Atrial Natriuretic Peptide Protects Hepatocytes Against Damage Induced by Hypoxia and Reactive Oxygen. Possible Role of Intracellular Free lonized Calcium

Peer Reviewe

Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children.

The study of cytotoxic T cell responses to measles antigens during infection and after vaccination may provide insight into the immunopathology of the infection. It will also provide a knowledge of the immunity conferred by wild or attenuated virus, which will help in the design of new vaccines. Direct cytotoxic T cell responses, which did not require in vitro restimulation, were measured from peripheral blood by a standard 51Cr-release assay in 35 patients with acute measles, using HLA class I matched allogeneic B cells as targets. 77% showed specific responses to measles fusion protein, 69% to the hemagglutinin, and 50% to the nucleoprotein. These responses, which were related to severity of disease and history of previous vaccination, had waned by 14-24 wk after measles when memory responses to the same antigens could be elicited by restimulation in 71% of the 13 patients tested. A similar pattern followed vaccination: direct cytotoxic responses to fusion and hemagglutinin proteins were shown in 70% of the 20 children tested while 50% responded to the nucleoprotein. These responses, which were mediated by both CD8(+) and CD4(+) cells, faded over 6 wk when memory responses could be restimulated. Thus, a vigorous cytotoxic T lymphocyte response to fusion, hemagglutinin, and nucleoproteins is important in both natural and vaccine-induced immunity to measles

Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone

Abstract Introduction The effects of parathyroid hormone were studied on Ca" fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na' gradient, and this was inhibited by the Na' ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2" exchange activity. There was a Na' gradient independent Ca2+ flux, but membrane binding of Ca2" was excluded from contributing to the Na' gradient-dependent efflux. The Na' gradient-dependent flux of Ca2" was very rapid, and even 2-and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2" and Na' with an apparent (5 s) K. for Na'-dependent Ca2" uptake of 10 ,uM, and an apparent The suspension was then further homogenized using a Polytron homogenizer (Brinkman Instruments, Westbury, NY), setting six for three bursts of 30 s. After samples were taken for analysis of protein and enzyme content, the homogenate was centrifuged at 2,500 g for 15 min and the pellet discarded. The suspension was then spun at 24,000 g for 20 min and the supernatant decanted. The soft portion of the pellet was resuspended in the homogenizing buffer to a value of 1.3 ml/g initial tissue and hand homogenized for seven strokes with a Dounce homogenizer. The suspension was then diluted and thoroughly mixed with 1.6 ml/g initial tissue of 100% Percoll (Pharmacia Fine Chemicals, Upsala, Sweden) and the homogenizing buffer, 17 ml/g initial tissue (final Percoll concentration 8%). The suspension was then centrifuged at 30,000 g for 35 min. This produced a density gradient of Percoll from 1.005 to 1.125 g/ml. The fraction corresponding to an opaque band and density 1.030-1.035 g/ml at volume 50-67% of the total was removed. For each set of experiments, the Percoll band containing BLMV was diluted in the solution for vesicle loading, generally 100 mM KCl, 20 mM Tris-Hepes, pH 7.5, unless otherwise indicated, at 14 ml/g initial tissue. BLMV were washed times two with centrifugation at 30,000 g for 30 min and resuspended in loading solution. The soft pellet was resuspended in the appropriate loading solution and aliquoted using a Hamilton syringe fitted with a 27-gauge needle (Hamilton Co., Reno, NV). Samples of suspension were taken for analysis of protein and enzyme content. In some experiments, the BLMV preparation was further purified by incubation in Sepharose-4B bound ATP (Pharmacia Fine Chemicals, Piscataway, NJ). After 30 min agitation at room temperature, the Sepharose-4B ATP and bound membranes were removed by centrifugation at 5,000 g, washed and recentrifuged at 5,000 g. The BLMV pellet was reformed by centrifuging the supernatant for 30 min at 30,000 g. Enzyme studies. Characterization of the BLMV by means of specific enzyme markers was performed as described previously (33, 34) using activities of Na+/K+-ATPase, a basolateral membrane marker (35); acid phosphatase, a lysosomal marker (36); cytochrome oxidase, a mitochondrial marker (37); maltase, a brush border marker (38); and NADPH-cytochrome-C reductase, an endoplasmic reticulum marker (39). Protein content was measured (40) using a bovine serum albumin standard. BLMV "sidedness. " The orientation of the membrane vesicles was assessed by latency of Na+/K+-ATPase activity and its ouabain sensitivity before and after two cycles of freezing and thawing during incubation with 0.4 mg/ml deoxycholate (DOC) (34, 41). Orientation was assessed by these maneuvers since the ouabain binding site of Na+/K+-ATPase is on the external membrane face and the ATP binding site is on the internal face of plasma membranes containing Na+/K+-ATPase activity. Thus, in a mixture of BLMV, inside out vesicles (IOV) showed Na+/ K+-ATPase activity not sensitive to ouabain before, but sensitive after DOC, and right side out vesicles (ROV) showed Na+/K+-ATPase activity only after DOC. Ouabain-sensitive Na+/K+-ATPase activity present before and after DOC was not an assignable orientation and may have represented poorly sealed vesicles or membrane sheets. These studies assumed that sealed BLMV were not permeable to ATP or ouabain and were completely disrupted by the DOC/freeze-thaw treatment. Electron microscopy. Samples of BLMV for transmission electron microscopy were fixed in 2.5% glutaraldehyde and post fixed in 1% osmium tetroxide. They were then dehydrated in a graded series of ethanols and embedded in D.E.R. 332-732 Embedding Media Formula B (Polysciences, Inc., Paul Valley Industrial Park, Warrington, PA). Ca2 efflux experiments. Vesicles prepared as described above with Parathyroid Hormone and Renal Calcium Transport 1097 100 mM KC1, 20 mM Tris-Hepes in the intravesicular space were divided into 10-Mu aliquots and incubated with 40 ul of a loading solution (100 mM, 20 mM Tris-Hepes, 25 MM 45CaC12, pH adjusted to 7.5) for 30 min at 30'C. Calcium efflux was initiated at 30'C with a solution of either 100 mM KCI (K+ efflux) or NaCi (Na' efflux) with 20 mM Tris-Hepes, and 1 mM EGTA, pH 7.5. To examine the effect of the Na' gradient, a NaCl efflux solution containing 5 gM monensin, a Na' ionophore, was used in addition to the normal Na' efflux solution. The reactions were stopped by dilution with either icecold 100 mM KCl or NaCI with 20 mM Tris-Hepes, pH 7.5, depending on the solution used to initiate efflux. The solutions were rapidly filtered through 0.65-MuM filters (Millipore; Millipore Corp., Bedford, MA) after addition of stop solutions. Radioactivity trapped on the filters was measured with a scintillation spectrometer (Hewlett-Packard Co., model 460-CD, Downers Grove, IL). All experimental points were done at least in triplicate. Variations. from these methods are explained with the relevan

CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver.

The success of orthotopic liver transplantation is dependent on multiple factors including MHC tissue compatibility and ischemic/reperfusion injury. Ischemic/reperfusion (I/R) injury in the liver occurs in a biphasic pattern consisting of both acute phase (oxygen free radical mediated) and subacute phase (neutrophil-mediated) damage. Although numerous studies have given insights into the process of neutrophil recruitment after I/R injury to the liver, the exact mechanism that initiates this subacute response remains undefined. Using a T cell-deficient mouse model, we present data that suggests that T-lymphocytes are key mediators of subacute neutrophil inflammatory responses in the liver after ischemia and reperfusion. To this end, using a partial lobar liver ischemia model, we compared the extent of reperfusion injury between immune competent BALB/c and athymic nu/nu mice. Studies evaluating the extent of liver damage as measured by serum transaminases (GPT) demonstrate similar acute (3-6 h) post-I/R responses in these two mouse models. In contrast, the subacute phase (16-20 h) of liver injury, as measured by both serum GPT levels and percent hepatocellular necrosis, was dramatically reduced in T cell-deficient mice as compared with those with an intact immune system. This reduction in liver injury seen in nu/nu mice was associated with a 10-fold reduction in hepatic neutrophil infiltration. Adoptive transfer of T cell-enriched splenocytes from immune competent mice was capable of reconstituting the neutrophil-mediated subacute inflammatory response within T cell-deficient nu/nu mice. Furthermore, in vivo antibody depletion of CD4+ T-lymphocytes in immune competent mice resulted in a reduction of subacute phase injury and inflammation as measured by serum GPT levels and neutrophil infiltration. In contrast, depletion of CD8+ T-lymphocytes had no effect on these indexes of subacute inflammation. Kinetic analysis of T cell infiltration in the livers of BALB/c mice demonstrated a fivefold increase in the number of hepatic CD4+ T-lymphocytes within the first hour of reperfusion with no significant change in the number of CD8+ T-lymphocytes. In summary, these results implicate CD4+ T- lymphocytes as key regulators in initiating I/R-induced inflammatory responses in the liver. Such findings have implications for therapy directed at the early events in this inflammatory cascade that may prove useful in liver transplantation

RP: Glucagon, catecholamine and pancreatic polypeptide secretion in type 1 diabetic recipients of pancreas allografts

After successful pancreas transplantation, recipients with type I diabetes mellitus achieve a return to normal fasting plasma glucose levels, normal oral glucose tolerance tests, and normal levels of HbA,C (1-7). However, whether recipients undergo alterations in counterregulatory hormonal responses to glycopenia, or improvement in counterregulation of hypoglycemia, has not been determined. These are important considerations because type I diabetic patients commonly have defective glucagon and epinephrine responses to hypoglycemia and, consequently, deranged counterregulation of hypoglycemia with its attendant risks (8-13). Therefore, presence or absence of improvement in these responses are highly relevant factors in the evaluation of the overall benefits of pancreas transplantation. Moreover, since the transplanted pancreas is denervated and since islet alpha cell and pancreatic polypeptide cell function is neurally modulated, investigation of pancreas recipients offers a unique opportunity to assess regulation ofglucagon and pancreatic polypeptide secretion from the pancreas in humans in the setting of absent central nervous system input into the islet. To evaluate hormonal counterregulation of hypoglycemia in patients with type I diabetes mellitus who have received successful pancreas allografts, we have examined glucose, glucagon, pancreatic polypeptide, and catecholamine responses during insulin-induced hypoglycemia. We have also assessed glucagon and pancreatic polypeptide responsivity to direct stimulators of alpha and pancreatic polypeptide cells (arginine and secretin, respectively) to ascertain whether possible alterations in secretory responses in pancreas recipients are specific for hypoglycemic signaling or more general in nature. In this study, the responses of pancreas recipients were compared with responses of patients with similar durations of type I diabetes mellitus, who had not received pancreas transplantation, and to nondiabetic control subjects. Methods Subjects. 38 type I diabetic recipients of pancreas allografts with iliac vessel anastomoses and with intact native pancreases who had been transplanted between 3 and 60 mo previously were studie