AKR1C3 Expression in T and B Acute Lymphoblastic Leukemia/Lymphoma for Clinical Use as a Biomarker

Objectives: To investigate aldo–keto reductase 1C3 (AKR1C3) expression in T and B acute lymphoblastic leukemia/lymphoma (ALL) patients.Methods: Three commercial antibodies were evaluated for AKR1C3 immunohistochemistry (IHC) staining performance: Polyclonal Thermofisher scientific (Clone#PA523667), rabbit monoclonal Abcam [EPR16726] (ab209899) and Sigma/Millipore anti-AKR1C3 antibody, mouse monoclonal, clone NP6.G6.A6, purified from hybridoma cell culture. Initial optimization was performed on cell line controls: HCT116 (negative control); genetically modified cell line HCT116 with AKR1C3 overexpression; Nalm and TF1 cell lines. Twenty normal bone marrows from archival B and T-ALL patient samples were subsequently examined. AKR1C3 expression levels in these samples were evaluated by immunohistochemistry, Protein Wes and quantitative RT-PCR.Results: Sigma/Millipore Anti-AKR1C3 antibody (mouse monoclonal, clone NP6.G6.A6) showed higher specificity compared to rabbit polyclonal antibody by immunohistochemistry. H-score was used to quantify percent of nuclear immunoreactivity for AKR1C3 with varying disease involvement. T-ALL samples had a higher H-score (172-190) compared to B-ALL cases (H-score, 30-160). The AKR1C3 expression in peripheral blood by Protein Wes and RT-qPCR showed concordance in relapsed/refractory and/or minimal residual T-ALL cases. Conclusions: Sigma/Millipore Anti-AKR1C3 antibody and mouse monoclonal, clone NP6.G6.A6 can be used to aid in AKR1C expression of T-ALL and in cases of relapsed/refractory and/or minimal residual disease.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

New Insights into Cellular Functions of Nuclear Actin

Actin is one of the most abundant proteins in eukaryotic cells. There are different pools of nuclear actin often undetectable by conventional staining and commercial antibodies used to identify cytoplasmic actin. With the development of more sophisticated imaging and analytical techniques, it became clear that nuclear actin plays a crucial role in shaping the chromatin, genomic, and epigenetic landscape, transcriptional regulation, and DNA repair. This multifaceted role of nuclear actin is not only important for the function of the individual cell but also for the establishment of cell fate, and tissue and organ differentiation during development. Moreover, the changes in the nuclear, chromatin, and genomic architecture are preamble to various diseases. Here, we discuss some of the newly described functions of nuclear actin.

Expression of DOCK9 and DOCK11 Analyzed with Commercial Antibodies: Focus on Regulation of Mutually Exclusive First Exon Isoforms

Dedicators of cytokinesis 9 and 11 (DOCK9 and DOCK11) are members of the dedicator of cytokinesis protein family encoding the guanosine nucleotide exchange factors for Rho GTPases. Together with DOCK10, they constitute the DOCK-D or Zizimin subfamily. Two alternative full-length amino terminal isoforms of DOCK9 are known, which we will call DOCK9.1 and DOCK9.2. In order to investigate the relevance of the presence of the alternative first exon isoforms within this family, and to lay the groundwork for future studies that seek to investigate their potential role as biomarkers of disease, the expression levels of DOCK9 and DOCK11 were measured by qRT-PCR in 26 human tissues and 23 human cell lines, and by Western blot analysis, using commercial antibodies in cell lines. DOCK9.1 and DOCK9.2 were widely distributed. High levels of expression of both isoforms were found in the lungs, placenta, uterus, and thyroid gland. However, only DOCK9.1 was significantly expressed in the neural and hematopoietic tissues. The unique first exon form of DOCK11 was highly expressed in hematopoietic tissues, such as the peripheral blood leukocytes, spleen, thymus, or bone marrow, and in others such as the lungs, placenta, uterus, or thyroid gland. In contrast to tissues, the expression of DOCK9.1 and DOCK9.2 differed from one another and also from total DOCK9 in cell lines, suggesting that the amino terminal isoforms of DOCK9 may be differentially regulated. This study demonstrates the usefulness of antibodies in investigating the regulation of the expression of DOCK9.1, total DOCK9, and DOCK11.

Adaptive immunity in the mucous membrane of the duodenum in neonatal sepsis

Aim. To assess of adaptive immunity of the duodenal mucosa in neonates with sepsis. Methods. A study of duodenal biopsy specimens obtained during duodenoscopy from neonates who had signs of suspected ulcerative lesions of the digestive tract. Five of them were obtained from infants with clinical and laboratory signs of neonatal sepsis (NS), two from infants without sepsis (comparison group). Immunohistochemical staining was performed using the Novolink Polymer Detection System imaging system with commercial antibodies to CD4, CD8, CD20 and Bcl-2, and the CSAII Biotin-free Tyramide Signal Amplification System imaging system with commercial antibodies to caspase-3 and caspase-9 in accordance with the manufacturers instructions. Results. It was found that the number of CD4+ T-lymphocytes of the duodenal mucosa in neonatal sepsis group in 4 of 5 infants did not differ from the control group. The number of CD8+ lymphocytes in neonates with sepsis in 3 of 5 cases was even higher than in the control group. The number of CD20+ B-lymphocytes in 4 infants with sepsis was significantly less compared with the control. Activation of apoptosis in mucosal cells was detected, which was manifested by a large number of caspase-3-positive cells (in 4 of 5 cases) in comparison with the control group. The number of caspase-9-positive cells in the studied groups was almost equal. A substantial decrease in the number of Bcl-2-positive mucosal cells in all 5 infants with sepsis was noted as compared with the control indicators. Conclusion. The study revealed moderate immunosuppression in the duodenal mucosa in neonates with sepsis, manifested by low values of CD20+ B-lymphocytes in the absence of a significant decrease in the number of CD4+ and CD8+ T-lymphocytes. The detected moderate activation of apoptosis processes against the background of reduced antiapoptotic potential creates the conditions for a possible translocation of the intestinal microbiota into the bloodstream.