CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression.

OBJECTIVE AND DESIGN: We have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression. METHODS: Using a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Delta 32 allele) was also determined. RESULTS: CCR5 density was stable over time on non-activated, HLA-DR(-)CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomatic and non-treated, we observed a correlation between CCR5 density on HLA-DR(-)CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Delta 32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002). CONCLUSION: These results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

Immunological aspects of psoriasis: V. T. cell subsets and suppressor cell functions regulating immune responses in peripheral blood.

T cell subsets bearing Fc-receptors for either IgG (TG) or IgM (TM), and suppressor cell activity of peripheral blood mononuclear cells on in vitro lymphoproliferative responses were studied in patients with untreated psoriasis. The proportions of TG and TM cells were unmodified in 15 patients compared to 15 control subjects studied in parallel experiments. The concanavalin A-induced suppressor cell activity, as well as the spontaneous suppressor cell function of in vitro short-lived adherent cells, were in the normal range for 7 out of 8 psoriatic patients investigated. The data argue against the possibility that a generalized suppressor cell defect occurs in psoriasis.

Pyruvate dehydrogenase activity is stimulated by growth hormone (GH) in human mononuclear cells: a new tool to measure GH responsiveness in man.

Human peripheral mononuclear cells (PMC) were used to examine the effects of hGH and insulin on the activity of the pyruvate dehydrogenase (PDH) complex. Incubation of PMC with 10(-7) mol/L hGH or insulin increased basal PDH activity. Hormonal effects were maximal (50-60% above control values) at 15 min. Later on, activation progressively decreased and was no longer detectable at 30 min. Total PDH activity was unaffected by hormonal treatment. PMC were subfractionated into lymphocytes and monocytes to assess the sensitivity of each cell types to the hormones. hGH significantly increased basal PDH activity in lymphocytes and monocytes (38% and 70% above control values, respectively), whereas insulin increased basal PDH activity only in monocytes (151% above control value). PMC from healthy subjects aged 1-45 yr were incubated for 15 min with 10(-7) mol/L hGH or insulin before PDH measurement. An increase of enzyme activity higher than 20% was observed in 26 patients out of 29 with hGH, and in 15 out of 18 with insulin. In conclusion, hGH is able to stimulate PDH activity of human mononuclear cells. This hormonal effect allows rapid evaluation of the cellular responsiveness of hGH in various pathophysiologic situations.

Human plasma growth hormone (GH)-binding proteins are regulated by GH and testosterone.

Possible regulation of GH-binding proteins (GH-BPs) in human plasma was examined. Eight children with isolated GH deficiency had a very low level of plasma GH-binding activity (10.2 +/- 1.1% of radioactivity). Under GH treatment the hormone binding to the high affinity BP (peak II-BP) increased in every patient to reach the mean value of 18.5 +/- 1.4%. In one patient, Scatchard plot analysis indicated that this increase was related to a higher binding capacity without any significant change in the binding affinity. A positive correlation existed between the GH-binding activity and insulin-like growth factor-I plasma levels. In nine boys with pubertal delay, the GH-specific binding to peak II-BP was normal (30.6 +/- 3.7% of radioactivity); it decreased significantly after testosterone treatment. In four boys with precocious puberty, the specific GH binding to peak II-BP was low (16.6 +/- 1.1%). It increased significantly to 21.6 +/- 1.1% of radioactivity after treatment with a LHRH analog. A negative correlation existed between plasma testosterone levels and GH binding to peak II-BP in boys presenting pubertal delay or precocious puberty. The high affinity GH-BP is regulated, and among the factors that play a role in this regulation, GH and testosterone have opposite effects.

Nonparallel changes of growth hormone (GH) and insulin-like growth factor-I, insulin-like growth factor binding protein-3, and GH-binding protein, after craniospinal irradiation and chemotherapy.

