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Biomedical subjects

R D Allen

Publications and source records attributed to R D Allen.

At least 19 recordsLinked to original sources
PubMed1992

Transplantation of the endocrine pancreas in 1992.

In 1992, transplantation of the endocrine pancreas is placed at the interface between laboratory research and clinical medicine. Unlike other forms of transplantation, it is not life saving, and yet its potential to treat all patients with insulin dependent diabetes is limited only by the availability of donated organs. Arguably, the long term glucose homoeostasis provided by vascularised pancreas transplantation is the standard by which other forms of endocrine pancreas transplantation (islet cell, fetal islet, and xenogeneic transplantation) must be judged. For the small number of type I diabetic Australians who develop end-stage renal failure and would otherwise require a renal transplant, the combined transplant procedure is a technically viable treatment option and, with some justification, the treatment of choice.

Diabetes Mellitus, Type 1↗
PubMed1992

Endosomal system of Paramecium: coated pits to early endosomes.

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/- 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/- 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

Acid Phosphatase↗
PubMed1991

Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes.

The expression of the alpha' and beta subunit genes of beta-conglycinin is differentially regulated during soybean embryo development. Although both are expressed solely in developing seeds during mid to late stages of embryo development, the alpha' subunit is expressed more highly on a per gene basis, and alpha' subunit mRNA begins to accumulate three to five days earlier than beta subunit mRNA. In cultured cotyledons, beta subunit gene(s) respond to changes in methionine or abscisic acid levels, whereas expression of the alpha' subunit gene(s) is unaffected by these changes. To investigate the mechanisms by which these genes are transcriptionally regulated, we examined the interactions of nuclear proteins with upstream sequences from the alpha' and beta subunit genes. Four distinct DNA binding factors were identified in nuclear extracts from developing soybean seeds. These factors are termed Soybean Embryo Factors (SEF) 1 through 4. SEF binding sites are distributed non-uniformly between the alpha' and beta subunit genes, and the amount of protein binding is modulated over the course of embryo development. DNA footprinting revealed the sequences recognized by three of these factors. Factors which behave in a manner similar to that of SEF3 were also identified in nuclear extracts from developing tobacco and sunflower seeds.

Antigens, Plant↗
PubMed1991

Percutaneous biopsy of bladder-drained pancreas transplants.

Percutaneous biopsy is a valuable investigation in the management of allograft rejection for all solid organs. Pancreas transplants have not been biopsed percutaneously, though open and percystoscopic biopsies have proved useful. We have compared percutaneous needle core biopsy with fine-needle aspiration cytology for the diagnosis of rejection in 18 patients receiving combined kidney and pancreas transplants and in one who was transplanted with the pancreas alone. Percutaneous needle core biopsy was successful in 37 of 40 attempts (93%), while fine-needle aspiration yielded diagnostic material on 33 of 47 attempts (70%). Transient hyperamylasemia occurred in 29%, returning to baseline in three days. One patient twice developed transient macroscopic hematuria. There was agreement between needle core biopsy and fine-needle aspiration on the diagnosis of rejection on six occasions and for the absence of rejection on 16. There was an 8% false-positive rate for fine-needle aspiration. In 13 instances of histologically proved renal rejection, concurrent pancreas biopsy revealed rejection in 69%. Pancreas rejection was not, however, seen in the absence of renal rejection. In this pilot study, percutaneous biopsy of the bladder-drained pancreas allograft was shown to be a practicable and valuable investigation without major complications.

Adult↗
PubMed1991

Identification of Porphyromonas gingivalis components that mediate its interactions with fibronectin.

Porphyromonas (Bacteroides) gingivalis W12 binds and degrades human plasma fibronectin. In the presence of the protease inhibitor N-alpha-p-tosyl-L-lysyl chloromethyl ketone, P. gingivalis cells accumulated substantial amounts of 125I-fibronectin as a function of incubation time. Fibronectin binding was specific, reversible, and saturable. The Kd for the reaction was estimated to be on the order of 100 nM, and there was an average of 3.5 x 10(3) fibronectin binding sites per cell. Unlabeled fibronectin inhibited the binding of 125I-fibronectin to bacteria; however, fibrinogen was an even more efficient inhibitor of 125I-fibronectin binding. Unrelated proteins were without effect on fibronectin binding. A fibronectin-binding component (Mr, 150,000) was identified in sodium dodecyl sulfate-solubilized P. gingivalis. Fibronectin was degraded into discrete peptides by P. gingivalis W12. The degradation of fibronectin was inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone. Two P. gingivalis components (Mrs, 120,000 and 150,000) degraded fibronectin in substrate-containing gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a previous study (M. S. Lantz, R. D. Allen, T. A. Vail, L. M. Switalski, and M. Hook, J. Bacteriol. 173:495-504, 1991), we found that the same strain of P. gingivalis bound and subsequently degraded human fibrinogen via apparently distinct cell surface components of molecular sizes similar to those of components now implicated in the binding and degradation of fibronectin. These results raise the possibility that the two ligands are recognized and modified by the same components on P. gingivalis W12. In support of this hypothesis, unlabeled fibrinogen effectively inhibited the binding of 125I-fibronectin to bacteria and blocked 125I-fibronectin binding to a P. gingivalis ligand-binding component (Mr, 150,000 immobilized on a nitrocellulose membrane.

