Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.     

Allograft rejection results from a failed attempt by the immune system to protect foreign tissue

The focus of our research is to understand the immune response to foreign tissue. We believe that adichotomy exists within the immune response to an allograft such that part of the response is dedicated to the protection of the graft. Nevertheless, in a dominantly graft-aggressive environment, rejection typically ensues. In this article, we describe models that have been set up to test directly the ability of potentially protective aspects of the immune response to prevent allograft rejection. We discussour data in the context of a growing body of exciting and often controversial literature.     

Persistent infection of SARS coronavirus in colonic cells in vitro

Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.     

Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

P-glycoprotein expression influences the result of 99mTc-MIBI scintigraphy in tertiary hyperparathyroidism

Precise localization of parathyroid glands using 99mTc-labeled hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) scintigraphy could be affected by various biological factors. There is increasing evidence that radiotracer retention could be controlled by members of multidrug resistance (MDR) system, especially P-glycoprotein (P-gp). Since the role of P-gp in tertiary hyperparathyroidism (T-HPTH) scintigraphic studies is poorly recognized, the aim of the study was to compare the correlation between parathyroid P-gp expression and results of their scintigraphy in T-HPTH versus primary hyperparathyroidism (P-HPTH). P-HPTH (n = 19) and T-HPTH (n = 18) patients were subjected to 99mTc-MIBI scintigraphy followed by surgical treatment. The parathyroid glands were assessed in routine hematoxylin-eosin staining and P-gp expression was analyzed using immunohistochemistry. Parathyroids collected during cadaver donor multi-organ harvesting were used as a control. It has been found that P-HPTH-derived parathyroid glands with predominating adenoma morphology expressed less P-gp, as compared to P-gp-rich T-HPTH glands, mainly displaying nodular or diffused hyperplasia phenotype. This finding reversely correlated with results of 99mTc-MIBI scintigraphy. However, we did not observe any difference in P-gp expression nor scintigraphy result between nodular or diffused hyperplasia. Altogether, these data suggest that P-gp overexpression in T-HPTH could be responsible for decreased sensitivity of 99mTc-MIBI scintigraphy in those patients. Therefore, the recently proposed reduced neck exploration or limited parathyroid resection on the basis of scintigraphy could create the risk of persisted/recurrent hyperparathyroidism. However, this problem requires further study.     

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le a antigen

Several lines of evidence indicate that sialosyl Le a , tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for a1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le a tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le a and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le a -negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metas-tases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le a antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.© Kluwer Academic Publishers 1998     

Increasing transplant mass results in long-term allograft survival and recovery from transplant vasculopathy

Late allograft rejection due to transplant vasculopathy continues to be a major clinical problem. Increasing the ratio of donor transplant size to recipient weight has been shown to reduce the incidence of late allograft failure. Using a murine pancreas transplant model we have tested the hypothesis that increasing the donor transplant size in a recipient can promote long-term allograft survival by promoting recovery from transplant vasculopathy. Recipients of an allograft that showed extensive vasculopathy were transplanted with a second donor transplant. The effect of the second allograft on the vasculopathy present in the first graft was measured. Transplanting a second allograft reversed all signs of ongoing rejection, including transplant vasculopathy, resulting in long-term survival of the first graft. Vasculopathy was only reversed if the first and second grafts were from the same mouse strain, suggesting an antigen-specific mechanism. However, the recovery of the first graft was not associated with antigen-specific peripheral tolerance.     

Fluorescence in situ hybridization investigation of cutaneous lesions in acute promyelocytic leukemia.

Cutaneous manifestations of acute promyelocytic leukemia are rare but well documented. Skin biopsies of leukemia can be difficult to confirm using morphology alone, and paraffin section immunophenotyping is not specific in separating acute promyelocytic leukemia from other acute myeloid leukemias involving the skin or inflammatory conditions, such as Sweet's syndrome and all-trans retinoic acid-associated genital ulcers, which may mimic leukemia cutis. Fluorescence in situ hybridization has been shown to be a fast and effective method of detecting the PML/RARA fusion gene characteristic of acute promyelocytic leukemia in fresh blood and bone marrow samples. Fluorescence in situ hybridization has also been demonstrated to be effective in detecting other chromosomal rearrangements in paraffin-embedded tissue. This retrospective study of cutaneous lesions from four patients with acute promyelocytic leukemia evaluates the utility of performing fluorescence in situ hybridization to confirm the presence of cutaneous manifestations of acute promyelocytic leukemia in formalin-fixed, paraffin-embedded skin biopsies. All patients had previous bone marrow findings of acute promyelocytic leukemia with characteristic morphology, immunophenotype, and cytogenetic studies, which detailed the presence of the t(15;17)(q22;q12) rearrangement. Two skin biopsies showed an infiltrate of blastic cells involving the dermis in a diffuse pattern and one biopsy had a perivascular/periadnexal pattern. The fourth case, involving the scrotum, showed a predominant neutrophilic infiltrate diffusely involving the dermis and epidermis with a subset of blastic cells. Nuclei were extracted from core biopsies of the formalin-fixed paraffin-embedded tissue and fluorescence in situ hybridization was performed using a dual color, dual fusion PML / RARA probe. All cases showed evidence of the t(15;17) rearrangement, with 90, 79, 51 and 16% positive signal patterns, each well above background limits. Fluorescence in situ hybridization appears to be a robust technique to detect cutaneous manifestations of acute promyelocytic leukemia in formalin-fixed paraffin-embedded skin biopsies.