Technical considerations when designing a gene expression panel for renal transplant diagnosis.

Gene expression analysis is emerging as a new diagnostic tool in transplant pathology, in particular for the diagnosis of antibody-mediated rejection. Diagnostic gene expression panels are defined on the basis of their pathophysiological relevance, but also need to be tested for their robustness across different preservatives and analysis platforms. The aim of this study is the investigate the effect of tissue sampling and preservation on candidate genes included in a renal transplant diagnostic panel. Using the NanoString platform, we compared the expression of 219 genes in 51 samples, split for formalin-fixation and paraffin-embedding (FFPE) and RNAlater preservation (RNAlater). We found that overall, gene expression significantly correlated between FFPE and RNAlater samples. However, at the individual gene level, 46 of the 219 genes did not correlate across the 51 matched FFPE and RNAlater samples. Comparing gene expression results using NanoString and qRT-PCR for 18 genes in the same pool of RNA (RNAlater), we found a significant correlation in 17/18 genes. Our study indicates that, in samples from the same routine diagnostic renal transplant biopsy procedure split for FFPE and RNAlater, 21% of 219 genes of potential biological significance do not correlate in expression. Whether this is due to fixatives or tissue sampling, selection of gene panels for routine diagnosis should take this information into consideration.

Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.