Mohammad Ameen Al-Aghbar

Background/Objectives: Antimicrobial resistance (AMR) is a major global health issue. Since community pharmacists are frontline health officials regarding the provision and management of antibiotics, it is of great importance to study the knowledge, attitudes, and practices (KAPs) of pharmacists with respect to antimicrobial stewardship (AMS) to formulate specific interventions. In Jordan, where dispensing antibiotics without a prescription is a common situation, this study was designed to assess the KAPs of community pharmacists in southern Jordan on AMR and AMS, the gap in the knowledge base and the practice, and the effect of other variables on antimicrobial education and responsible dispensing. Results: Participant pharmacists (n = 383) confirmed a moderate or lack of knowledge in antibiotic choice, resistance mechanisms, and the basis of stewardship. Despite positive attitudes for AMS, important practice gaps occurred: 38.6% infrequently dispensed antibiotics without prescriptions, 67.4% mistakenly believed that antibiotics may cure viral infections (flu/common cold), and only 33.4% firmly rejected non-prescription antibiotic requests. Knowledge scores were significantly higher among bachelor’s degree pharmacists, public university pharmacists, and urban-working pharmacists. Practice scores were better among master’s degree holders and urban practitioners. Conclusions: This work indicates that the AMS knowledge and practices of pharmacists in the southern Jordanian community are lacking in a severe way, with almost 4 out of 10 having poor behaviors including dispensing non-prescription antibiotics.

With the exception of the T-helper 2 (Th2) subset, cytokine driven pathways provide a robust mechanistic explanation for the observed outcomes of CD4+ T-cell differentiation. Using a quantitative model of activation, we studied the integration of TCR-signal-strength with cytokine signalling during Th2 differentiation. Upon the initial activation of Th-naïve cells, TCR signalling was found to set early expression levels for the master regulators of differentiation Tbet and GATA3, independent of the presence of polarizing cytokines. Subsequently cytokine stimuli modulated transcription factor (TF) expression levels to tune the outcome of differentiation. Here, weak TCR signalling was sufficient to drive the early upregulation of GATA3 and induce Th2 differentiation, in an IL-4 independent manner. Th1 differentiation was however shown to require additional cytokine signalling input, either in the form of autocrine IFNγ or exogenous IL-12. Using mathematical modelling we demonstrate that T-helper differentiation occurs along a continuum of states. Set by the relative co-expression of regulatory TFs, where effector cytokine production is controlled in a probabilistic manner determined by the relative levels GATA3 and Tbet expressed. Together, our data indicate TCR signalling inputs drive an early bifurcation in the T-helper differentiation pathway. Together, the integration of TCR signal strength with cytokine inputs act as a mechanism for the detection of immuno-evasive parasitic infections, whilst providing an additional checkpoint to prevent aberrant Th1 associated immunopathology.

The activation of CD4+ T‐cells in a T cell receptor (TCR)‐dependent antigen‐specific manner is a central characteristic of the adaptive immune response. In addition to ensuring that CD4+ T‐cells recognise their cognate antigen during activation, TCR‐mediated signalling can also direct the outcome of differentiation. In both in vivo and in vitro model systems, strong TCR signalling has been demonstrated to drive Th1 differentiation, whereas weak TCR signalling drives Th2 responses. During the process of differentiation, TCR signal strength acts as a quantitative component in combination with the qualitative effects imparted by cytokines to polarise distinct T‐helper lineages. Here, we investigated the role of interleukin 2 (IL‐2) signalling in determining the outcome of TCR‐dependent differentiation. IL‐2 production was initiated as an early response to TCR‐induced activation and was regulated by the strength of TCR signalling initially received. In the absence of IL‐2, TCR dependent differentiation was found to be abolished. However, proliferative responses and early markers of activation were maintained, including the upregulation of GATA3, Tbet and Foxp3 at 24 h post‐stimulation. Demonstrating that IL‐2 signalling has a key role in stabilising and amplifying lineage‐specific transcirption factor expression during differentiation. Further, activation of IL‐2‐deficient T‐cells in the presence of exogenous cytokines was sufficient to restore differentiation whilst maintaining transcriptional signatures imparted during initial TCR signalling. Combined, our data demonstrate that the integration of quantitative TCR‐dependent signalling and qualitative IL‐2 signalling is essential for determining the fate of CD4+ T‐cells during differentiation.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing pancreatic β-cells by the immune system. Although conventional therapeutic modalities, such as insulin injection, remain a mainstay, recent years have witnessed the emergence of novel treatment approaches encompassing immunomodulatory therapies, such as stem cell and β-cell transplantation, along with revolutionary gene-editing techniques. Notably, recent research endeavors have enabled the reshaping of the T-cell repertoire, leading to the prevention of T1D development. Furthermore, CRISPR–Cas9 technology has demonstrated remarkable potential in targeting endogenous gene activation, ushering in a promising avenue for the precise guidance of mesenchymal stem cells (MSCs) toward differentiation into insulin-producing cells. This innovative approach holds substantial promise for the treatment of T1D. In this review, we focus on studies that have developed T1D models and treatments using gene-editing systems.

