The role of donor and recipient factors in initial renal graft non-function

ATN is a deleterious problem in the outcome of kidney transplantation. This complication is usually related to multiple factors including donor parameters, surgical technique, ischemic time, and recipient variables. In order to develop prophylactic measures, out of 430 kidney transplants performed in our Department, a series of 90 consecutive cadaveric renal allografts has been considered in this study. The overall incidence of IGNF was 23/90 (25.5%). Kidneys from MOD revealed a lower rate of IGNF (7/35 = 20%) when compared with organs from SOD (16/55 = 29%, P = NS). No difference was noted when kidneys were removed together with heart and/or liver and/or pancreas. Out of the donor factors, only CID was significant (17 +/- 9 hours in IGNF v 11 +/- 10 hours in patients with IGF, P = less than .05). Analysis of data concerning the fate of paired kidneys revealed two cases of IGNF in both kidneys from the same donor v 14 cases of IGNF in only one of the two paired grafts (P = NS). We conclude that: 1. Donor factors are clearly associated with a minority of IGNF. 2. The introduction of multiorgan procurement programs does not complicate early function. 3. Recipient factors (immunological events and intraoperative fluid management) provides important additive effects on initial graft nonfunction.     

Fluorocytometric analysis of anti-HLA class 2 autoantibodies in patients with lupus nephritis

Systemic lupus erythematosus (SLE) patients are known to produce a variety of autoantibodies (AAb), some of which may be directed against immunocompetent cells. Anti-B cell autoimmunity may encompass reactivity against HLA-class 2 molecules, which are also expressed on kidney tissue. We studied 15 patients with moderate to severe renal involvement and 5 lupus patients with no clinical renal disease, in order to detect the presence of anti-HLA class 2 AAb. Flow cytometry was employed in an inhibitory assay using patient sera, autologous cells and two anti-class 2 monoclonals, to establish the specificity of anti-B cell AAb. Seven out of 15 nephritis patients had detectable anti-class 2 AAb with an epitopic heterogeneity, as demonstrated by different degrees of inhibition on the binding of non-overlapping monoclonals. The specificity of the reaction was confirmed by the lack of inhibition of non-class 2 antibody binding. The presence of such AAb was not correlated with disease activity but with the presence of a diffuse proliferative glomerulonephritis on renal biopsy. Anti-class 2 AAb may be a marker of SLE diffuse proliferative nephritis.     

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A₂ sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens representing the major etiological agents of hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease characterized by hemolytic anemia, thrombocytopenia, and renal failure (19). The infection correlates with ingestion of contaminated meat or vegetables but is also transmitted by water or even person-to-person contact (8, 14, 44). Sporadic or massive outbreaks have been reported in several developed countries but, in Argentina, HUS is endemic and represents a serious public health problem with high morbidity and mortality rates (29, 40). Production of verocytotoxin or Shiga-like toxin (Stx) is the basis of EHEC pathogenesis (18, 20). The toxin is formed by a single A subunit, which possesses N-glycosidase activity to the 28S rRNA and promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells (9, 28). Although two major types (Stx1 and Stx2) and several subtypes have been described, Stx2 and Stx2c are the most frequently found toxins in severe HUS cases among EHEC-infected subjects (12, 41). The degree of antigenic cross-reactivity between Stx2 and Stx1 is low, and several authors have reported that the two toxins do not provide heterologous protection, particularly concerning the B subunits (45, 47). On the other hand, Stx2c and Stx2d variants are readily neutralized by antibodies against Stx2 (27).Despite the magnitude of the social and economic impacts caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. So far, attempts to develop vaccine formulations against EHEC-associated sequelae have relied mainly on induction of serum anti-Stx antibody responses. Several approaches have been pursed to generate immunogenic anti-Stx vaccine formulations and include the use of live attenuated bacterial strains (2, 32), protein-conjugated polysaccharides (21), purified B subunit (33, 48), B-subunit-derived synthetic peptides (15), and mutated Stx1 and Stx2 nontoxic derivatives (5, 6, 13, 16, 37, 39, 42, 45).In a previous report we described anti-Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N-terminal amino acid of the A₁ subunit (8). The DNA vaccine encoding the hybrid protein elicited Stx-specific immune responses in mice and partial protection to Stx2 challenge (1, 33). Recent data have indicated that epitopes leading to generation of Stx-neutralizing antibodies are present on both the B as well as the A subunit (34, 45, 46). In addition, further evidence indicates that the A₂ subunit contains some of the most immunogenic epitopes of the Stx2 toxin (4). Thus, in line with such evidence, we attempted the construction of a new DNA vaccine encoding the last 32 amino acids from the A₂ subunit, in addition to the complete B subunit of Stx2, as separated polypeptides which could enhance the immunogenicity and protective effects of the vaccine formulation. In the present report, we describe the generation of a new DNA vaccine encoding both Stx2 A2 and B subunits as an approach to elicit protective antibody responses to Stx2. The results obtained demonstrate that immunization with this vaccine formulation results in systemic antibody responses to Stx2 A and B subunits and toxin neutralization activity both in vitro and in vivo.     

