Attitude and Acceptance towards COVID-19 Booster Doses among Literacy Advantaged Population in Pakistan: A Cross-Sectional Study.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has affected billions of lives and is expected to impose a significant burden on the economy worldwide. Vaccination is the only way to prevent the infection. However, convincing people to get themselves vaccinated is challenging in developing countries such as Pakistan. Therefore, a cross-sectional questionnaire-based study was conducted (n = 982 participants) all over Pakistan to evaluate the perception, knowledge, attitude, and acceptance of the general public towards the SARS-CoV-2 vaccine, in general, and a booster dose of SARS-CoV-2, in particular. The highest number of participants were from the province of Punjab (84.5%), followed by Islamabad (3.8%), Sindh (3.7%), Khyber Pakhtunkhwa (2.7%), Baluchistan (2.6%), Gilgit Baltistan (1.4%), and Azad Jammu and Kashmir (1.4%). A total of 915 participants were vaccinated against COVID-19, out of which 62.2% received one booster dose, followed by double booster doses (25.5%) and single vaccine shots (12.3%). The highest number of vaccinated participants were from Punjab (85.8%), followed by Islamabad (3.9%), Sindh (2.8%); Khyber Pakhtunkhwa (2.6%); Baluchistan (2.3%); Gilgit-Baltistan (1.3%); and Azad, Jammu, and Kashmir (1.2%). Among the vaccinated individuals, 71.4% were unemployed, 27.4% were employed (653), and 1.2% were retired from service. However, no significant association was observed among genders and educational levels in regard to acceptance of the booster vaccine. The outcomes of the study revealed that the increased acceptance of booster doses of the SARS-CoV-2 vaccines among the public was associated with the intent of personal and family protection. Moreover, individuals with low socioeconomic status and pregnant females showed the least acceptance towards the vaccine inoculation. The study also revealed a decline trend of accepting SARS-CoV-2 vaccine among children.

Molecular Epidemiological Investigations of Localized SARS-CoV-2 Outbreaks-Utility of Public Algorithms.

The recent rapid expansion of targeted viral sequencing approaches in conjunction with available bioinformatics have provided an effective platform for studying severe acute respiratory syndrome coronavirus-2 (CoV-2) virions at the molecular level. These means can be adapted to the field of viral molecular epidemiology, wherein localized outbreak clusters can be evaluated and linked. To this end, we have integrated publicly available algorithms in conjunction with targeted RNASeq data in order to qualitatively evaluate similarity or dissimilarity between suspect outbreak strains from hospitals, or assisted living facilities. These tools include phylogenetic clustering and mutational analysis utilizing Nextclade and Ultrafast Sample placement on Existing tRee (UShER). We herein present these outbreak screening tools utilizing three case examples in the context of molecular epidemiology, along with limitations and potential future developments. We anticipate that these methods can be performed in clinical molecular laboratories equipped with CoV-2-sequencing technology.

Similarity Index-Probabilistic Confidence Estimation of SARS-CoV-2 Strain Relatedness in Localized Outbreaks.

Outbreaks of SARS-CoV-2 can be attributed to expanding small-scale localized infection subclusters that eventually propagate into regional and global outspread. These infections are driven by spatial as well as temporal mutational dynamics wherein virions diverge genetically as transmission occurs. Mutational similarity or dissimilarity of viral strains, stemming from shared spatiotemporal fields, thence serves as a gauge of relatedness. In our clinical laboratory, molecular epidemiological analyses of strain association are performed qualitatively from genomic sequencing data. These methods however carry a degree of uncertainty when the samples are not qualitatively, with reasonable confidence, deemed identical or dissimilar. We propose a theoretical mathematical model for probability derivation of outbreak-sample similarity as a function of spatial dynamics, shared and different mutations, and total number of samples involved. This Similarity Index utilizes an Essen-Möller ratio of similar and dissimilar mutations between the strains in question. The indices are compared to each strain within an outbreak, and then the final Similarity Index of the outbreak group is calculated to determine quantitative confidence of group relatedness. We anticipate that this model will be useful in evaluating strain associations in SARS-CoV-2 and other viral outbreaks utilizing molecular data.