Asthma is ranked among the most common chronic conditions and has become a significant public health issue due to the recent and rapid increase in its prevalence. Investigations into the underlying genetic factors predict a heritable component for its incidence, estimated between 35% and 90% of causation. Despite the application of large-scale genome-wide association studies (GWAS) and admixture mapping approaches, the proportion of variants identified accounts for less than 15% of the observed heritability of the disease. The discrepancy between the predicted heritable component of disease and the proportion of heritability mapped to the currently identified susceptibility loci has been termed the ‘missing heritability problem.’ Here, we examine recent studies involving both the analysis of genetically encoded features that contribute to asthma and also the role of non-encoded heritable characteristics, including epigenetic, environmental, and developmental aspects of disease. The importance of vertical maternal microbiome transfer and the influence of maternal immune factors on fetal conditioning in the inheritance of disease are also discussed. In order to highlight the broad array of biological inputs that contribute to the sum of heritable risk factors associated with allergic disease incidence that, together, contribute to the induction of a pro-atopic state. Currently, there is a need to develop in-depth models of asthma risk factors to overcome the limitations encountered in the interpretation of GWAS results in isolation, which have resulted in the missing heritability problem. Hence, multiomics analyses need to be established considering genetic, epigenetic, and functional data to create a true systems biology-based approach for analyzing the regulatory pathways that underlie the inheritance of asthma and to develop accurate risk profiles for disease.

The chain of events that leads to the sensitization of the immune system to environmental antigens, resulting in the onset of allergic disease, has been studied in great detail over the past 30 years. However, during this time, the rate of allergic diseases has increased exponentially, indicating the need to concentrate our studies on host-environmental factors that contribute to the onset of disease. Monocyte-derived dendritic cells (DCs) play a key role in driving localized and systemic immune responses. In this study, we developed a platform for screening the molecular signature and phenotypic profile of DCs activated by allergenic stimuli, including TSLP, IL-25, IL-33, IL-1a, Vit-D3 (1α,25-Dihydroxyvitamin D3), PAR1-AP Peptide, Papain, and recombinant human DerP1 protein to induce a type II associated inflammatory signature. Following activation with allergenic stimuli, modulated DCs are subjected to deep phenotyping via flow cytometry for surface and intracellular markers to detect and/or validate immunomodulatory properties. RNA sequencing is further used to compare the gene expression profiles of DCs responding to either allergenic or microbial stimuli, including the TLR3 agonist dsRNA Poly I:C and TLR4 agonist LPS. In our study, we aimed to identify key molecular signatures of DCs involved in the development of asthma and allergy based on their comparative activation with this broad panel of allergens. We expect to determine central control modules of transcription factors in DCs associated with Th2 induction.

Allergic diseases constitute significant health and economic issues in both developed and developing nations, with epidemiological studies demonstrating a rapid increase in the global prevalence of food allergy among the pediatric population. Cow milk protein allergy (CMPA), one of the most common forms of food allergies observed in early childhood, affects between 2%–6% of infants and children under 3 years of age. CMPA can present as either an IgE-mediated atopic allergy or a non-IgE mediated allergic response. Antigen-specific T cells play a pivotal role in directing the type of inflammatory immune response that occurs as well as in the formation of immunological memory. IgE-mediated CMPA is thought to develop because of an abnormal expansion of allergen-specific type-2 helper T (Th2) cells and a corresponding deficiency in immune regulation by regulatory T cells (Tregs), thereby altering the Th2/Treg balance. The gut microbiota, established very early during childhood through host-microbe interactions, can influence the incidence of allergic diseases. In this study, we aimed to analyze both the microbiome composition and CD4+T cell differentiation patterns in pediatric patients with and without cow milk allergy to establish the association between these factors. Using 16S rRNA sequencing, we analyzed the microbiome composition in stool samples of allergic and non-allergic pediatric patients aged between 1–4 years and identified the microbial species abundant in IgE and non-IgE mediated cow milk allergies. To assess the CD4+T cell differentiation patterns, peripheral blood mononuclear cells (PBMCs) from these patients were re-stimulated with cow milk antigen, and T cell subsets were assessed using flow cytometry. Antigen-specific CD4+T cells were identified and sorted for high throughput sequencing and subsequent gene expression analysis. The CD4+T cell differentiation patterns of the total and antigen-specific T cells were analyzed and statistically compared with controls. The identification of the correlation between the CD4+T cell differentiation patterns and species-specific microbial abundance in IgE and non-IgE mediated cow milk allergies can help in determining how the gut microbiome influences the CD4+T cell immune compartment development, ultimately leading to the development of cow milk allergy in pediatric patients.

