Using Clustered Regularly Interspaced Short Palindromic Repeats gene editing to induce permanent expression of fetal hemoglobin in ß-thalassemia and sickle cell disease: A comparative meta-analysis.

ß-hemoglobinopathies like sickle cell disease (SCD) and ß-thalassemia are characterized by differing mutations in the hemoglobin subunit beta gene (HBB). These disorders vary in phenotypic presentation and severity, with more severe manifestations leading to transfusion dependence along with associated complications such as infection and iron overload. ß-hemoglobinopathies symptoms rapidly worsen after birth as the levels of fetal hemoglobin (HbF) begin to decline. To reverse this decline, current treatment plans typically involve the use of pharmacological agents such as hydroxyurea to raise expression levels of HbF. However, these treatments only result in transient effects and must be consistently administered. Gene editing technologies such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated protein) offer the opportunity to create novel treatments which can raise HbF expression with potential permanent effects. Two gene targets, B-cell lymphoma/leukemia 11A gene (BCL11A) and the promoter regions of gamma globin genes (HBG1/2), have been identified to significantly increase HbF protein expression. In order to differentiate the effectiveness of BCL11A and HBG1/2 editing, a meta-analysis was performed by first identifying 119 studies for inclusion based on the search terms terms "ß-Thalassemia," "beta-thal" "sickle cell disease," "SCD," and "CRISPR." Following application of exclusion and inclusion criteria, we performed analysis on 8 peer-reviewed published studies from 2018 to 2021 were included in the study. Forest plots were generated using R (version 4.1.2). Primary comparative analysis shows HBG1/2 had a significantly (p < 0.01)greater impact on induction of HbF expression compared to BCL11A. This analysis leads us to conclude that HBG1/2 merits further investigation as a possible gene editing target for treatment of SCD and ß-thalassemia.

Attitudes and Knowledge of Medical Students Towards Healthcare for Lesbian, Gay, Bisexual, and Transgender Seniors: Impact of a Case-Based Discussion With Facilitators From the Community.

BACKGROUND:  Lesbian, gay, bisexual, and transgender (LGBT) seniors are generally a medically underserved population that faces unique healthcare challenges. When compared to younger patients, LGBT seniors are at a greater risk for social isolation and have higher rates of smoking, disability, physical and mental distress, and lack of access to healthcare services. They are often reluctant to discuss their sexual orientations and gender identities with healthcare providers due to fear of discrimination and receiving inferior care based on prior unsatisfactory experiences with untrained or insensitive healthcare providers. Furthermore, recent research has revealed that only about 50% of primary care providers indicated confidence in providing culturally competent LGBT healthcare, highlighting the need for more LGBT proficiency training in medical school curricula. OBJECTIVES: The aim of this study was to provide early intervention training to first-year medical students regarding best practices for equitable healthcare for LGBT seniors through integrative, small group, case-based discussions. The impact of this activity on the knowledge and attitudes of medical students regarding LGBT healthcare was also assessed. METHODS: First-year medical students participated in a two-hour small group, case-based discussion. Each group consisted of seven to eight students with one of seven facilitators who were invited members of the LGBT community. Students were provided with two clinical case scenarios related to treatment of LGBT senior patients. Students were given a pre/post-session knowledge and attitude survey to assess the impact of the session on their attitudes and understanding of the importance of providing equitable healthcare to LGBT patients. A rubric was also used by facilitators to evaluate level of student engagement and professionalism. RESULTS: A total of 51 first-year medical students attended the session and 38 (74.5%) completed the pre/post surveys. There was diverse representation in our student demographic with 5.2% of respondents identifying as LGBT. Survey results showed a significant increase in knowledge confidence and attitudes following the session. Students' attitudes regarding determinants of health status changed significantly for nine of the 13 (69%) survey items. In addition, their confidence in knowledge regarding healthcare barriers, health issues, and practices for LGBT culturally competent care significantly increased post-session. Data from our assessment rubrics also show that students were highly professional and engaged with the LGBT facilitators. CONCLUSION: Our study provides some evidence that case-based training of medical students regarding issues that affect health of LGBT seniors can improve attitudes and sensitize them to the unique needs of this population. Through this activity, the students indicated their desire to learn more about the topics covered and to receive further training in this field of study. While the study was somewhat limited by a small participant number, the significance of the data demonstrates the effectiveness of the approach involving members of the LGBT community as facilitators. Future work with these students as part of a longitudinal curriculum will include additional LGBT proficiency training to be offered in the subsequent blocks of instruction. Additionally, this intervention could potentially be adapted by other medical schools.

Leishmania parasitophorous vacuole membranes display phosphoinositides that create conditions for continuous Akt activation and a target for miltefosine in Leishmania infections.

Miltefosine is an important drug for the treatment of leishmaniasis; however, its mechanism of action is still poorly understood. In these studies, we tested the hypothesis that like in cancer cells, miltefosine's efficacy in leishmaniasis is due to its inhibition of Akt activation in host cells. We show using pharmacologic agents that block Akt activation by different mechanisms and also using an inducible knockdown approach that miltefosine loses its efficacy when its access to Akt1 is limited. Interestingly, limitation of Akt activation results in clearance of established Leishmania infections. We then show, using fluorophore-tagged probes that bind to phosphoinositides, that Leishmania parasitophorous vacuole membranes (LPVMs) display the relevant phosphoinositides to which Akt can be recruited and activated continuously. Taken together, we propose that the acquisition of PI(4) P and the display of PI (3,4)P2 on LPVMs initiate the machinery that supports continuous Akt activation and sensitivity to miltefosine.

Defensin DEFB103 bidirectionally regulates chemokine and cytokine responses to a pro-inflammatory stimulus.

Human ß defensin DEFB103 acts as both a stimulant and an attenuator of chemokine and cytokine responses: a dichotomy that is not entirely understood. Our predicted results using an in silico simulation model of dendritic cells and our observed results in human myeloid dendritic cells, show that DEFB103 significantly (p < 0.05) enhanced 6 responses, attenuated 7 responses, and both enhanced/attenuated the CXCL1 and TNF responses to Porphyromonas gingivalis hemagglutinin B (HagB). In murine JAWSII dendritic cells, DEFB103 significantly attenuated, yet rarely enhanced, the Cxcl2, Il6, and Csf3 responses to HagB; and in C57/BL6 mice, DEFB103 significantly enhanced, yet rarely attenuated, the Cxcl1, Csf1, and Csf3 responses. Thus, DEFB103 influences pro-inflammatory activities with the concentration of DEFB103 and order of timing of DEFB103 exposure to dendritic cells, with respect to microbial antigen exposure to cells, being paramount in orchestrating the onset, magnitude, and composition of the chemokine and cytokine response.