobulated ovoid lesion in the third ventricle with minimal extension into the right lateral ventricle through the foramen of Monro, causing mild ventricular dilatation. Surgical resection was performed and histopathology revealed choroid plexus papilloma.

Zinner Syndrome Unmasked by Workup for Renal Colic and Uncontrolled Hypertension.

Zinner syndrome is a rare hereditary disorder of the mesonephric duct. The triad of the absence of one kidney, ipsilateral cystic dilatation of the seminal vesicle, and ejaculatory duct obstruction makes the diagnosis. Mostly, it is asymptomatic. However, genitourinary manifestations and workup for the incidental absence of one kidney often uncover the disease. Ultrasound and CT scan can identify the absence of a kidney and seminal vesicle cyst, while MRI is the gold standard for diagnostic elaboration of the pelvic anatomy. In this article, we have presented a 51-year-old male patient who presented with renal colic and hypertension. Radiological investigations for the renal colic uncovered the diagnosis of Zinner syndrome incidentally. This case highlights the incidental nature, variability in the clinical presentation, and the diagnostic challenges of this rare disorder. It also emphasizes on the radiologist for a careful evaluation of the pelvic images in patients with unilateral absence of a kidney.

Phytotoxic and genotoxic effect of Aluminum to date palm (Phoenix dactylifera L.) in vitro cultures.

BACKGROUND: Al is a common metallic element found in earth's crust and is a toxic pollutant present at high concentrations in acidic soil, thus affecting plant growth. Despite being well studied as a toxic element, the effects of Al on date palm have not been investigated. This study aimed to assess the toxic effects of different Al concentrations on the development and growth of date palm callus and evaluate the biochemical and molecular response of date palm cells under Al stress. RESULTS: Our study revealed the phytotoxicity of Al concentrations (50, 100, 150 and 200 mg.l-1) on date palm callus. The fresh and dry weight and the number of produced embryos were significantly decreased in response to Al concentration. At 150 mg.l-1, the embryo number decreased to 1.66 compared with the 19.33 in the control treatment. At high Al concentration (200 mg.l-1), the callus failed to produce any embryo. Biochemical analysis revealed that Al exposure had negative effect on callus. Total soluble carbohydrates, total soluble protein and free amino acids were decreased in plants receiving 200 mg.l-1 Al treatment compared with those in the untreated ones. A similar decline was observed in total soluble protein and free amino acid in response to Al treatment. Significant accumulations of malondialdehyde, H2O2 and peroxidase activity accompanied the increase in Al concentration in cultured tissues, revealing the generation of toxic reactive oxygen species in affected cultures. The genotoxic effect of Al at high concentrations (150 and 200 mg.l-1) was revealed by protein patterns. CONCLUSION: Our findings revealed for the first time the phytotoxicity of Al to date palm callus. At 200 mg.l-1, Al prevented the embryo production of date palm callus. At 50, 100, 150 and 200 mg.l-1, Al negatively affected the biochemical characteristics of date palm callus. At 150 and 200 mg.l-1, Al induced changes in protein expression. These data showed that the tissue culture technique can be used as a valuable approach in heavy metal toxicity studies.