An immunopathologic study of a 330-kD protein defined by monoclonal antibodies and reactive with anti-RTE alpha 5 antibodies and kidney eluates from active Heymann nephritis

There is evidence indicating that the glomerular Ig deposits of Heymann's nephritis (HN)--a model of epimembranous glomerulonephritis--may be formed at least in part in situ by binding of free circulating antibody with brush border (BB) antigen expressed by glomerular epithelial cells. In this work, we provide evidence that a 330-kD protein defined by seven monoclonal antibodies is responsible for HN. 1) Ig eluted from glomeruli of rats with HN induced classically with crude BB preparation bind specifically the 330-kD antigen; 2) passive immunization with monoclonal antibodies induces epimembranous glomerular Ig deposits; 3) active immunization with the 330-kD antigen induces proteinuric glomerulonephritis; 4) the 330-kD antigen was present in the nephritogenic preparation purified by Edgington, Glassock, and Dixon, because it was identified by the corresponding heterologous antisera. These results, obtained by a completely different approach, confirm and extend those of Kerjaschki and Farquhar and provide a link with the classical studies on HN.     

Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.     

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Exosomes are tiny vesicles (30–150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.     

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.     

Biological effects of anti-idiotypic antibodies on lymphocyte function: I. Analysis of the effects on B lymphocytes of combining site and framework-directed anti-T-15 idiotypic antibodies

Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALB/c origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine- HCl, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e., binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in vitro production of antiphosphorylcholine antibody in culture.     

The content of the chief amount of myoglobin in the blood serum as an immune complex

Pooled serum of health blood donors was tested for myoglobin and naturally occurring antibodies to myoglobin containing by competitive enzyme immunoassay. Data obtained show that myoglobin and autoantibodies of myoglobin are present in serum in the form of circulating immunocomplexes. Based on these results the suggestion about elimination of the main quantities of myoglobin by reticuloendothelial system is arisen. Estimated level of myoglobin concentration in the sera of health individuals established by different methods (80 ng/ml) is concerned apparently to free-circulated myoglobin and is not adequate to absolute myoglobin quantities.     

The total protein content in the blood serum of vertebrates

The total protein content in the blood serum of 416 species and subspecies of vertebrates is determined by the microbiuret method. Generally speaking the total protein content of the serum shows an increase with a higher evolutionary level. The difference between the mammals and the other groups is particularly large. This increase is probably attributable to a number of factors, for instance adaptation to living on land, development of the circulatory system, the development of adaptive immunity and the forming of specific transport proteins. A connection between albumin content and level of development can also be noticed.     

Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood

Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me₂SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.     

Iron content of the blood and saturation of serum transferrin with iron as affected by roentgen rays

Total-body X-irradiation of rabbits with a dose of 4.5 Gy caused appreciable changes in the iron content of blood and iron saturation of blood serum transferrin over a period from 30 min to 30 days. The pattern of changes in the indices under study depended upon the time lapsed after irradiation.     

The glycosphingolipid composition of the placenta of a blood group P fetus delivered by a blood group Pk1 woman and analysis of the anti-globoside antibodies found in maternal serum

To further define the molecules that may mediate spontaneous abortion due to maternal-fetal blood group incompatibility within the P blood group system, we have examined the fine specificities of maternal antibodies and the glycolipid antigens from the placenta of a P infant born to a Pk1 mother. Maternal antibodies obtained during therapeutic plasmapheresis were analyzed to determine their reactivities with placental glycolipid extracts on thin-layer plates. Second antibodies specific for IgM, IgG, and IgA revealed immunoglobulins of all of these classes strongly reactive with one major placental glycolipid that comigrates with globoside. GC/MS analysis confirmed that the major P-active pentaglycosylceramide of placenta has the same structure as that previously shown for the P antigen of red blood cells: GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. Serum antibodies partially purified by affinity chromatography on globoside-octyl-Sepharose specifically recognize glycolipids that contain terminal GalNAc beta 1-3Gal . . . residues and also recognize the same sequence as an internal determinant in some, but not all, glycolipids with extended globoside core regions. Thus, in the blood group P incompatible fetus, the major P antigen present in placenta has the same carbohydrate structure as the P antigen present in fetal and adult erythrocytes and might be a target for the maternal immune system.     

Modifications of plasma prolactin levels and catecholamine content in an ectopic anterior pituitary gland transplanted under the kidney capsule

In order to study the possible role on prolactin secretion of the catecholamines present in ectopic pituitaries, female rats bearing an anterior pituitary graft under the kidney capsule since day 30 of life and their sham-operated controls, were sacrificed at 1, 2, 4, 7, 15, 30, 45 and 60 days after the operation. Data obtained showed a significant increase in plasma prolactin levels in grafted rats versus controls from the 4th day on after the grafting (p less than 0.01) until the 60th day (p less than 0.001). Dopamine content in the ectopic pituitary of grafted rats was higher than in their own in situ pituitaries or on those of sham-operated rats until day 45 being similar to them afterwards. Norepinephrine was also present in the pituitary graft but was not detected in the in situ pituitaries. The grafting of an anterior pituitary gland in an ectopic location was able to induce changes in the local catecholaminergic control of the prolactin secretion.