Quantitation of apolipoprotein epsilon gene expression by competitive polymerase chain reaction in a patient with familial apolipoprotein E deficiency.

A simple method of obtaining semiquantitative and reliable data on apolipoprotein (apo) sigma gene expression is described. We detected apo sigma specific sequences by reverse transcription (rT)-PCR. For quantitative measurement, an apo sigma DNA standard was produced allowing the development of a competitive PCR-method. The efficiency of RNA extraction and cDNA synthesis was controlled by quantitation of a housekeeping gene (glyceraldehyde-3-phosphatedehydrogenase, G3PDH) in separate reactions. To imitate a defined induction of apo sigma gene expression, serial twofold dilutions of total RNA were reversely transcribed and the respective cDNAs used to perform a competitive apo sigma and G3PDH PCR. The change in apo sigma cDNA and G3PDH cDNA was 1.7-2.3-fold with an expected value of 2.0-fold. Standard deviations in three independently performed experiments were within a range of < 15% of the mean, indicating low intra-assay variation and high reproducibility. To illustrate this method, apo sigma gene expression was measured in a patient with complete lack of functional active apo E in comparison to healthy controls. The method presented here might be valuable in assessment of apo sigma gene expression in human disease.

Differential expression of bcl-2 and susceptibility to programmed cell death in lymphocytes of HIV-1-infected individuals.

The bcl-2 protooncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death. There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. In this study, we examined bcl-2 expression in lymphocytes from human immunodeficiency virus type 1 (HIV-1)-infected and healthy subjects by flow cytometry. bcl-2 expression was detected in more than 97% of peripheral blood lymphocytes in both healthy and HIV-infected individuals. It was consistently observed that CD4+ lymphocytes from HIV-1-infected individuals with less than 200 CD4+ cells/microliter expressed significantly less bcl-2 than healthy controls. In contrast, bcl-2 expression in CD8+ lymphocytes of these patients was significantly enhanced. No significant alteration of bcl-2 expression was found when lymphocytes of healthy individuals were polyclonally activated in the presence of various regulatory cytokines. Cells undergoing apoptosis showed significantly lower bcl-2 expression than viable cells. Staining of apoptotic cells revealed that lymphocytes from HIV-1-infected subjects were characterized by an increased susceptibility to programmed cell death which was not restricted to a particular lymphocyte subset. Despite significantly different bcl-2 expression in CD4+ and CD8+ lymphocytes of HIV-1-infected individuals with less than 200 CD4+ cells/microliter, no difference could be observed concerning their susceptibility to undergo apoptosis. Therefore, we conclude that sensitivity or resistance to in vitro induction of apoptosis does not directly correlate with bcl-2 expression.

Mechanism of gamma sigma T-cell-mediated inhibition of stem cell differentiation in vitro: possible relevance for myelosuppression in HIV-infected individuals.

We investigated whether gamma delta T cells contribute to the suppression of myelopoiesis in HIV infection. Freshly isolated gamma delta T cells from HIV seropositive patients suppressed CFU-GM growth in vitro. Preactivation of gamma delta T cells with IL-2 and/or IL-15 further reduced the number of CFU-GM. Natural killer cells and to a lower extent CD4+ and CD8+ cells also inhibited CFU-GM growth. In contrast to gamma delta T cells, this effect was not dependent on IL-15 or IL-2 preactivation. Moreover, no enhanced inhibitory effect of CD56+ and CD4+ cells was observed in HIV+ subjects compared to HIV- donors. The myelosuppressive effect of supernatants of gamma delta T cells could be inhibited by antibodies against IFN-gamma or TNF-alpha. Accordingly, we found increased numbers of TNF-alpha or IFN-gamma-secreting CD8+ gamma delta T cells in HIV+ patients. We conclude that the increased fraction of activated gamma delta T cells producing myelosuppressive cytokines might contribute to the dyshematopoiesis frequently observed in HIV-infected individuals.

Increase in Vdelta1+ gammadelta T cells in the peripheral blood and bone marrow as a selective feature of HIV-1 but not other virus infections.

