Immunological and molecular assessment of HIV-1 mutations for antiretroviral drug resistance in Saudi Arabia.

Human Immunodeficiency Virus (HIV) is a significant threat to public health. HIV genotyping and antiretroviral resistance testing may have contributed to improved non-treated management. Immune markers might assist HIV-1 diagnosis and drug-resistant variant identification. HIV-1 immunogenicity and molecular characteristics of antiretroviral drug resistance are evaluated in 56 treatment-naive HIV patients. DNA sequencing and retroviral resistance testing identified HIV-1 genotypes. 55.4% of patients were susceptible to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) antiretroviral drugs, whereas 44.6% had drug-resistance mutations against at least one antiretroviral drug. 3.6% of cases had PI-resistant mutations, while 30.4% had NRTI-resistant mutations, and 30.4% had NNRTI-resistant mutations. In patients who are susceptible to PI, the mean value of human plasma sCD80 is 2.11 ± 0.65 ng/mL; in patients with mutations, it is 3.93 ± 2.91 ng/mL. Individuals who are susceptible to PI have plasma sCD27 levels of 78.7 ± 63.2 U/mL, whereas individuals who are mutant have levels of 56.5 ± 32.1 U/mL. IP-10's mean value was 363 ± 109.2 pg/mL for the susceptible patients and 429 ± 20.7 pg/mL for the mutated patients. In susceptible patients, the plasma sCD4 level is 0.163 ± 0.229 ng/mL; in mutant patients, it is 0.084 ± 0.012 ng/mL. The data showed a relative relation between immunological parameters such as sCD80, sCD27, sCD4, and IP-10 and mutation for drug resistance.

Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia.

Herpesvirus entry mediator (HVEM) is a molecular switch that can modulate immune responses against cancer. The significance of HVEM as an immune checkpoint target and a potential prognostic biomarker in malignancies is still controversial. This study aims to determine whether HVEM is an immune checkpoint target with inhibitory effects on anti-tumor CD4+ T cell responses in vitro and whether HVEM gene expression is dysregulated in patients with acute lymphocytic leukemia (ALL). HVEM gene expression in tumor cell lines and peripheral blood mononuclear cells (PBMCs) from ALL patients and healthy controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor cells were left untreated (control) or were treated with an HVEM blocker before co-culturing with CD4+ T cells in vitro in a carboxyfluorescein succinimidyl ester (CFSE)-dependent proliferation assay. HVEM expression was upregulated in the chronic myelogenous leukemia cell line (K562) (FC = 376.3, p = 0.086) compared with normal embryonic kidney cells (Hek293). CD4+ T cell proliferation was significantly increased in the HVEM blocker-treated K562 cells (p = 0.0033). Significant HVEM differences were detected in ALL PBMCs compared with the controls, and these were associated with newly diagnosed ALL (p = 0.0011) and relapsed/refractory (p = 0.0051) B cell ALL (p = 0.0039) patients. A significant differentiation between malignant ALL and the controls was observed in a receiver operating characteristic (ROC) curve analysis with AUC = 0.78 ± 0.092 (p = 0.014). These results indicate that HVEM is an inhibitory molecule that may serve as a target for immunotherapy and a potential ALL biomarker.

Interaction between Gut Microbiota and Dendritic Cells in Colorectal Cancer.

Colorectal cancer (CRC) is a malignancy that manifests in serial stages and has been observed to have an escalating incidence in modern societies, causing a significant global health problem. The development of CRC is influenced by various exogenous factors, including lifestyle, diet, nutrition, environment, and microbiota, that can affect host cells, including immune cells. Various immune dysfunctions have been recognized in patients with CRC at different stages of this disease. The signature of microbiota in the development of CRC-inflammation related to obesity, diet, and reactive host cells, such as dendritic cells (DCs)-has been highlighted by many studies. This study focuses on DCs, the primary cellular mediators linking innate and adaptive immune responses against cancer. In addition, this review focuses on the role of microbiota in dysbiosis and how it affects DCs and, in turn, the immune response and progression of CRC by stimulating different sets of T cells. Additionally, DCs' role in protecting this delicate balance is examined. This is to determine how gene yields of commensal microbiota may be critical in restoring this balance when disrupted. The stages of the disease and major checkpoints are discussed, as well as the role of the C-type lectin receptor of immature DCs pattern recognition receptor in CRC. Finally, based on a thorough examination of worldwide clinical studies and recent advancements in cancer immunotherapy, it is recommended that innovative approaches that integrate DC vaccination strategies with checkpoint inhibitors be considered. This approach holds great promise for improving CRC management.

Optimizing the generation of mature bone marrow-derived dendritic cells in vitro: a factorial study design.