Bacteroides↗
PubMed1991

Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen.

Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

Bacterial Proteins↗
PubMed1991

Longitudinal histopathologic assessment of rejection after bladder-drained canine pancreas allograft transplantation.

In preparation for assessment of percutaneous biopsies in our clinical pancreas transplant program, a working knowledge of the histopathologic changes after transplantation was obtained in a longitudinal open biopsy study of 16 dogs receiving bladder-drained whole pancreas allografts. Edema, extravasation of polymorphs, and lymphocytes associated with focal parenchymal injury were early, invariable, and probably nonspecific findings. The initial feature of unmodified rejection was the appearance of capillary and small vein endothelial changes with mainly perivascular inflammatory cell infiltration. Acinar cell loss occurred early and was progressive, whereas islets and ducts were relatively preserved, indicating that acinar tissue may be more vulnerable to lytic necrosis when damaged. Functional rejection, determined by fasting urinary amylase levels, was at a stage of extensive and irreversible necrosis. Functioning grafts in immunosuppressed dogs had minor and transient endothelial changes with absence of class II antigen staining of parenchymal cells.

Acute Disease↗
PubMed1990

Differences defined by bone marrow transplantation suggest that lpr and gld are mutations of genes encoding an interacting pair of molecules.

Homozygosity for either of the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations of mice causes the development of systemic lupus erythematosus-like autoimmune syndromes that are characterized by severe lymphadenopathy and highly elevated serum immunoglobulin levels. Although the mutations are nonallelic, analysis of homozygous lpr/lpr and gld/gld mice on the same strain background has indicated that the pathology and severity of the autoimmune syndromes induced by these mutations are indistinguishable. To explain this, it has previously been suggested that lpr and gld may represent mutations in molecules involved in sequential steps of an intracellular metabolic pathway of T cells. We have now investigated the behavior of both lpr and gld in a variety of bone marrow chimeras and have found that functional differences between lpr and gld become apparent after bone marrow transfer. Transfer of lpr/lpr bone marrow to irradiated congenic +/+ recipients caused the development of a graft-vs.-host-like lymphoid wasting syndrome, whereas transfer of gld/gld bone marrow to +/+ recipients resulted in development of a gld-like autoimmune syndrome. Additionally, gld/gld hosts behaved like +/+ hosts irrespective of the genotype of the donor bone marrow, whereas lpr/lpr hosts behaved unlike +/+ hosts when reconstituted with either lpr/lpr, gld/gld, or +/+ bone marrow. These are the first clear differences between these two mutations yet described. Our studies indicate that the molecule altered by the gld mutation is expressed only by bone marrow-derived cells, whereas the molecule altered by the lpr mutation is expressed by both bone marrow-derived cells and by one or more peripheral radioresistant cell populations. To reconcile these differences with the fact that homozygous lpr/lpr and gld/gld mice are indistinguishable, we suggest an alternative model for the relationship between the lpr and gld mutations in which the two molecules affected represent an interacting ligand-receptor pair expressed by different cells.

Animals↗
PubMed1990

Bone marrow transplantation from mutant lpr/lpr mice. Functional abnormalities rather than alloantigenic differences appear to determine the development of a graft-vs.-host-like syndrome.

Transfer of bone marrow (BM) from autoimmunity-prone mice homozygous for the lymphoproliferation (lpr) mutation to irradiated congenic +/+ recipients has previously been shown to result in a syndrome similar to chronic graft-vs.-host (GVH) disease. It has been suggested that this syndrome may be due to an antigenic difference caused by the lpr mutation itself or to antigenic differences at loci closely linked to the lpr locus (Theofilopoulos, A. N. et al., J. Exp. Med. 1985. 162:1; Mosbach-Ozmen, L. and Loor, F., Thymus 1987. 9:197). However, the results presented here indicate that alloantigenic differences do not play a role in this syndrome. Instead, the chronic disease observed in lpr/lpr----(+/+) BM chimeras appears to develop as a result of a functional defect associated with the lpr mutation which is expressed shortly after transfer of lpr/lpr BM to irradiated recipients. This defect causes an increase in the levels of serum IgG1 and IgG2, which peak at 4-5 weeks post-transfer and then decline to normal levels by 9-10 weeks post-transfer. Inflammation similar to that observed in classic GVH reactions accompanies excess IgG production in congenic +/+ recipients but not in lpr/lpr recipients of lpr/lpr BM. We demonstrate that the GVH-like response occurring in lpr/lpr----(+/+) chimeras is dependent on mature T cells, but that either lpr/lpr or (+/+) T cells can support this reaction. These results suggest that transfer of lpr/lpr BM to normal mice causes immunoregulatory disturbances which lead to nonspecific activation of T cells. We speculate that lpr/lpr BM causes a GVH-like reaction in +/+ recipients but a systemic lupus erythematosus-like syndrome in lpr/lpr recipients because of intrinsic differences in the +/+ and lpr/lpr host environments. Considering these findings, the lpr/lpr----+/+ GVH model may be useful for analysis of factors capable of inducing undesirable reactions in clinical BM transplantation between nominally histocompatible donors and recipients, in addition to being informative about the nature of the lpr mutation itself.

Animals↗