Dysregulation of T-cell receptor (TCR) alphabeta bearing lymphocytes and an increase in Vdelta1+ gammadelta T cells are typical features of HIV-1 infection. However, the role of gammadelta T cells remains unclear. Therefore, peripheral blood mononuclear cells (PBMC) of 103 HIV-1-infected patients were investigated with respect to expression of Vdelta1. These results were compared to the Vdelta1 expression of bone marrow mononuclear cells (BMMC). In contrast to healthy controls, Vdelta1+ cells dominated among both PBMC and BMMC in HIV-1-infected patients. Analysis of the coexpression of CD25, CD8, HLA-DR and CD45RO revealed a high prevalence of Vdelta1/CD45RO and Vdelta1/HLA-DR double-positive PBMC only in HIV-1-infected patients but not in healthy donors. Furthermore, analysis of the gammadelta TCR repertoire in patients infected with hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV)-1 and HSV-2 showed that the selective enhancement of Vdelta1+ cells was restricted to HIV infection and not observed in other virus diseases. Our data provide further support for the involvement of gammadelta T cells in immunosuppression and progression of HIV infection.

Demonstration of the Th1 to Th2 cytokine shift during the course of HIV-1 infection using cytoplasmic cytokine detection on single cell level by flow cytometry.

OBJECTIVE: To characterize changes of Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) that occur during the course of HIV infection by cytoplasmic cytokine staining on single cell level. DESIGN AND METHODS: Mitogen-stimulated PBMC from 16 healthy donors, 18 HIV-1-infected individuals without AIDS and 14 patients with AIDS were stained intracellularly with fluorescein-labelled MAb against interleukin (IL)-2, IL-4, IL-10 and interferon (IFN)-gamma. Additionally, co-staining of CD4+ T-cell, CD8+ T-cell, natural killer (NK) cell, B-cell and monocytic markers was performed. Fluorescence staining was analysed by three-colour flow-cytometry. RESULTS: A reduced percentage of IL-2 and IFN-gamma (Th1 type)-producing cells among CD4+ T cells from HIV-1-infected individuals could be demonstrated. There was a continuous decrease of IFN-gamma-producing CD4+ T cells in the course of HIV infection and a dramatic reduction of IL-2-expressing cells among CD4+ T cells in patients with AIDS. In contrast to Th1 cytokines, the frequency of Th2 cytokine expressing cells among CD4+ T cells increased in HIV-infected individuals. The maximum frequency of IL-4-expressing cells among CD4+ T cells was seen in HIV-infected individuals without AIDS, whereas the rate of IL-10-producing cells was highest in patients with AIDS. In HIV-infected individuals no significant proportion of Th0 cells expressing both Th1 and Th2 cytokines was detectable. In CD8+ T cells the percentage of IL-2 was expressing cells decreased continuously accompanied by a strong increase of the frequency of IFN-gamma-producing cells. CONCLUSION: The decreased percentage of cells expressing IL-2 and IFN-gamma in conjunction with an increased proportion of IL-4- and IL-10-producing cells among the CD4+ T cells in HIV-1-infected individuals demonstrate a Th1 to Th2 cytokine shift in the course of HIV infection on a single cell level. There was no evidence of a Th1 to Th0 cytokine shift. In addition to the loss of CD4+ T cells in HIV infection, the qualitative changes of Th1/Th2 cytokine expression may serve as a marker for progressive failure of cell-mediated immunity.

Genetics of type III hyperlipoproteinemia.

One hundred forty-seven relatives of 43 patients with "classical" type III hyperlipoproteinemia (HLP) having the apolipoprotein (apo) E2/2 phenotype were studied to determine the occurrence of hyperlipidemia and the presence of further possible genes for lipoprotein disorders in these families. In 12 pedigrees primary dyslipidemia was prevalent among patients and respective blood-relatives. In these kindreds the coexistent presence of genes for familial combined hyperlipidemia (n = 6), familial hypertriglyceridemia (n = 5), and familial hypercholesterolemia (n = 1), respectively, was supposed. Our results, therefore, confirm and extend previous data on the multifactorial genesis of the diseases. Besides homozygosity for a receptor binding-defective isoform of apo E (apo E2), additional genes for familial lipoprotein disorders might operate in the pathogenesis of type III HLP. This is the largest family study performed so far in this primary lipoprotein disorder.

