Umbilical Cord Blood-Derived Therapies as a Treatment for Graft-Versus-Host Disease

Umbilical cord blood (UCB) has been increasingly used as a source of haematopoietic stem cells (HSCs) for transplantation. UCB transplantation (UCBT) has some advantages such as less stringent human leucocyte antigen (HLA) matching and lower impact of graft-versus-host disease (GvHD). UCBT is also characterised by a high rate of infections, graft failure, delayed engraftment and slow recovery of the immune system. UCB contains HSC as well as immune cells that could be considered to develop new treatments for the main complications post-UCBT but also to treat other diseases. GvHD remains a major complication post-CBT and post-haematopoietic stem cell transplantation (HSCT). In view of their ability to induce tolerance and suppress the functions of effector T cells, regulatory T (Treg) cells have been proposed as an adoptive therapy to modulate GvHD post-HSCT. In addition, we showed that UCB contains soluble NKG2D ligands that can modulate the functions of NKG2D expressing cells, making UCB plasma a product of interest to modulate inflammation and in particular skin GvHD. Here, we aim to describe some of the therapies currently developed using UCB, focusing on Treg cells and UCB plasma for the treatment of GvHD

Feasibility of using quadriceps-strengthening exercise to improve pain and sleep in a severely demented elder with osteoarthritis – a case report

BACKGROUND: Osteoarthritis (OA) of the knee, which is prevalent among older adults in nursing homes, causes significant pain and suffering, including disturbance of nocturnal sleep. One nonpharmacologic treatment option is quadriceps-strengthening exercise, however, the feasibility of such a treatment for reducing pain from OA in severely demented elders has not been studied. This report describes our test of the feasibility of such an exercise program, together with its effects on pain and sleep, in a severely demented nursing home resident. CASE PRESENTATION: The subject was an elderly man with severe cognitive impairment (Mini-Mental Status Exam score 4) and knee OA (Kellgren-Lawrence radiographic grade 4). He was enrolled in a 5-week, 10-session standardized progressive-resistance training program to strengthen the quadriceps, and completed all sessions. Pain was assessed with the Western Ontario and MacMaster OA Index (WOMAC) pain subscale, and sleep was assessed by actigraphy. The patient was able to perform the exercises, with a revision to the protocol. However, the WOMAC OA pain subscale proved inadequate for measuring pain in a patient with low cognitive functioning, and therefore the effects on pain were inconclusive. Although his sleep improved after the intervention, the influence of his medications and the amount of daytime sleep on his nighttime sleep need to be considered. CONCLUSIONS: A quadriceps-strengthening exercise program for treating OA of the knee is feasible in severely demented elders, although a better outcome measure is needed for pain

The mechanism of Ca2+ transport by a Ca2+ pump

Skeletal muscle sarcoplasmic reticulum Ca2+-ATPase pumps two Ca2+ ions across the membrane of the sarcoplasmic reticulum for each molecule of ATP hydrolysed. Recent experiments suggest that transport of Ca2+ involves transfer to two bound Ca2+ ions from a pair of binding sites exposed to the cytoplasm to a pair of sites exposed to the lumen. The nature of this transfer process was studies using rapid kinetic measurements of phosphorylation-induced release of Ca2+. It was found that at alkaline pH values, the rate of release of Ca2+ from the phosphorylated ATPase decreases with increasing Mg2+ concentration and that at high concentrations of Mg2+ release of Ca2+ is biphasic. The results are consistent with sequential release of Ca2+ from the phosphorylated ATPase. The effect of Mg2+ on the rate of release of the first Ca2+ could follow from binding to a gating site known to affect the binding of Ca2+ to the cytoplasmic sites. To study the order in which Ca2+ is transferred between the cytoplasmic and lumenal sites, use was made of the observation that modification of the ATPase with N-ethylmaleimide (NEM) slows the rate of release of Ca2+. It was found that the two bound Ca2+ ions become randomized during the transfer process. Using fluorescence studies it was found that NEM modification of the ATPase does not affect Ca2+ binding to the cytoplasmic sites or phosphorylation of the ATPase by ATP. NEM was found to modify a number of cysteine residues on the ATPase. Modification of the ATPase by NEM in the presence of 4-(bromomethyl)-6,7-dimethoxycoumarin (Br-DMC), a fluorescent probe known to label Cys-344, was also studied. It was found that one of the cysteine residues on the ATPase modified by NEM is Cys-344 and this prevents Br-DMC labelling at this site.</p

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

Abstract Background Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. Results The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants. Conclusion This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.</p

Clinical Grade Regulatory CD4+ T Cells (Tregs): Moving Toward Cellular-Based Immunomodulatory Therapies

Regulatory T cells (Tregs) are CD4+ T cells that are key players of immune tolerance. They are powerful suppressor cells, able to impact the function of numerous immune cells, including key effectors of inflammation such as effector T cells. For this reason, Tregs are an ideal candidate for the development of cell therapy approaches to modulate immune responses. Treg therapy has shown promising results so far, providing key knowledge on the conditions in which these cells can provide protection and demonstrating that they could be an alternative to current pharmacological immunosuppressive therapies. However, a more comprehensive understanding of their characteristics, isolation, activation, and expansion is needed to be able design cost effective therapies. Here, we review the practicalities of making Tregs a viable cell therapy, in particular, discussing the challenges faced in isolating and manufacturing Tregs and defining what are the most appropriate applications for this new therapy