BACKGROUND: Factorial design is a simple, yet elegant method to investigate the effect of multiple factors and their interaction on a specific response simultaneously. Hence, this type of study design reaches the best optimization conditions of a process. Although the interaction between the variables is widely prevalent in cell culture procedures, factorial design per se is infrequently utilized in improving cell culture output. Therefore, we aim to optimize the experimental conditions for generating mature bone marrow-derived dendritic cells (BMDCs). Two different variables were investigated, including the concentrations of the inducing factors and the starting density of the bone marrow mononuclear cells. In the current study, we utilized the design of experiments (DoE), a statistical approach, to systematically assess the impact of factors with varying levels on cell culture outcomes. Herein, we apply a two-factor, two-level (22) factorial experiment resulting in four conditions that are run in triplicate. The two variables investigated here are cytokines combinations with two levels, granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or with interleukin-4 (IL4). The other parameter is cell density with two different concentrations, 2 × 106 and 4 × 106 cells/mL. Then, we measured cell viability using the trypan blue exclusion method, and a flow cytometer was used to detect the BMDCs expressing the markers FITC-CD80, CD86, CD83, and CD14. BMDC marker expression levels were calculated using arbitrary units (AU) of the mean fluorescence intensity (MFI). RESULTS: The current study showed that the highest total viable cells and cells yield obtained were in cell group seeded at 2 × 106 cells/mL and treated with GM-CSF and IL-4. Importantly, the expression of the co-stimulatory molecules CD83 and CD80/CD86 were statistically significant for cell density of 2 × 106 cells/mL (P < 0.01, two-way ANOVA). Bone marrow mononuclear cells seeded at 4 × 106 in the presence of the cytokine mix less efficiently differentiated and matured into BMDCs. Statistical analysis via two-way ANOVA revealed an interaction between cell density and cytokine combinations. CONCLUSION: The analysis of this study indicates a substantial interaction between cytokines combinations and cell densities on BMDC maturation. However, higher cell density is not associated with optimizing DC maturation. Notably, applying DoE in bioprocess designs increases experimental efficacy and reliability while minimizing experiments, time, and process costs.

Dendritic Cell-Based Immunity: Screening of Dendritic Cell Subsets in Breast Cancer-Bearing Mice.

Background: Breast cancer (BC) is the most devastating disease, particularly the lethal invasive form. It is the most underlying cause of death among women worldwide. The expansion of BC is controlled by a variety of alterations in the tumor cells themselves, in addition to the state of the immune system, which has a direct influence on the tumor microenvironment. Numerous receptors expressed by T-cells interact with ligands on antigen-presenting cells to provide activation signals results in mounting effector anti-tumor T-cell responses. On the other hand, there is a dearth of information about the actual interactions and reactions of T-cells and dendritic cells (DCs) all through the progression of tumor development. Aim: Immune system response against BC was investigated through tumor induction in mice. The size and volume of the tumor were calculated. Moreover, the phenotypical profile of T-cells and DCs from lymph nodes (LN) and spleens of BC-bearing mice was investigated. In addition, the levels of Transforming growth factor-ß, Interferon-gamma (IFN-γ), Interleukin IL-2, IL-10, IL-4, IL-12, and tumor necrosis factor (TNF)-α were determined. Materials and Methods: MDA231 cells were utilized to induce BC in 30 white BALB/C mice, whereas the other 30 mice acted as healthy controls and were not treated with any cancer-causing agents. The impact of malignancy was evaluated using flow cytometry based on the marking surface molecules, as well as the titer of specific cytokines of the mice's LN culture using the ELISA method. These cytokines included transforming growth factor-ß (TGF-ß), IFN-γ, IL-2, IL -10, IL -4, IL -12, and TNF-α. Results: The findings showed that the maturation of DCs was inhibited, followed by an accumulation of immature DCs. These immature DCs increase the release of TGF-ß and cytokines like IL-10 and inhibit the release of IFN-γ and IL-12 in the culture supernatant of nodal lymph and spleen suspension of BC-bearing mice compared to control. In addition, there was a low expression of CD80 and CD86 on DCs, which indicates a low maturation process. Conclusion: According to the findings, the tumor microenvironment may have been responsible for preventing the maturation of DCs. This, in turn, weakened the immune response and facilitated the ability of the tumor to proliferate. Furthermore, the tumor microenvironment increased the number of immature DCs by inhibiting their stimulation by overexpression of TGF-ß-produced by regulatory T lymphocytes and stimulation of tumor cells. In addition, the tumor microenvironment stimulated the secretion of cytokines such as IL-10, and CD4 and decreased the secretion of IFN-γ-and IL-12 in tumor-induced mice cultured LN and spleen.

Herpesvirus entry mediator as a potential biomarker in breast cancer compared with conventional cytotoxic T­lymphocyte­associated antigen 4.