We studied the GH-insulin-like growth factor-I (IGF-I) axis serially over 24-36 months in six patients with medulloblastoma who underwent surgical removal of the tumor followed by craniospinal irradiation therapy for 6 weeks and then chemotherapy for 42 weeks. Eighteen and 24 months after beginning irradiation there was a decline in the peak GH secretory response to acute stimulation with arginine/insulin hypoglycemia. Six months after irradiation and during chemotherapy there was a transient decline in IGF-I, IGF binding protein-3 (IGFBP-3), and GH-BP values (respective mean values of 56.1 +/- 9.0 ng/mL, 1.1 +/- 0.2 microgram/mL, and 7.6 +/- 3.3% of radioactivity as compared to time 0 values: 13%%o/- 15 ng/mL, 2.2 +/- 0.2 micrograms/mL, and 20.0 +/- 4.0%, P < 0.001), although provoked GH secretion was normal at this time. The IGF-I, IGFBP-3, and GH-BP returned to pretreatment ranges by 12-36 months after initiation of the study. There was also a decline in body mass index and serum protein values at 6 months, suggesting suboptimal nutrition during this period. Six months after irradiation in ligand and immunoblot analysis there was a decline in IGFBP-3 and an abnormal electrophoretic mobility of IGFBP-2 that were both normalized at 36 months. In one patient we observed a high level of IGFBP-3 proteolysis at this time. This study demonstrates that before the decrease of GH secretion in patients receiving cranial irradiation there is a transient phase of GH insensitivity that may be characteristic of the acute therapeutic phase including the chemotherapy. This partial insensitivity may explain the early growth retardation observed in these patients.

Evaluation of the growth hormone-binding proteins in human plasma using high pressure liquid chromatography gel filtration.

A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.

[Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]. / Influence de l'état thyroïdien sur le profil électrophorétique des protéines mitochondriales de foie de rat

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.

[Effects of thyroidectomy of the rat on the structure and functions of skeletal muscle mitochondria]. / Effets de la thyroïdectomie du rat sur la structure et les fonctions des mitochondries des mescles squelettiques

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.

Human placenta-eluted gammaglobulins in immunomodulating treatment of rheumatoid arthritis.

Thirty-one patients with severe rheumatoid arthritis were treated with intravenous perfusion of human placenta-eluted gammaglobulins. These gammaglobulins, which are IgG eluted from placental tissue, have strong immunomodulating properties in vitro. Several clinical trials were tested to find the optimal useful dosage. A 50 percent improvement was considered a good result and was obtained in 60 percent of patients with rheumatoid arthritis. The best results were obtained in patients receiving 1,500 mg daily seven days each month. Six subjects had a long remission of their disease after the end of treatment. The side effects were usually minor. In all patients, an immunostimulation of lymphocyte function was shown, even when they had no improvement. A control group of patients underwent perfusion with IgG from placental blood without any clinical or immunologic effect. It is suggested that the in vivo effects of placenta-eluted gammaglobulins might be mediated by polyspecific anti-HLA-DR antibodies.

Molecular cloning, functional expression, pharmacological characterization and chromosomal localization of the human metabotropic glutamate receptor type 3.

Glutamic acid is the major excitatory amino acid of the central nervous system which interacts with two receptor families, the ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are coupled to G proteins and can be divided into three subgroups based on their sequence homology, signal transduction pathway and pharmacology. In this study, we describe the cloning of the cDNA encoding the human metabotropic glutamate receptor type 3 (HmGluR3). It was obtained by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to highly conserved sequences between rat mGluRs. The receptor shows 879 amino acids with 96% amino acid sequence identity with rat mGluR3. It is strongly expressed in fetal and adult whole brain, especially in caudate nucleus and corpus callosum. The gene was identified by fluorescence in situ hybridization on chromosome 7 band q22. Activation of the human mGluR3, permanently expressed in Baby Hamster Kidney (BHK) cells, by excitatory amino acid inhibits the forskolin-stimulated accumulation of intracellular cAMP. The rank order of potency is L-glutamic acid > or = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD) >> ibotenic acid > quisqualic acid. (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mM] is without effect on inhibition of forskolin-induced cAMP accumulation by L-glutamic acid.

Low dose antithymocyte globulins in renal transplantation: daily versus intermittent administration based on T-cell monitoring.

