Effect of cytokines on HIV-induced depletion of thymocytes in vivo.

BACKGROUND: Cytokines play an important role in the differentiation of thymocytes into mature T cells; consequently, certain cytokines could be useful for immune reconstitution after HIV infection without increasing viral load. OBJECTIVE: To investigate whether cytokines affect immune depletion caused by HIV infection with a CXCR4-tropic strain in SCID-hu mice implanted with human fetal thymus and liver (thy/liv) tissue. METHODS: The thy/liv implants were either mock infected or infected with HIV-1 NL4-3, a CXCR4-tropic molecular clone. Interleukin (IL)-2, IL-4, IL-7, interferon-gamma (IFN-gamma) or diluent was administered to the mice during the second and third week postinfection. Viral load and immunophenotype were determined in thymocytes. RESULTS: Thymocyte subset distributions at 3 weeks postinfection were significantly influenced by treatment with certain cytokines. In particular, IL-2 caused the infected mice to retain a thymocyte profile that was more similar to that in mock-infected mice than that in diluent-treated infected mice, in that the percentages of immature CD4+CD8+ and CD5+CD1+ cells were slightly higher and much less variable than in diluent-treated infected mice. The effect of IFN-gamma treatment was similar to IL-2 but did not reach statistical significance. However, after IFN-gamma treatment, normal percentages of mature CD3+CD69+ cells were maintained whereas this population was relatively increased in diluent-treated infected mice. Although treatment with IL-4 and IL-7 delayed depletion of immature thymocytes, these cytokines increased viral load. CONCLUSIONS: Cytokines such as IL-2 and IFN-gamma maintain immature thymocytes without increasing viral load and may be useful as adjuncts to improve immune reconstitution after HIV infection.

Human immunodeficiency virus type 1-induced hematopoietic inhibition is independent of productive infection of progenitor cells in vivo.

Human immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viral vpr gene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34(+) progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34(+) cells in vivo.

Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4(+) lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.

Transient renewal of thymopoiesis in HIV-infected human thymic implants following antiviral therapy.

Stem cell gene therapy strategies for AIDS require that differentiation-inducing stromal elements of HIV-infected individuals remain functionally intact to support the maturation of exogenous progenitor cells into mature CD4+ cells. To investigate the feasibility of stem cell reconstitution strategies in AIDS, we used the SCID-hu mouse to examine the ability of HIV-infected CD4+ cell-depleted human thymic implants to support renewed thymopoiesis. Here we report that following treatment of these implants with antiretroviral drugs, new thymopoiesis is initiated. This suggests that antiviral therapies might allow de novo production of T lymphocytes and provides support for the concept of therapeutic strategies aimed at reconstitution of the peripheral CD4+ T-cell compartment.

Human immunodeficiency virus 1 envelope proteins induce interleukin 1, tumor necrosis factor alpha, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain.

Although microglia are the only cells found to be productively infected in the central nervous system of acquired immunodeficiency disease syndrome (AIDS) patients, there is extensive white and gray matter disease nonetheless. This neuropathogenesis is believed to be due to indirect mechanisms other than infection with human immunodeficiency virus 1 (HIV-1). Cytokines and toxic small molecules have been implicated in the clinical and histopathological findings in CNS AIDS. Previously, we have demonstrated in rodent glial cultures the presence of biologically active epitopes of gp120 and gp41 that are capable of inducing interleukin 1 and tumor necrosis factor alpha. In this study, we map the HIV-1 envelope epitopes that induce nitric oxide, inducible nitric oxide synthase, interleukin 1, and tumor necrosis factor alpha in human glial cultures. Epitopes in the carboxy terminus of gp120 and the amino terminus of gp41 induce these proinflammatory entities. In addition, we compare HIV-1 infection and pathology in glial cells derived from human brain taken at different states of maturation (fetal, neonatal, and adult brain) in an effort to address some of the clinical and histological differences seen in vivo. This study demonstrates that, in the absence of virus infection and even in the absence of distinct viral tropism, human glia respond like rodent glia to non-CD4-binding epitopes of gp120/gp41 with cytokine and nitric oxide production. Differences among fetal, neonatal, and adult glial cells' infectivity and cytokine production indicate that, in addition to functional differences of glia at different stages of development, cofactors in vitro and in vivo may also be critical in facilitating the biological responses of these cells to HIV-1.

The mapping of HIV-1 gp160 epitopes required for interleukin-1 and tumor necrosis factor alpha production in glial cells.

