AL-amyloidosis and light-chain deposition disease light chains induce divergent phenotypic transformations of human mesangial cells

Human mesangial cells (HMCs) are injured by either excessive amounts or abnormal light chains (LCs), or a combination of both in patients with plasma cell dyscrasias. Consequently, these HMCs undergo phenotypic transformations. HMCs were incubated with eight different light-chains (LCs) for 96 h. These cells, in addition to 51 patient samples from patients with AL-amyloidosis (AL-Am), light-chain deposition disease (LCDD), myeloma cast nephropathy (MCN) and controls were analyzed by immunohistochemistry for CD68, muscle-specific actin (MSA), smooth muscle actin (SMA), CD14, and Ham56 protein expressions. All samples were also studied using electron microscopy. Greater staining (four- and three-fold) expressions of CD68 and Ham56, respectively, were observed in the HMCs incubated with AL-Am-LCs compared to those with LCDD-LCs and control. SMA expression levels were five-fold higher in LCDD-LC-treated cells compared to the other categories of LC-treated and control cells. Similar results were obtained in the renal specimens, however, CD68 levels were 12-fold higher in the AL-Am cases compared to the LCDD cases, respectively. Conversely, MSA and SMA levels were three fold higher in the LCDD cases than in the AL-Am ones. No CD14 expression was noted in any of the samples and CD-34 staining of HMCs treated with the various LCs only showed rare positive cells. Dynamic real-time studies to visualize the rough endoplasmic reticulum (RER) and lysosomal compartments in HMCs incubated with LCDD and AL-Am-LCs showed striking expansion of each of the above-mentioned compartments, respectively. This indicates the presence of more RER in the LCDD-LC-treated HMCs and a striking increase in lysosomes noticeable in the AL-Am-LC-treated cells. Data obtained in this study highlighted that HMCs incubated with LCDD-LCs undergo a myofibroblastic phenotypic transformation, while AL-Am-LCs induce a macrophage-like phenotype in these cells.     

New STR at the D5S373 locus and its relevance in human population studies

The genetic analysis of a new trinucleotide repeat (D5S373) was carried out with a view to its application in both individual genetic profiling and human population genetics. In a screening analysis from seven world populations (n = 706) and after nucleotide sequence analysis, up to nine alleles were found corresponding to 8–13 repetitions of a TAA motif. This analysis shows He values ranging between 0.689–0.762. D5S373 reveals interpopulational variability which leads to specific frequency profiles in the major human groups, with alleles 8, 11, l2, and 13 being particularly informative, which suggests the that this marker may be of interest in the biological study of human populations. Am. J. Hum. Biol. 14:347–350, 2002. © 2002 Wiley‐Liss, Inc.     

Expression and one-step purification of Plasmodium proteins in dictyostelium

Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.     

Identification of macrophages and smooth muscle cells with monoclonal antibodies in the human atherosclerotic plaque

Summary Sections of human atherosclerotic plaques, obtained from 21 autopsy cases with various degrees of atherosclerosis, were stained with the indirect immunoperoxidase technique using specific monoclonal antibodies against macrophages and smooth muscle cells. Distinctive results were found in differing stages: Single blood monocytes were observed in diffuse intimal thickening and the foam cells seen in fatty streaks were mostly identified as mature tissue macrophages, while only very few blood monocytes were present. The spindle cells observed in fibroelastic plaques showed positive reactions to antibodies against desmin, which points to their derivation from smooth muscle cells, whereas only a few macrophage-derived foam cells were seen in these lesions. In the complicated lesions the majority of foam cells were macrophage-derived, but there was also a small number of foam cells positive to antibodies against desmin, suggesting a smooth muscle cell derivation. - Our results confirm that in human atherosclerotic plaques the majority of the foam cells are obviously macrophage-derived, which emphasizes the important role of macrophages in the morphogenesis of these lesions.Supported by Landesamt für Forschung Nordrhein-Westfalen     

Ultrastructural immunolabeling: a general overview of techniques and applications.

Ultrastructural immunolabeling techniques combine the advantages of routine electron microscopy and detection of antigenic epitopes, the latter of which is customarily done by immunocytochemistry at the light microscopic level. In surgical pathology, immunocytochemistry has become routine to approach the differential diagnosis of difficult cases. Immunoelectron microscopy has been used primarily for research purposes or for addressing specific questions in diagnostic pathology. Ultrastructural immunolabeling is extremely useful in those instances in which the ultrastructural and immunocytochemical findings are not pathognomonic and are subject to interpretation. Preembedding and postembedding labeling techniques have been described in the literature. Preembedding techniques are not as applicable to diagnostic work, however. Their use remains rather limited to applications in which the antigen to be labeled cannot maintain its viability when exposed to fixatives. Even in such cases a new methodology--the LifeCell process--has emerged; this technique cryofixes tissues, thereby maintaining antigenic integrity. After cryofixation, a postembedding labeling technique can be utilized. Immunogold and peroxidase methods are used for labeling. Immunogold methods elegantly mark reaction sites with preservation of underlying morphology. Postembedding immunogold methods are used by most individuals working in the field. Ultrastructural labeling techniques are rapidly moving from classification as exclusively research tools to the diagnostic arena.     

The administration of 20 ppm of inhaled nitric oxide produces a faster response than the inhalation of 5 ppm in adult respiratory distress syndrome

OBJECTIVES: To compare the effects of 5 and 20 ppm of nitric oxide, evaluating time until response for each dose, in patients with adult respiratory distress syndrome (ARDS). PATIENTS AND METHODS: The study was prospective, controlled and random. Fifteen patients assigned to two groups received either 5 ppm (8 patients) or 20 ppm (7 patients) from November 96 to July 97. The main variables analyzed were PaO2/FiO2 and pulmonary vascular resistance index. We also studied etiology, severity of pulmonary damage as reflected by the Lung Injury Score, age, sex, Apache II prognostic score and exitus. Outcome was considered good if PaO2/FiO2 increased and/or pulmonary vascular resistance index decreased by more than 30% from the initial level (before inhalation of nitric oxide). RESULTS: The mean lung injury score was 2.9 +/- 0.4 and the two groups were homogeneous. Time until response to nitric oxide was significantly less in the 20 ppm group. Both PaO2/FiO2 and pulmonary vascular resistance index improved significantly in both groups whereas Qs/Qt improved only in the 20 ppm group. We also found that cardiac index and oxygen transport increased, the latter significantly only in the 20 ppm group. NO2 formation was less than 2 ppm and methemoglobin levels did not rise above 2%. CONCLUSIONS: Inhaled nitric oxide significantly improves oxygenation and decreases pulmonary vascular resistance without altering systemic vascular resistance during treatment of ARDS. The final outcomes were similar for both doses, but the 20 ppm dose produced a significantly faster response as well as a significant decrease in Qs/Qt.