Perceived risk and perceptions of COVID-19 vaccine: A survey among general public in Pakistan.

BACKGROUND: The coronavirus disease has become a global pandemic, and it continues to wreak havoc on global health and the economy. The development of vaccines may offer a potential eradication of COVID-19. This study evaluated the general knowledge, attitude, and perception of COVID-19 vaccines in the Pakistani population. METHODS: A self-reporting e-survey and questionnaire-based survey from vaccination centers of different cities of Pakistan among 502 participants were conducted. The questionnaire comprised four sections inquiring demographics, vaccination status, and perception or attitude towards the vaccine. Univariate logistic regression was applied to predict the knowledge, attitude and behavior of participants. RESULTS: The mean age of participants was 50.8±20.3 years. 53% of the participants have both doses of vaccine administered. Pain on the site of injection (49.8%) was the most common symptom, followed by asthenia (43.0%), muscle pain (29.5%), and swelling (24.5%) on the site of vaccine administration. Females complain of more symptoms than males. More severe symptoms were reported after the first dose of vaccine administration; these symptoms subsided within a week for most participants. Overall, the respondents have a positive attitude towards the vaccine. 47.4% are sure about the vaccine's efficacy, 48.6% said getting vaccinated was their own decision, and 79.9% also recommended others to get vaccinated. CONCLUSION: The study concluded that the Pakistani population has a positive attitude but inadequate knowledge towards COVID-19 vaccines. Immediate awareness and vaccination education programs should be conducted by the authorities to complete the mass vaccination schedule.

Decidualization score identifies an endometrial dysregulation in samples from women with recurrent pregnancy losses and unexplained infertility.

OBJECTIVE: To study decidualization-associated endometrial factors. DESIGN: Retrospective cohort study to compare endometrial gene expression patterns in women experiencing reproductive failure including recurrent pregnancy loss or unexplained infertility versus fertile controls. SETTING: University Reproductive Medicine Center. PATIENTS: Women experiencing recurrent reproductive failure including recurrent pregnancy loss or unexplained infertility (n = 42) and fertile controls (n = 18). INTERVENTIONS: Endometrial biopsy samples were analyzed with targeted ribonucleic acid sequencing via next-generation sequencing. MAIN OUTCOME MEASURES: The primary end point measurements were the expression of genes important for endometrial transformation during decidualization measured singly and in a combined/cumulative score approach. The secondary end point measurements were receiver operating curve analysis and comparisons between the specific biomarkers. RESULTS: The comparison revealed differential expression of factors associated with decidualization, tissue homeostasis, and immune regulation: FOXO1, GZMB, IL15, SCNN1A, SGK1, and SLC2A1. A combined evaluation of these 6 signature factors was designated as a decidualization score in which the maximal score was "6" and the minimal was "0". Among controls, 89% of the samples had a score ≥5 and 11% had a score of "4". A total of 76% of samples in the patient group had scores ≤4 and 19% had the lowest score of "0". A decidualization score <4 provided evidence of abnormality in the decidualization process with a sensitivity of 76% (95% CI 61%-88%) and specificity of 89% (95% CI 65%-99%). CONCLUSIONS: Decidualization scoring can determine whether the endometrial molecular profile is implantation-friendly. Further validation of this testing approach is necessary to determine a particular patient population in whom it could be used for selecting patients that require therapeutic actions to improve endometrial conditions prior to the in vitro fertilization procedure.

Epidemiological and Clinical Characteristics of COVID-19: A Retrospective Multi-Center Study in Pakistan.