Breast cancer (BC) is the most common cancer in women worldwide, with 2.3 million cases recorded in 2020. Despite improvements in cancer treatment, patients with BC still succumb to the disease, due to regional and distant metastases when diagnosed at later stages. Several immune checkpoint inhibitors have been approved for BC treatment, based on their expression and role in maintaining immunosurveillance against tumors. The present study aimed to evaluate the expression of 12 immune checkpoints in patients with BC, and assess their role as diagnostic and therapeutic markers. Expression levels were measured using reverse transcription-quantitative polymerase chain reaction. Among the 12 immune markers, herpesvirus entry mediator (HVEM) was found to be significantly upregulated in patients with malignant BC compared to non-malignant controls, with a relative fold change (FC) of 1.46 and P=0.012. A similar finding was observed for cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; FC=1.47 and P=0.035). In addition, receiver operating characteristic curve analysis revealed that HVEM expression allowed significant differentiation between groups, with an area under the curve of 0.74 (P=0.013). Upregulation in both HVEM and CTLA4 was revealed to be significantly associated with the human epidermal growth factor receptor-2 (HER2)-enriched phenotype (FC=3.53, P=0.009 and FC=5.98, P=0.002, respectively), while only HVEM was significantly associated with the triple-negative phenotype (FC=2.07, P=0.016). Furthermore, HVEM was significantly higher in patients with grade III tumors (FC=1.88, P=0.025) and negative vascular invasion (FC=1.67, P=0.046) compared with non-malignant controls. Serum protein levels were assessed by multiplex immunoassay, and a significant increase in HVEM was detected in patients with malignant BC compared with that in non-malignant controls (P=0.035). These data indicated that HVEM may serve as a potential biomarker and target for immunotherapy, especially for certain types of BC.

Gender Differences in Response to COVID-19 Infection and Vaccination.

Since COVID-19 first appeared, a number of follow-up events have taken place. In an effort to find a solution to this catastrophe, a great deal of study and analysis has been conducted. Because of the high morbidity and exceptionally large losses, scientists are being pushed to conduct more research and find vaccination and treatments. The virus has a wide range of effects, one of which is how it affects sexual activity in both men and women. The impact of the cardiovascular system and susceptibility to embolism, lung stress, and infection heightens the probability of hospitalization in the intensive care unit for pregnant women who have contracted COVID-19. There is no evidence of infection being passed from mother to child. In the current review, the role of COVID-19 infection and vaccination on male and female sexual activity, hormones, and the menstrual cycle for females, as well as on male sex hormones and sexual activity during infection and after vaccination, are being investigated. There are no reports of the virus being isolated from the semen of an infected patient or recently recovered patients. A recent investigation on the influence of the virus on gender susceptibility to sexual organs and function has been uncovered throughout this study.

Genotyping and phylogenetic analysis of canine parvovirus circulating in Egypt.

AIM: This study aimed to detect and characterize current genotypes of canine parvovirus (CPV) in Egypt during 2018. MATERIALS AND METHODS: A total of 50 fecal swabs were collected from clinically infected domestic dogs of 2-5 months of age, suspected to suffer from CPV infection, from Cairo and Giza Governorates. The samples were subjected to qualitative antigen detection using the rapid test, followed by isolation on Madin-Darby Canine Kidney (MDCK) cells, molecular characterization with partial amplification of VP2 gene using polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis. RESULTS: Out of 50 fecal samples, 20 samples were positive (40%) by Rapid CPV/canine coronavirus Ag Test Kit. These positive samples were cultured successfully on MDCK cells. Nine randomly chosen samples out of 30 apparently negative samples were amplified using PCR with primers Hfor and Hrev to yield a typical 630 bp fragment. Then, six randomly chosen samples out of nine were amplified using PCR with primers Pbs and Pbas to yield a typical 427 bp fragment. Sequencing, BLAST analysis and assembly of the two fragments (630 bp and 427 bp) to produce 912 bp fragments, in the six samples, revealed two serotypes CPV-2b and CPV-2c. The obtained strains were submitted to GenBank and given accession numbers MK642272, MK642273, MK642274, MK642275, MK642276, and MK642277. Phylogenetic analysis of the Egyptian strains serotype 2b illustrated that they were closely related to Thailand strains (accession numbers KP715709, KP715694, KP715701, and KP715700); while Egyptian strains serotype 2c was closely related to Thailand strains (accession numbers MH711894 and MH711902), Taiwanese strain (KU244254), Chinese strain (MF467242), and Vietnamese strain (accession number LC216910). CONCLUSION: The current research recommends further epidemiological studies to assess the extent of the occurrence of different serotypes of CPV in Egypt and the efficiency of imported and locally produced vaccines in protection against CPV infection.