Flupirtine protects both neuronal cells and lymphocytes against induced apoptosis in vitro: implications for treatment of AIDS patients.

In the present study we demonstrate that flupirtine, an already clinically used, centrally acting, non-opiate analgesic agent, protects rat cortical neurons against HIV-gp120 induced apoptotic cell death. The drug was active at concentrations between 1 and 10 microg/ml. Furthermore we show inhibition of in vitro induced apoptosis in human blood mononuclear cells, using flupirtine. Induced apoptosis in peripheral blood mononuclear cells from healthy individuals and HIV-1 infected patients was reduced to approximately 50% after in vitro preincubation with flupirtine at concentrations between 0.1 and 10 microg/ml. The anti-apoptotic effect of flupirtine was restricted to CD3+ lymphocytes and in particular to CD4+ cells. Flupirtine does not affect uninduced apoptosis in human lymphocytes in vitro. The selective potential of flupirtine to reduce apoptosis without influencing uninduced apoptosis may qualify this compound as a potential drug in the therapy of HIV-1 infected patients.

Substitution of testosterone in a HIV-1 positive patient with hypogonadism and Wasting-syndrome led to a reduced rate of apoptosis.

Peripheral blood mononuclear cells from HIV infected individuals develop in vitro apoptosis to a much higher extent than healthy donors. Aside from the direct cytopathic effect of HIV, programmed cell death can be induced by such cytokine system imbalance as seen with increased levels of TNF-alpha or the Th1-->Th2-cytokine shift. However, wasting syndrome, which occurs in the majority of AIDS patients is associated with an enhanced expression of TNF-alpha and IL 6 as well. A 37-year-old AIDS patient suffering from wasting syndrome and hypogonadism was treated with 1 alpha-dihydrotestosterone. The rate of apoptotic peripheral blood mononuclear cells was determined before, during and after this therapy. After three weeks of androgen substitution therapy, the rate of spontaneous apoptosis was reduced to 34% and the ionomycin induced apoptosis to 52% of the rate of apoptotic cells at the beginning of the therapy. Moreover, the general and nutritional condition improved remarkably. Thus, we suggest that the use of anabolic drugs for the treatment of AIDS-associated wasting-syndrome would not only improve their general and nutritional condition, but might also prevent the loss of CD4+ T-cells through an inhibition of apoptosis.

Ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in HIV infection.

Programmed cell death (apoptosis) of T-lymphocytes observed in human immunodeficiency virus (HIV) infected individuals could be linked to oxidative stress. Therefore, we have investigated whether reactive oxygen species (ROS) induce apoptosis, which might contribute to the cell loss during progression of HIV-1 infection. ROS were generated in peripheral blood mononuclear cells (PBMC) obtained from HIV-1-positive patients and from healthy controls by stimulation with bacteria or by treatment with hypoxanthine/xanthine oxidase, which has been shown to generate ROS without direct involvement of cytokines. A dose-dependent inhibition of ROS formation correlated with the reduction of apoptosis induced by both bacterial and hypoxanthine/xanthine oxidase stimulation, suggesting that ROS generation was responsible for the induction of apoptosis. In addition, hydrogen peroxide (H2O2) rather than superoxide (O2.-) was observed to induce apoptosis. ROS-dependent apoptosis was shown to be independent of cytokines such as tumor necrosis factor-alpha (TNF-alpha). ROS-induced apoptosis was significantly enhanced in HIV-infected subjects even in the very early stages after infection. Moreover, ROS-mediated apoptosis was not restricted to a particular lymphocyte subset. In view of the diminished oxidative resistance of HIV-infected individuals, our results suggest that ROS-mediated apoptosis might contribute to the deletion of lymphocytes and to the pathogenesis of the disease.

Unusual xanthomas in a young patient with heterozygous familial hypercholesterolemia and type III hyperlipoproteinemia.