The pathology in central nervous system (CNS) AIDS suggests that direct infection with HIV-1 is not required for changes in glia and neurons. Induction of a variety of pathological responses in vitro in rodent brain cultures also suggests that CD4 is not the receptor for HIV-1 in the brain, given that human and rodent CD4 are not homologous. This implies that the epitopes on HIV-1 which bind glia and activate them are novel, non-CD4-binding domains. We have therefore mapped the envelope (env) regions required for production in rat glial cultures of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) which we hypothesize are important in CNS AIDS. Serially truncated deletion mutants from the gp120/gp41 carboxy terminus representing folded, glycosylated recombinant env proteins were expressed in HeLa cells via a vaccinia virus vector. These proteins, linear gp120/gp41 peptides, as well as polyclonal and monoclonal antibodies reactive to defined regions of gp120/gp41 were used to map the epitopes involved in production of IL-1 and TNF alpha. Compared to HeLa cell and wild-type vaccinia virus controls, the vaccinia recombinant env protein gp160 containing cleaved gp120 and gp41 induced both IL-1 and TNF alpha. If gp160 was not cleaved into gp120 and gp41, IL-1 but not TNF alpha induction was reduced. Peak production of TNF alpha by gp120/gp41 was at 4 h while IL-1 production was still significantly elevated at 44 h at the highest concentrations of env protein. Using the truncation deletions, the V3 loop of gp120 appeared to be critical for IL-1. Glycosylation and folding of V3 is probably important in IL-1 induction since a V3 peptide was not as active. While removal of glycosylated, folded V4 and C4 regions had no effect on IL-1, linear peptides in the region from the V4 loop to the C4 domain were strong inducers of IL-1. Non-glycosylated, linear V4 loop peptide induced more IL-1 than the V4 in protein generated in HeLa cells, suggesting that glycosylation and/or conformational structures sequester V4 inducer epitopes. Using the truncation deletions, the carboxy terminus region (V4-C5) of gp120 as well as gp41 were shown to be critical for TNF alpha production. Peptides representing linear epitopes in the V3 loop, C5 domain of gp120, and the ectodomain of gp41 were all strong inducers of TNF alpha; a protein representing almost the entire gp41 was the strongest inducer of TNF alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of IgA anti-HLA class I antibodies in kidney transplant survival.

The unusually high 88% one-year cadaver kidney graft survival rate in patients with IgA nephropathy (IgAN) prompted us to investigate the influence of IgA anti-HLA class I antibodies on subsequent graft survival. We found that patients with various original diseases with IgA antibodies to the HLA molecule had high 91% one-year graft survival compared with 58% one-year survival for those who did not have preformed IgA antibodies against the HLA molecule prior to transplantation (P < 0.0005). The IgA antibodies were detected by reaction with class I HLA molecules isolated by capture with monoclonal antibody and detected with an enzyme-linked immunosorbent assay. In contrast, IgG antibodies to the HLA molecule resulted in a lower one-year graft survival rate (74%) than in those patients without IgG antibodies (87%) (p = 0.08). IgA antibodies to the HLA molecule, when present, tended to react at a high frequency on a random lymphocyte panel. These findings suggest that sensitization resulting in IgA anti-HLA antibodies may counteract the deleterious effect of an IgG antibody response in clinical kidney transplantation.

In vitro studies to explain high renal allograft survival in IgA nephropathy patients.

The pretransplant sera of 27 IgA nephropathy (N) kidney transplant patients were investigated for antibodies to the HLA molecule, together with 104 sera from non-IgA N patients and 60 controls. IgA antibodies to HLA were found in 61% of IgA N patients and 2% of non-IgA N patients. IgA N patients with IgA antibodies to HLA had a 100% 2-year cadaver donor graft survival rate compared with 70% in those without IgA antibodies. Patients with IgG antibodies to HLA without accompanying IgA antibodies had the worst graft survival rates. We propose that IgA anti-HLA contributes to the high kidney graft survival in IgA N patients by blocking IgG antibodies or inhibiting cellular immune response.

trans-Activation of genes encoding activation-associated human T lymphocyte surface proteins by murine retroviral sequences.

The mechanisms whereby RNA leukemia viruses cause T lymphocyte leukemias or lymphomas after a long latent period are not understood. We report here that infection of human T lymphocyte lines with a murine leukemia virus results in up-regulation of a number of lymphocyte-specific cell surface Ag. These proteins include CD2, CD3, CD4, the TCR, and MHC class I Ag. The expression of other cell surface proteins, such as LFA-3, are unaffected by the presence of the retrovirus. This up-regulation occurs at the level of the mRNA transcripts encoding these proteins, and is the result of increased transcription of the respective genes. The increases in transcription are the result of a trans-activation process by the leukemia virus. The transient introduction of chimeric genes consisting of MHC class I gene promoter sequences attached to the reporter gene CAT into human T cells containing murine retrovirus produces stimulated transcription of the reporter gene. Subgenomic portions of the murine leukemia virus containing the long terminal repeats and the 5' untranslated region are sufficient to produce transactivation of the same set of T cell genes as the whole leukemia virus. The finding that murine leukemia viruses enhance transcription and expression of a group of T cell surface proteins, all of which have been reported to be capable of transducing an activating signal to the lymphocyte, may be relevant to the pathophysiologic mechanisms whereby these viruses induce leukemias and lymphomas.

Sex and age effects in renal transplantation.