The emergence of a pathogen responsible for a mysterious respiratory disease was identified in China and later called a novel coronavirus. This disease was named COVID-19. The present study seeks to determine the epidemiological and clinical characteristics of COVID-19 in Pakistan. This report will exhibit a linkage between epidemiology and clinical aspects which in turn can be helpful to prevent the transmission of the virus in Pakistan. A retrospective, multiple center study was performed by collecting the data from patients' with their demographics, epidemiological status, history of co-morbid conditions, and clinical manifestations of the disease. The data was collected from 31 public-sector and 2 private hospitals across Pakistan by on-field healthcare workers. A Chi-square test was applied to assess the relationship between categorical data entries. A total of 194 medical records were examined. The median age of these patients was found to be 34 years. A total of 53.6% active cases were present including 41.2% males and 12.4% females till the end of the study. Adults accounted for most of the cases (94.3%) of COVID-19. Fever (86.60%), cough (85.05%), fatigue (36.60%), dyspnea (24.74%), and gastrointestinal discomfort (10.31%) were among the most frequently reported signs and symptoms by the patients. However, 4.12% of the total patient population remained asymptomatic. The median duration of hospital stay was found to be 14 (0-19) days. The earliest source of the spread of the virus may be linked to the foreigners traveling to Pakistan. Spread among men was more as compared to women. A few cases were found to be positive, due to the direct contact with pets or livestock. Hypertension (7.73%), diabetes (4.64%), cardiovascular conditions (2.58%) were the most common co-morbidities. The percentage mortality was 2.50% with the highest mortality among elders.

Human BM-MSC secretome enhances human granulosa cell proliferation and steroidogenesis and restores ovarian function in primary ovarian insufficiency mouse model.

Primary ovarian insufficiency (POI) is defined as the loss of ovarian function before 40 years of age. It clinically manifests as amenorrhea, infertility, and signs of estrogen insufficiency. POI is frequently induced by chemotherapy. Gonadotoxic chemotherapy reagents damage granulosa cells, which are essential for follicular function and development. Our recently published studies demonstrated that intraovarian transplantation of human mesenchymal stem cells (hMSCs) can restore fertility in a chemotherapy-induced POI mouse model. However, the regenerative mechanism underlying the hMSC effect in POI mice is not fully understood. Here, we report that the hMSC secretome increased the proliferation of human granulosa cells (HGrC1). We showed by FACS analysis that treatment of HGrC1 cells with hMSC-conditioned media (hMSC CM) stimulates cellular proliferation. We also demonstrated that the expression of steroidogenic enzymes involved in the production of estrogen, CYP19A1 and StAR, are significantly elevated in hMSC CM-treated HGrC1 cells. Our data suggest that hMSC CM stimulates granulosa cell proliferation and function, which may explain the therapeutic effect of hMSCs in our chemotherapy-induced POI animal model. Our findings indicate that the hMSC secretome may be a novel treatment approach for restoring granulosa cell and ovarian function in patients with POI.

Clinical molecular genetics evaluation in women with reproductive failures.

Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)-based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection-based abnormalities such as human herpesvirus-6 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.

Attitude, Perception, and Knowledge of COVID-19 Among General Public in Pakistan.

The World Health Organization has acknowledged coronavirus disease 2019 (COVID-19) disease as a pandemic. Efforts are being made all over the world to raise awareness to prevent the spread of the disease. The goal of this study was to assess the attitude, perception, and knowledge of Pakistani people toward COVID-19 disease. We conducted a cross-sectional survey in which a questionnaire of 17 questions was transformed online on Google forms and was sent to random individuals online. A total of 1,000 questionnaires from individuals throughout Pakistan were evaluated. The results revealed that 42.9% of the participants knew about COVID-19 through social media, the largest source of information. Most of the participants (48.3%) started working from home amid the lockdown; 39.9% of the participants reported that they wash their hands every hour, and 56.9% participants are using a surgical mask. About thermal scanners, 30.5% of the people answered they may be effective, and 46.0% of the people think COVID-19 is a bioweapon; 59% of the participants think everyone is susceptible, whereas 83.9% of the people recognize fever as a primary symptom; 65.2% of the people are practicing social distancing, whereas 85.1% of the people think social gatherings causes spread of the disease. In general, participants had a good knowledge about the disease and a positive attitude toward protective measures. The effective measures are being taken by the government and the public; still, there remains a need for further awareness campaigns and knowledge of safe interventions to combat the spread of disease.