We report on a 20-year-old man with the combination of two independent familial lipoprotein disorders: heterozygous familial hypercholesterolemia (FH) and type III hyperlipoproteinemia (HLP). Familial hypercholesterolemia was diagnosed by elevated total and low density lipoprotein cholesterol levels and family history. By denaturing gradient gel electrophoresis, DNA sequencing and restriction fragment length polymorphism analysis, a G --> A splice donor mutation in intron 3 of the proband's low density lipoprotein receptor gene was identified as the underlying molecular defect. This mutation was described previously as a receptor-negative founder mutation in Norway (FH-Elverum) and subsequently in 6 unrelated heterozygous English patients, creating a severe phenotype of familial hypercholesterolemia. Type III HLP was confirmed by homozygosity for apolipoprotein (apo) E2 and an elevated ratio of very low density lipoprotein cholesterol to serum triglycerides (0.40; normal ratio about 0.20). The patient has unusual flat xanthomas in the interdigital webs of the hands which are normally not found in either disease. These dermatological findings might therefore be indicative of the rare combination of both disorders of lipoprotein metabolism in one individual.

Apolipoprotein E5 (Glu212-->Lys): increased binding to cell surface proteoglycans but decreased uptake and lysosomal degradation in cultured fibroblasts.

A new apolipoprotein (apo) E variant, apoE5 (Glu212-->Lys) was identified in a Turkish family. The variant was due to a point mutation (CAG-->AAG) at the first nucleotide position of the codon encoding amino acid residue 212 of the mature apoE. The 23-year-old index patient was heterozygous for the mutation. Examination of the proband's kindred revealed six heterozygous and two homozygous mutation carriers. Compared to non-carriers, carriers of the mutation had slightly higher triglycerides (1.25 versus 1.11 g/l) and lower HDL cholesterol (0.36 versus 0.41 g/l). Very low density lipoproteins (VLDL) from an apoE5 (Glu212-->Lys) homozygote displayed enhanced binding (+17%, P < 0.05), but decreased uptake (-35%, P < 0.0001) and degradation (-51%, P < 0.0001) in cultured fibroblasts, compared to E3/3-VLDL. The region of the apoE molecule surrounding residue 212 contains a heparin binding domain. Consistently, the enhanced cell surface binding of E5/5-VLDL was observed in "wild-type" Chinese hamster ovary cells (+19%, P < 0.05), but not in proteoglycan-deficient cells. The binding of E5/5-VLDL to heparin was increased (+22%, P < 0.05). As the endocytosis of apoE-containing particles involves the transfer of proteoglycan-bound ligands to lipoprotein receptors, the stronger binding of apoE5 (Glu212-->Lys) to proteoglycans could reduce the rate at which the mutant is finally delivered to endocytotic pathways. These data may provide evidence for a functionally important heparin binding site around amino acid residue 212 of the apoE molecule in vivo.

TNF-alpha mediated apoptosis of CD4 positive T-lymphocytes. A model of T-cell depletion in HIV infected individuals.

Apoptosis has been suggested to account for loss of CD4+ T-cells in HIV infected individuals. Aside from MHC II dependent superantigens no mediator for preferential apoptosis of CD4+ T-cell has been described. However, the expression of TNF-alpha is increased in HIV+ patients. Additionally, TNF-alpha is known as a potent inducer of apoptosis in a variety of cell types. We therefore investigated the capacity of TNF-alpha to mediate apoptosis in vitro preferentially in CD4+ T-cells from HIV+ individuals. In the presence of TNF-alpha, CD4+ T-cells from HIV+ individuals with more than 200 CD4+ T-cells/microl (classified CDC A1, A2, B1, B2) could be significantly depleted by apoptosis without a reduction of CD8+ T-cells. In cells from patients with less than 100 CD4+ T-cells/microl (classified CDC C3), TNF-alpha mediated apoptosis was not apparent due to an already immensely elevated rate of apoptosis observed in the absence of TNF-alpha. Here we demonstrate cord blood mononuclear cells as a model for apoptosis since these cells develop apoptosis at a similar rate as that of PBMC in HIV+ patients. More than 50% of TNF-alpha stimulated CD4+ cord blood T-cells were depleted within 3 days by apoptosis, whereas CD8+ T-cells, B-cells and NK-cells were not affected. In PBMC from healthy adults, a preferential loss of CD4+ T-cells mediated by TNF-alpha was not observed. A reduced production of IFN-gamma was observed in mononuclear cells from newborns and from HIV+ patients. Moreover, IFN-gamma and IL-2 could prevent TNF-alpha mediated apoptosis. Therefore, a reduced Th1-cell-function may contribute to TNF-alpha mediated apoptosis of CD4+ T-cells from these donor groups. Taken together, the data suggest that TNF-alpha probably is a mediator of the loss of CD4+ T-cells in HIV infected patients in vivo.