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.

Cytotoxic T cells (CTLs) and B cells engage distinct interactions in GVHD patients' blood and tissues, detectable in regular flow-cytometry screenings, by size and by double positive CD19-CD8 antibody markers (Deola, BMT 2017). B-CTL couplets are formed by alpha-betaTCR+ CD8+ CTLs preferentially targeting CD27+ CD19+ cells displaying an activated CD80 and CD86 phenotype. Interactions may last from 5 minutes to roughly 1 hour, and release a pattern of T cell attracting chemokines, as IP10, MIG, ITAC, which are also known GVHD biomarkers. To further unravel the mechanism of this cell interaction, we built an in-vitro model where human PBMCs cells are expanded with cognate peptides and IL2 for 1-2 weeks, then immune-selected for CD8 antigen by Miltenyi microbeads negative-selection and incubated (2-18 hours) with fresh autologous CD19-B cells, immune-selected with the same method. The interactions are studied under confocal microscope video-imaging (Zeiss LSM 880+Imaris 3D analysis software) and in flow-cytometry (SymphonyA5 BD) after deep phenotype antibody staining. The intensity of interaction, measured by fluorescence interference on cell membranes, revealed an active engagement of CD19 and CD8 antigens. CD19 antigen penetrates deeper in contacting T cells, than CD8 on B cells, and consistently with this finding, after the interactions there is an antigen exchange between cells with CD19 antigen actively transferred in CD8 cells (p value =<0.001), but not the contrary. We already proved that this type of B-T interaction is not antigen specific in CTL-to-B direction (Deola et a, JI 2008) but to exclude cross-presentation from B to CTLs and to unravel the role of CD8, we interfered by antibody blocking of MHC class I pathway on B cells and CD8 on CTLs. B-T cell interactions are not abolished after MHC-I or CD8 blocking, the intensity of coupling is unchanged after MHC-I block, and is higher after blocking CD8 (p value=<0.001). In particular, by blocking CD8 molecule, T cells target preferentially CD19+/CD27- cells rather than CD19+/27+ cells. Interestingly, B cell engagement follows 2 repetitive patterns of interaction: a high intensity interaction that visually corresponds to tight coupling cells with high CD19 penetration in T cells, and a low-intensity continuous interaction, visually measurable by cells "sniffing" each other. Both patterns correspond to diverse Calcium flux activation on T cells and B cells, suggesting functional different pathways triggered by the 2 type of interactions. Deep phenotype flow cytometry analyses after coupling reveals distinct programs triggered by the contact in both B cells and T cells. While after the interaction CTLs double their pool of perforin bearing effectors and their fraction of CD45RA-/CD27+ memory CTLs, CD19 preferentially undergo a deletion of IgD- CD27- (DN) cells (13,85%+/-1,1 and 22,95%+/-4,5 CD95/Fas+, respectively in B cells alone and B+CTLs, n=2) and a rescue of affinity mature CD27+ IgD- cells (39.8%+/-25,47 and 21,2%+/- 29% CD95/Fas+ in the same groups) CTLs are the ultimate line of "tissue attack" in GVHD and several diseases, as autoimmune diseases, cancer, viral diseases, sharing a common pathological program definable as "immune rejection". B cells are key players in immune rejection, but a link between these 2 types of cells is still unclear. Our findings enforce the hypothesis of a program of peripheral tolerance/activation triggered directly between B cells and activated CTLs in the context of inflammation and of GVHD. Disclosures No relevant conflicts of interest to declare.

How T cell receptors (TCRs) are triggered to start signaling is still not fully understood. It has been proposed that segregation of the large membrane tyrosine phosphatase CD45 from engaged TCRs initiates signaling by favoring phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3 molecules. However, whether CD45 segregation is important to initiate triggering is still uncertain. We examined CD45 segregation from TCRs engaged to anti-CD3 scFv with high or low affinity and with defined molecular lengths on glass-supported lipid bilayers using total internal reflection microscopy. Both short and elongated high-affinity anti-CD3 scFv effectively induced similar calcium mobilization, Zap70 phosphorylation, and cytokine secretion in Jurkat T cells but CD45 segregated from activated TCR microclusters significantly less for elongated versus short anti-CD3 ligands. In addition, at early times, triggering cells with both high and low affinity elongated anti-CD3 scFv resulted in similar degrees of CD3 co-localization with CD45, but only the high-affinity scFv induced T cell activation. The lack of correlation between CD45 segregation and early markers of T cell activation suggests that segregation of CD45 from engaged TCRs is not mandatory for initial triggering of TCR signaling by elongated high-affinity ligands.