Seasonal and interannual variability of climate and vegetation indices across the Amazon

Drought exerts a strong influence on tropical forest metabolism, carbon stocks, and ultimately the flux of carbon to the atmosphere. Satellite-based studies have suggested that Amazon forests green up during droughts because of increased sunlight, whereas field studies have reported increased tree mortality during severe droughts. In an effort to reconcile these apparently conflicting findings, we conducted an analysis of climate data, field measurements, and improved satellite-based measures of forest photosynthetic activity. Wet-season precipitation and plant-available water (PAW) decreased over the Amazon Basin from 1996−2005, and photosynthetically active radiation (PAR) and air dryness (expressed as vapor pressure deficit, VPD) increased from 2002–2005. Using improved enhanced vegetation index (EVI) measurements (2000–2008), we show that gross primary productivity (expressed as EVI) declined with VPD and PAW in regions of sparse canopy cover across a wide range of environments for each year of the study. In densely forested areas, no climatic variable adequately explained the Basin-wide interannual variability of EVI. Based on a site-specific study, we show that monthly EVI was relatively insensitive to leaf area index (LAI) but correlated positively with leaf flushing and PAR measured in the field. These findings suggest that production of new leaves, even when unaccompanied by associated changes in LAI, could play an important role in Basin-wide interannual EVI variability. Because EVI variability was greatest in regions of lower PAW, we hypothesize that drought could increase EVI by synchronizing leaf flushing via its effects on leaf bud development.     

Flow cytometry to estimate circulating hematopoietic progenitors for autologous transplantation: comparative analysis of different CD34 monoclonal antibodies.

We report that the 2 fluorescein (FITC) conjugated CD34 monoclonal antibodies available to date, namely FITC-8G12 and FITC-QBEND10, exhibit different capabilities of detecting circulating hematopoietic progenitors (CHP) as cells expressing the CD34 antigen (CD34+) by direct immunofluorescence flow cytometry. Mean fluorescence intensity conferred by FITC-QBEND10 to CD34+ CHP is 2.8-4.3 times lower than that conferred by FITC-8G12. By indirect immunofluorescence, native unconjugated QBEND10 and 8G12 antibodies detect CD34+ CHP in a comparable manner, thus indicating that the decrease in QBEND10 affinity is due to FITC-conjugation. Utilization of FITC-QBEND10 instead of FICT-8G12 to estimate infrequent (usually less than or equal to 5%) CD34+ CHP for clinical decision-making in autologous transplantation of CHP exposes clinicians to the risk of either overestimating (false positive) or underestimating (false negative) these cells in peripheral blood. Recently published guidelines for large-scale collection of CHP for autologous transplantation in cancer patients were based on data obtained with FITC-8G12. The same guidelines cannot be considered valid if FITC-QBEND10 is employed. We recommend FITC-8G12 as the optimal reagent to date for standardizing results of autologous CHP transplantation in cancer therapy.