BACKGROUND: Despite the long history of use of antithymocyte globulins (ATG) in renal transplantation, ideal doses and duration of ATG administration based on the monitoring of T lymphocytes have yet to be defined. METHODS: Two immunosuppressive regimens based on low-dose rabbit ATG (Thymoglobuline; Imtix-Sang-stat, Lyon, France) were assessed during the first year after transplantation: daily ATG (DATG; n=23) where 50 mg of ATG was given every day and intermittent ATG (IATG; n=16) where similar doses of ATG were given for the first 3 days and then intermittently only if CD3+ T lymphocytes (measured by flow cytometry) were > 10/mm3. Both groups received steroids, azathioprine, and cyclosporine. RESULTS: ATG-induced depletion was similar for peripheral blood lymphocytes and T cells in both groups: it began at day 1 after transplantation, was submaximal at day 3, and reached maximum intensity between days 6 and 8, from which time cell counts progressively increased. However, T-cell depletion was still present at day 20. The total ATG dose per patient (381.5+/-121 vs. 564+/-135 mg/patient) and the mean cumulative daily dose of ATG (0.60+/-0.17 vs. 0.80+/-0.14 mg/kg/day) were significantly lower in the IATG group (P=0.0001 and 0.0006, respectively). The overlap of ATG and cyclosporine treatment was 6.7+/-3 vs. 7.4+/-4.3 days (P=NS), and the mean duration of ATG therapy was 11.3+/-3.2 vs. 11.6+/-2.7 days in the IATG and DATG groups, respectively (P=NS). ATG was given in an average of one dose every 1.6 days in the IATG group compared with one dose daily in the DATG group (P=7 x 10(-7)). There was no significant difference in renal graft function, the number of acute graft rejections, or ATG-related side effects and complications. Despite the daily immunological follow-up, there was a net saving of $760/patient in the cost of treatment in the IATG group. CONCLUSION: IATG had the advantage of a reduction in the dose of ATG and in the cost of treatment, while offering similar T-cell depletion and effective immunosuppression. This approach could be proposed as an induction protocol, particularly for patients with poor graft function in whom cyclosporine introduction has to be delayed or those with increased risk of cytomegalovirus infections or secondary malignancies.

Natural killer cell and alphabeta and gammadelta lymphocyte traffic into the liver graft immediately after liver transplantation.

BACKGROUND: The persistence and migration of donor leukocytes has been well established, but cellular kinetics immediately after revascularization and the potential relevance of these different lymphocyte populations to spontaneous tolerance remain unclear. During the early hours of revascularization, there is a transitory "congestion" of the liver graft, which is evidence of an early phase that we have termed "first cellular contact." METHODS: We have carried out by flow cytometry a prospective comparative study of the peak kinetics of lymphocyte subpopulations contained in: (a) peripheral blood and liver grafts at the time of multi-organ extraction from 14 brain-dead donors, (b) recipient peripheral blood before transplantation, and (c) recipient peripheral blood and liver grafts after (t=2 h) declamping and vascularization of the liver graft. RESULTS: Before transplantation, the liver grafts contained large numbers of natural killer (NK) and NK-like cells with early lymphocyte activation. Immediately after revascularization, there was an influx of recipient NK and NK-like cells into the liver. CONCLUSIONS: NK and CD3+CD56+ (NK-like) cells flooding into the liver graft immediately after revascularization could rapidly destroy allogeneic cells. However, spontaneous tolerance and the persistence of donor lymphocytes after orthotopic liver transplant could be a result of donor TCRalphabeta NK1.1 liver graft lymphocytes, which may be involved in the destruction of CD8+ T lymphocytes that would have received the apoptosis signal, and to NK and NK-like cell inhibition via inhibitory NK receptors. The decrease in gammadelta T lymphocytes in the two compartments suggests a mechanism of recirculation and capture in other lymphoid organs.

Description of a polymorphism in the regulatory region of the HLA-DRA gene.

It was previously reported that the cell membrane expression of HLA class II molecules is tissue specific and variable among individuals. This variation could be explained at the level of gene regulation. Due to the fact that a coordinate regulation of the HLA-DR genes seems to exist, we focused on the HLA-DRA monomorphic gene. In order to study the polymorphism of the HLA-DRA gene regulatory region, we used restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified genomic DNA. The DdeI and PvuII digestions of the amplified DNA permitted the definition of the two alleles according to the absence (allele A) or the presence (allele B) of the two polymorphic restriction sites. The presence of these sites was confirmed by direct sequencing after PCR. Using homozygous typing cells, a close relationship between the HLA-DRB coding region and the HLA-DRA regulatory region polymorphisms was shown. Furthermore, a linkage between the HLA-DRA regulatory region and DRB3 gene coding region polymorphisms could be established. These results suggested a structural argument for different levels of HLA class II genes and, consequently, of cell-surface expression of class II antigens according to the allelic specificities of DRA, DRB1, and DRB3.

Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology.