A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease.

Prevalence and association of atherosclerosis at three different arterial sites in patients with type III hyperlipoproteinemia.

We have examined the prevalence of clinically significant atherosclerosis in 78 patients with type III hyperlipoproteinemia (HLP) and homozygosity for apolipoprotein (apo) E2. Forty-six of these individuals (59%) had no atherosclerosis, 32 patients (41%) had atherosclerosis, i.e., atherosclerosis of the extracranial carotid arteries (CAA), coronary arteries (CAD) or/and peripheral arteries of the legs (PVD), either singly or in combination. No association could be shown with respect to the co-prevalence of atherosclerotic lesions at these different arterial sites, except for the high predictive value (pv = 0.88, P = 0.006) of CAA for the presence of PVD. Hence, documentation of atherosclerosis under clinical aspects at one of these exposed arterial territories does not allow a reliable prediction of generalised atherosclerosis or local atherosclerosis at other sites of the arterial tree in individuals with this familial lipoprotein disorder. Therefore, assessment of the extent of clinically significant atherosclerosis in type III HLP patients should include careful and thorough examination of the extracranial carotid arteries, the coronary arteries, and the peripheral arteries of the legs.

Decreased function of monocytes and granulocytes during HIV-1 infection correlates with CD4 cell counts.

Monocytes and neutrophils are involved in the primary immune response against opportunistic infections that occur during the progression of human immunodeficiency virus (HIV) infection towards development of acquired immune deficiency syndrome (AIDS). Phagocytic cells operate through the generation of reactive oxygen species which may be toxic for fungi, bacteria and viruses. In the present study we evaluated the function of monocytes and granulocytes in whole blood samples of 16 healthy controls, 12 HIV infected subjects who had not undergone significant infections and of 17 individuals with AIDS. Using flow cytometric methods we were able to determine phagocytosis and respiratory burst under conditions that reflect the normal environment of these cells. Compared with results in samples from controls, granulocytes and monocytes from asymptomatic HIV infected patients exhibited a significantly increased capacity to phagocytose bacteria. The production of reactive oxygen intermediates was in the normal range. In comparison to asymptomatic HIV infected individuals, patients with AIDS showed a significant reduction of phagocytosis and respiratory burst which correlated with the number of CD4+ cells. In comparison to controls, patients infected with HIV, whether they were symptomatic or not, revealed a significantly diminished number of oxygen radical producing cells compared with the number of phagocytic cells. These results indicate that monocytes and granulocytes show reduced antimicrobial activity even in early stages of HIV infection. This defect is only partly due to the HIV infection itself as neutrophils are not target cells for HIV.

Serum amyloid A protein (SAA): a marker for liver allograft rejection in humans.

Serum amyloid A protein (SAA) concentrations were monitored in 12 consecutive liver transplant recipients until the 70th postoperative day. Fourteen rejection episodes were identified histologically in 42 liver biopsies of the grafts. Of 12 rejections 8 (66.7%) were characterized by pronounced simultaneous increases in SAA concentrations in plasma, the mean peak value being 16.94 +/- 8.82 mg/dl (range 4.58-28.55 mg/dl) compared with a mean normal value of 0.98 +/- 0.42 mg/dl in healthy controls. Of 42 biopsies 28 did not show histological evidence of graft rejection. Of 25 negative biopsies 24 (96.0%) were not accompanied by a parallel SAA increase in plasma. These findings demonstrate that measurements of SAA concentrations may provide a valuable noninvasive aid in identifying acute liver allograft rejection in humans.