In vivo anti-V-ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses.

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

PD-1 and PD-L1 expression on T-cell subsets in women with unexplained recurrent pregnancy losses.

PROBLEM: Does programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) expression on the T-cell subsets such as T helper (Th) 1, Th17, and Treg cells differentiate women with recurrent pregnancy losses (RPL) from normal fertile women? METHOD OF STUDY: The study was designed as a prospective cohort study. Forty-five women with two or more RPL of unknown etiology and twenty fertile women who had at least one or more live-born infants were enrolled prospectively from Jan 2017 to Jul 2019. PD-1 and PD-L1 expression on T-cell subsets were measured by flow cytometric analysis. RESULTS: The proportions of PD-1+ Th1 (CD4+ /IFN-γ+ /CD279+ and CD4+ /TNF-α+ /CD279+ ) and PD-1+ Th17 cells (CD4+ /IL17+ /CD279+ ) were significantly lower in RPL group than those of controls (P < .05, respectively). The proportion of PD-1+ Tregs (CD4+ /CD25+ /CD127dim/- /CD279+ ) in RPL group was not different from that of controls. The proportion of PD-L1+ Th17 cells (CD4+ IL17+ CD274+ ) was significantly lower as compared with that of /controls (P < .05). However, the proportions of PD-L1+ Th1 (CD4+ /IFN-γ+ /CD274+ and CD4+ /TNF-α+ /CD274+ ) and PD-L1+ Treg (CD4+ /CD25+ /CD127dim/- /CD274+ ) cells were not different between the RPL group and controls (P > .05, respectively). In Th1, Th17 and Treg cells, the proportions of PD-L1+ (CD274+ ) cells were significantly higher than those of PD-1+ (CD279+ ) cells in both RPL group and controls (P < .05, respectively). CONCLUSION: PD-1 and PD-L1 expressions on Th17 cells as well as PD-1 expression on Th1 cells were significantly downregulated in women with RPL, which may lead to increased Th1 and Th17 immunity, and imbalance between Th17, Th1, and Treg cells in women with RPL.

Adult anthropometry in Type 2 diabetic population: A case-control study.

OBJECTIVES: This study was aimed to compare the body mass index (BMI) and waist-to-hip ratio (WHR) in their ability to predict type 2 diabetes risk in a large prospective cohort of men and women in Pakistan. METHODS: This was a case-control study conducted at Diabetic and medical OPD of GTTH. Anthropometric measures including BMI and WHR were analyzed. Student's t-test, Chi-squared test along with Cramer's V value, was applied to evaluate association between variables. Receiver operating curve (ROC) was used to assess anthropometric measures. RESULTS: The study included 804 diabetics and 396 non-diabetics between 30-60 years of age. Comparing the BMI parameters it was found that 717 (89.2%) in diabetic group were overweight or obese (p-value < 0.001). On comparing the WHR, 97.9% diabetics had increased WHR (p-value <0.001). Both BMI & WHR were further compared using ROC curve which found out that WHR had an area under ROC of 0.720 & BMI has 0.680, suggesting that WHR is more better predictor of diabetes as compared to BMI. CONCLUSIONS: Both BMI and WHR were strong discriminators of T2DM but WHR was found superior according to ROC value. Family history is significantly associated in patients with diabetes.

Mast cell-induced immunopathology in recurrent pregnancy losses.

