Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis

GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.     

Dynamic patterns of colocalization of calbindin, parvalbumin and GABA in subpopulations of mouse basolateral amygdalar cells during development

Calbindin cells represent a major interneuron subtype of the cortical/pallial regions, such as the basolateral amygdala, which are often analyzed in studies of tangential migration of interneurons from the subpallial ganglionic eminences to the pallium/cortex. However, previous evidence suggests that during development the calbindin cells may include more than one of the interneuron subtypes found in the adult pallium/cortex. Furthermore, in the adult basolateral amygdala, calbindin cells include a subpopulation of non-GABAergic (non-interneuron) cells. To better characterize these cells throughout development, in the present study we investigated the colocalization of calbindin, parvalbumin and GABA in cells of the mouse basolateral amygdala during late embryonic (E16.5) and several postnatal ages from birth until 4 weeks after birth (P0, P10 and P28). Our results indicate that CB, PV and GABA show a dynamic pattern of colocalization in cells of the mouse basolateral amygdalar nucleus throughout development. From E16.5 through P28, the majority of CB+ neurons and virtually all PV+ neurons are GABAergic. However, after P10, the percentage of GABAergic CB+ cells decline from 96% to 70%. Furthermore, while only 9% of CB+ neurons are PV+ at P10, this percentage raises to 42% at P28. At all postnatal ages studied, the majority of the PV+ cells are CB+, suggesting that PV+ interneurons develop postnatally mainly as a subpopulation within the CB+ cells of the basolateral amygdalar nucleus. These results are important for interpreting data from interneuron migration.     

Waiting list outcome of Peld/Meld exceptions: A single‐center experience in Argentina

As PELD/MELD‐based allocation policy was adopted in Argentina in 2005, a system of exception points has been in place in order to award increased waitlist priority to those patients whose severity of illness is not captured by the PELD/MELD score. We aimed to investigate the WL outcome of patients with granted PELD/MELD exceptions. A retrospective cohort study was conducted in children under 18 years old. WL outcomes were evaluated using univariable analysis. From 07/2005 to 01/2014, 408 children were listed for LT. There were 304 classified by calculated PELD/MELD. During this time, 85 (30%) PELD/MELD exceptions were granted. In this cohort, 89.4% (76 of 85) were transplanted and 7.1% (6 of 85) died while on the WL. The remaining 3 pts (3.5%) were removed from the WL due to other causes. We compared the impact of PELD/MELD exceptions in those 85 patients to outcomes in 87 non‐exception patients with PELD/MELD ≥19 points. Patients with the exception had significantly better access to WL and lower WL mortality. Our data suggest that children listed by PELD/MELD exceptions had an advantage compared to children with CLD with equivalent PELD/MELD listing priorities.     

Analysis of null deletion polymorphism of glutathione S-transferase theta (GSTT-1), associated with anti-GSTT-1 antibodies development in transplantation

Glutathione S-transferase theta 1 (GSTT1) is an enzyme involved in phase II biotransformation processes and a member of a multigene family of detoxifying and clearing reactive oxygen species. GSTT1 is polymorphic like other biotransforming enzymes, allowing variability in hepatic conjugation processes. Immunological recognition of the GSTT1 alloantigen, as evidenced by donor-specific antibodies formation, has previously been observed in recipients lacking GSTT1 protein (called GSTT1−, GSTT*0, null phenotype or homozygous for the GSTT1 deletion) who receive liver or kidney transplants from GSTT1+ donors and is a risk factor for the development of de novo hepatitis following liver transplants from a GSTT1 expressing donor. Antibodies against GSTT1 are demonstrated in patients who are GSTT1 null and received a transplant from a GSTT1+ donor. Understanding the local population frequency of the GSTT1 deletion is of value in understanding the potential clinical risk of developing post-transplant complications, which can be attributed to the nonexpression of GSTT1. A population of 173 healthy donors of the Murcia Region in Southeast Spain was evaluated for a null allele of GSTT1 (n = 173). DNA was extracted, and GSTT-1 null allele detection was performed by real-time polymerase chain reaction. The frequency of the null GSTT1 genotype (nonexpression or deletion of the homozygous polymorphism of the GSTT1 protein) was 17.9% (n = 31 null allele GSTT1/173 total individuals). Our data suggest that the frequency of null GSTT1 mutations in our population in Southeast Spain is 17.9%, lower than in other Caucasoid populations. This would convert our recipient population into more susceptible to nonlocal potential organ donors and less susceptible to local donors. All recipients bearing this GSTT1 deletion homozygous would be without the protein and triggering an alloantigen in the case of transplantation with a donor without deletion.