HLA-DRB1 allelic specificities can be determined using SSOs annealing to their complementary PCR-amplified target DNA. To perform HLA-DR oligotyping routinely for donors and recipients of bone marrow transplantation, a "reverse" dot-blot technique has been developed that consists in the hybridization of labeled PCR-amplified target DNA to SSOs that have been first attached to nitrocellulose membranes. The 15 oligonucleotides chosen enabled the following HLA-DRB1 "generic" specificities to be defined: DR1, BON, 2, 3, 4, 11, 11 JVM, 12, 13, 13 HAG, 14, 7, 8, 9, 10. The genomic DNA was amplified by asymetric PCR with incorporation of biotinylated deoxynucleotides predominantly to generate labeled single-stranded DNA. Hybridization between specific immobilized oligoprobes and target DNA was nonradioactively detected by a colorimetric reaction using alkaline phosphatase. The reverse dot-blot methodology was successfully tested, first, for the determination of HLA-DR4 subspecificities, and then the procedure was routinely applied to the generic HLA-DR oligotyping of bone-marrow donors and recipients.

Pharmacology of ribosomal immunotherapy.

The use of immunostimulating drugs is one way to intervene in the immune system. Many of these agents are of bacterial origin and most are able to stimulate the nonspecific immune response by acting on polymorphonuclear cells (PMNs) and macrophages. Ribosomal immunotherapy ('Ribomunyl') contains both proteoglycans from Klebsiella pneumoniae and ribosomes from 4 different bacterial strains. It can stimulate not only macrophages but also specific antibody production. 'Ribomunyl' has been shown to stimulate many of the functions of PMNs, specifically the formation of oxygenated free radicals, chemotaxis and adhesion. The effect of 'Ribomunyl' immunostimulant on the properties of macrophages is of special interest, as these cells participate in both the nonspecific immune response (phagocytosis, proinflammatory cytokine production) and the specific immune response (antigen processing and presentation, lymphocyte proliferation). 'Ribomunyl' has been shown to increase the production of many cytokines [interleukin (IL)-1, IL-6, IL-8, tumour necrosis factor-alpha and colony-stimulating factor], leading to the activation of the cytokine network. 'Ribomunyl' was also able to stimulate natural killer cells involved in viral immunity. Because of the presence of ribosomes from 4 frequently encountered bacterial strains, 'Ribomunyl' has specific immunostimulant properties. This has been clearly demonstrated in animals and humans, where specific antibody-forming B cells were found in the tonsils after oral administration. However, specific T-cell response has not been reported, suggesting that 'Ribomunyl' could act directly on B cells such as T-cell-independent bacterial antigens.

Effect of thyroidectomy on oxidative phosphorylation mechanisms in rat liver mitochondria.

(1) In the presence of succinate, 2,4-dinitrophenol raised the oxygen consumption rate of state 3 liver mitochondria, whatever the thyroid state. The rise was clearly greater in thyroidectomized than in normal rats, but the uncoupled state 3 mitochondria of thyroidectomized rats nevertheless consumed oxygen more slowly than normal rat mitochondria. Thyroidectomy therefore weakened respiratory chain activity and phosphorylation reactions. (2) With beta-hydroxybutyrate as substrate, oxygen consumption V of state 3 mitochondria was greatly diminished in thyroidectomized rats, but the KM remained unchanged. In the presence of succinate, state 3 respiration was not affected by thyroidectomy when substrate concentrations were low, but diminished at concentrations above 3 mM. (3) Respiratory chain activity was estimated by determining the succinate cytochrome c reductase activity. After thyroidectomy, catalytic efficiency (V/KM) dropped by 60% in intact mitochondria at high concentrations of the substrate, but only by 30% when concentrations were low. In submitochondrial particles, thyroidectomy reduced V without changing KM. These results suggest that in thyroidectomized rats, succinate penetration is faster when substrate concentrations are low. (4) Mg2+ -stimulated ATPase activity dropped by 20% in thyroidectomized rats. In the presence of increasing concentrations of oligomycin, inhibition of ATPase activity was greater in normal than in thyroidectomized rat mitochondria. Thyroidectomy reduced by over 30% the ATPase activity actually involved in oxidative phosphorylation.

Early effects of thyroidectomy and triiodothyronine administration on rat-liver mitochondria.

This work was undertaken to study the action exerted by thyroid hormones on mitochondria. By day 6 after thyroidectomy, the respective activities of two inner-membrane enzymes--succinate and beta-hydroxybutyrate cytochrome c reductases--had already dropped by 32 and 50%, whereas, in the outer membrane, the activity of rotenone-insensitive NADH-cytochrome c reductase did not change significantly. The decrease in the activity of the inner-membrane enzymes closely followed the disappearance of T3 and T4 from serum. 10 h after administration of 25 micrograms/100 g T3 to thyroidectomized rats, the activity of succinate and beta-hydroxybutyrate cytochrome c reductases and the oxygen consumption rate with succinate or beta-hydroxybutyrate were significantly increased, while, in the outer membrane, the activity of monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase remained unchanged. In the thyroidectomized rat, L-[3H]leucine incorporation in vivo is diminished in all the liver mitochondrial proteins, and especially in two constituents of MW 19 000 and 28 000. The radioactivity of these two components is also decreased in the normal rat treated with chloramphenicol, a specific inhibitor of mitochondrial protein synthesis. L-[14C]leucine incorporation in isolated liver mitochondria was significantly increased in the thyroidectomized rat, 10 h after T3 treatment. Thus, thyroid hormones have an early and preferential action on the mitochondrial protein synthesizing system and on the inner-membrane enzyme activities.