PROBLEM: Mast cells (MC) have been known to play an important role in inflammation and angiogenesis by secreting numerous mediators, such as proteases, gelatinases, and proteoglycans. Three different MC subtypes were found in the endometrial layers of the uterus. In this study, we aim to investigate the role of endometrial MCs in recurrent pregnancy losses (RPL). METHOD OF STUDY: Endometrial biopsy was performed 5-7 days post-ovulation (implantation window) in women with a history of two or more RPL (n = 46) and normal fertile women (n = 10). Quantitative RT-PCR was performed to detect the expression of various mast cell mediators. Endometrial samples were evaluated using immunohistochemistry for c-kit receptor (CD117) and tryptase (MC activation marker). RESULTS: Mast cells were present throughout the entire layers of the endometrium; their count was elevated in RPL patients as compared to controls. The gene expression of c-Kit receptor was not different between the study groups. There are significant increases in the mRNA expression of various mediators, that is, stem cell factor (P = 0.029), tryptase (P = 0.024), heparan sulfate (P = 0.0005), and MMP-2 (P < 0.0001) in women with RPL as compared to normal controls. Chymase gene expression was not detected in most of the endometrial samples. CONCLUSION: This study has shown that MCs are overactive in RPL patients by creating a pro-inflammatory milieu, suggesting a novel role in the immunopathology of RPL. Future studies are needed to better understand the role of MC in implantation and placental angiogenesis.

Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells.

The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.

Prevalence of HHV-6 in endometrium from women with recurrent implantation failure.

PROBLEM: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. METHOD OF STUDY: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. RESULTS: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. CONCLUSION: HHV-6 infection is an important factor in RIF.

A Role for Iodide and Thyroglobulin in Modulating the Function of Human Immune Cells.

Iodine is an essential element required for the function of all organ systems. Although the importance of iodine in thyroid hormone synthesis and reproduction is well known, its direct effects on the immune system are elusive. Human leukocytes expressed mRNA of iodide transporters (NIS and PENDRIN) and thyroid-related proteins [thyroglobulin (TG) and thyroid peroxidase (TPO)]. The mRNA levels of PENDRIN and TPO were increased whereas TG transcripts were decreased post leukocyte activation. Flow cytometric analysis revealed that both PENDRIN and NIS were expressed on the surface of leukocyte subsets with the highest expression occurring on monocytes and granulocytes. Treatment of leukocytes with sodium iodide (NaI) resulted in significant changes in immunity-related transcriptome with an emphasis on increased chemokine expression as probed with targeted RNASeq. Similarly, treatment of leukocytes with NaI or Lugol's iodine induced increased protein production of both pro- and anti-inflammatory cytokines. These alterations were not attributed to iodide-induced de novo thyroid hormone synthesis. However, upon incubation with thyroid-derived TG, primary human leukocytes but not Jurkat T cells released thyroxine and triiodothyronine indicating that immune cells could potentially influence thyroid hormone balance. Overall, our studies reveal the novel network between human immune cells and thyroid-related molecules and highlight the importance of iodine in regulating the function of human immune cells.

Optimization of methods for the genetic modification of human T cells.

CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production.

GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

GADS is required for TCR-mediated calcium influx and cytokine release, but not cellular adhesion, in human T cells.

GRB2 related adaptor protein downstream of Shc (GADS) is a member of the GRB2 family of adaptors and is critical for TCR-induced signaling. The current model is that GADS recruits SLP-76 to the LAT complex, which facilitates the phosphorylation of SLP-76, the activation of PLC-γ1, T cell adhesion and cytokine production. However, this model is largely based on studies of disruption of the GADS/SLP-76 interaction and murine T cell differentiation in GADS deficient mice. The role of GADS in mediating TCR-induced signals in human CD4+ T cells has not been thoroughly investigated. In this study, we have suppressed the expression of GADS in human CD4+ HuT78 T cells. GADS deficient HuT78 T cells displayed similar levels of TCR-induced SLP-76 and PLC-γ1 phosphorylation but exhibited substantial decrease in TCR-induced IL-2 and IFN-γ release. The defect in cytokine production occurred because of impaired calcium mobilization due to reduced recruitment of SLP-76 and PLC-γ1 to the LAT complex. Surprisingly, both GADS deficient HuT78 and GADS deficient primary murine CD8+ T cells had similar TCR-induced adhesion when compared to control T cells. Overall, our results show that GADS is required for calcium influx and cytokine production, but not cellular adhesion, in human CD4+ T cells, suggesting that the current model for T cell regulation by GADS is incomplete.