Implementation of an evidence-based guideline on fluid resuscitation: lessons learnt for future guidelines

Introduction  

There is little experience with the nationwide implementation of an evidence-based pediatric guideline on first-choice fluid for resuscitation in hypovolemia.     

Transient CRISPR-Cas Treatment Can Prevent Reactivation of HIV-1 Replication in a Latently Infected T-Cell Line

Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application.     

CD25+ cell depletion hastens the onset of severe disease in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen‐induced arthritis (CIA).

Methods

We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII‐specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens.

Results

CD25+ cell–depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen‐specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen.

Conclusion

These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.