Increased expression of liver PKC alpha in hypoinsulinemic diabetic rats: a post-translational effect.

Ca2+-dependent protein kinase C (cPKC) activity and expression have been studied in livers from hypoinsulinemic streptozotocin (STZ)-induced diabetic and untreated control rats. In diabetic rats, cPKC activity was slightly decreased in liver total particulate and nuclear fractions but was unchanged in mitochondrial-lysosomal, microsomal and cytosolic fractions. On Western immunoblot analysis, PKC alpha was identified as two distinct proteins of 90 and 81 kDa. In diabetic rats, the abundance of the 90 kDa protein was increased in most subcellular fractions with a maximum in the cytosolic and microsomal fractions (180%) but that of the 81 kDa protein was unchanged. PKC beta2 was detected as a single 81 kDa protein in cytosolic and microsomal fractions with unchanged levels in diabetic rats. Liver PKC alpha mRNA levels as measured by reverse transcription and competitive PCR amplification were similar in diabetic and control rats. The increased expression of PKC alpha protein in diabetic rats was reversed by insulin but not by phlorizin, suggesting that it did not result from hyperglycemia. We conclude that STZ-induced diabetes induces the expression of a biologically inactive form of PKC alpha which differs from active PKC alpha by an undefined post-translational modification, possibly an increase in phosphorylation state.

Characterization of insulin degradation products generated in liver endosomes: in vivo and in vitro studies.

The degradation products generated from A14 and B26 125I-labelled insulins in liver endosomes in vivo and in vitro have been isolated by high-performance liquid chromatography and cleavages in the B chain have been identified by automated radiosequence analysis. In rats sacrificed various times after injection of each of the 125I-labelled insulins, two major degradation products slightly less hydrophobic than intact iodoinsulins were identified; these accounted, at 8 min. for about 45% (A14 125I-labelled insulin) and 15% (B26 125I-labelled insulin) of the total radioactivity recovered, respectively. The products generated from A14 125I-labelled insulin contained an intact A chain, whereas those generated from B26 125I-labelled insulin contained a B chain cleaved at the B16-B17 bond. With B26 125I-labelled insulin, two minor products, with cleavages at the B23-B24 and B24-B25 bonds, were also observed. In vivo chloroquine treatment did not alter the nature but caused a decrease in the amount of insulin degradation products associated with endosomes. When endosomal fractions isolated from iodoinsulin injected rats were incubated at 30 degrees C in isotonic KCl, a rapid degradation of iodoinsulin, maximal at pH 6, was observed. With A14 125I-labelled insulin, the two major degradation products identified in vivo were generated along with monoiodotyrosine, but with B26 125I-labelled insulin monoiodotyrosine was the main product formed. Addition of ATP, presumably by decreasing the endosomal pH, shifted the medium pH for maximal iodoinsulin degradation to about 7-8. These studies have allowed a direct identification of two previously suggested cleavage sites in the B chain. They have also shown that the degradation products generated in cell-free endosomes under conditions that promote endosomal acidification are similar to those identified in vivo.

Increase of class II HLA molecules on the membrane of B lymphocytes from patients with rheumatoid arthritis.

Using a novel cytofluorometric method of cellular antigen quantification, we examined peripheral blood mononuclear cells (PBMC) from patients suffering from rheumatoid arthritis (RA) for quantitative modification of class II human leucocyte antigen (HLA) molecules expressed on the surface. Class II HLA molecules were detected by indirect immunofluorescence with a monomorphic monoclonal antibody. No change was observed in the density of class II HLA molecules at the surface of monocytes of RA patients as compared to that of paired healthy subjects. We confirmed that the percentage of class II HLA-bearing T cells was slightly increased in RA patients versus controls, but the density of class II antigens per cell could not be determined accurately. An increase in the density of class II HLA molecules on RA B cells was shown, suggesting that a chronic activation stage of this population contributes to the disease.