1. The age and sex of the recipient were not significant factors in transplant outcome. The age and sex effects observed were all associated with the kidney donor. 2. The 1-year graft survival rates for male and female donor kidneys were 78% and 76%, respectively in first cadaver transplants and 91% and 88%, respectively, in parent donor first transplants. The donor sex had no significant effect on survival of transplants from sibling donors, irrespective of HLA match. 3. Long-term survival rates, reflected in transplant half-lives, were also significantly better in recipients of male cadaver (8 years) or paternal donor (13 years) first transplants than in recipients of female cadaver (6 years) or maternal donor (9 years) kidneys. 4. A higher percentage of HLA-A,B matched cadaver kidneys than mismatched organs were transplanted to sensitized recipients. Despite a higher percentage of sensitized female recipients, there was no difference in first cadaver transplant survival comparing males and females. 5. A positive flow cytometry crossmatch (FCXM) was associated with poor 3-month cadaver retransplant survival in both males (54%) and females (56%) compared with 83% and 76%, respectively, for FCXM-negative males and females. 6. The impact of preformed antibodies on first cadaver transplant outcome differed between males and females. Among sensitized recipients, females had 83% and 81% 3-month graft survival with a positive and negative FCXM, respectively, whereas positive FCXM male patients had 71% vs 86% for FCXM-negative males. 7. Graft survival ranged from 76%-79% in first transplant recipients aged 1-5, 6-14, 15-55, and over 55 when the cadaver donor age was 15-55. Poorer survival rates in pediatric and older recipients were associated with "age-matching" the donor kidney. Nearly 40% of pediatric patients received kidneys from pediatric donors that did poorly in all recipient age groups. More than 20% of kidneys from donors over 55 were transplanted to older recipients. These older donor kidneys also had uniformly poor survival in all recipient age groups. 8. When death was excluded as a cause of graft loss in first cadaver transplants, patients over 55 had an 80% 1-year graft survival rate. Although death was clearly a factor for older patients, it was interesting that survival including death was 76% when the donor age was 15-55 and 63% when the donor was over 55.

Sensitization and crossmatching in renal transplantation.

1. Presensitization in first cadaver kidney recipients can lead to increased risk of graft failure by hyperacute rejection, or delayed function up to 1 month. Fifty percent of the hyperacute rejections occurred in nonsensitized recipients. The number of "classical" hyperacute rejections was small, but they have been occurring at a rate of about 10 per year. 2. One-year graft survival of nonsensitized recipients of first and second cadaver transplants was about the same. One-year graft survival of broadly sensitized recipients of first and second cadaver transplants was 8% lower than those who were moderately sensitized. One-year graft survival of second cadaver transplants in all sensitized recipients was significantly lower (9-13%) than in first cadaver transplants. 3. The proportion of transfused recipients was 89% in parous females, 84% in nulliparous females, and 80% in males. Pretransplant transfusions also increased sensitization of males and females awaiting their first kidney transplant. Females were significantly more sensitized than males, whether they were transfused or not. 4. One-year graft survival rates of transfused recipients were 5-9% higher than nontransfused recipients. Highly sensitized patients who were transfused had the same 1-year graft survival as nontransfused, nonsensitized recipients. 5. Patients in Southern California waiting for a second transplant were more broadly sensitized than those waiting for a first kidney. A higher proportion of sensitized patients were waiting more than 3 years for a second transplant than for a first. 6. Patients waiting for a first transplant were more sensitized than those transplanted for the first time. The highest number of waiting or transplanted patients was blood group O. 7. A significantly greater proportion of sensitized patients with blood groups A and B was waiting than those with blood type O. The type O patients were transplanted at the same rate as they entered the waiting list. It is possible that sensitized type O patients were being discouraged from entering the waiting list. 8. A significantly smaller proportion of broadly sensitized SLE patients was waiting for a first transplant since 1988, although SLE patients were more broadly sensitized compared to those with other diseases and waiting since 1981 to 1987. This further confirms that many SLE patients are transplanted, as their sensitization is more often associated with autoantibody. 9. The highest proportion of currently sensitized recipients occurred in the transplants with 0 mismatches for the HLA-A,B specificities of the donor kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased expression of CD4 molecules on Jurkat cells mediated by human immunodeficiency virus tat protein.

The tat gene of the human immunodeficiency virus, tat-III, when introduced into T-lymphoblastoid Jurkat cells by a Moloney retroviral recombinant DNA vector expressed high levels of the functional tat protein as measured by the chloramphenicol acetyltransferase assay. Immunofluorescence analysis with CD4-specific monoclonal antibodies demonstrated that the cell surface levels of the CD4 antigen were increased by 5- to 10-fold in the tat-III-infected Jurkat cells. Cellular cytoplasmic RNA analysis indicated that the enhanced CD4 expression was mediated at the mRNA level. Our findings suggest that the single expression of the human immunodeficiency virus tat protein in the absence of the other viral proteins causes an upregulation of CD4 gene expression on helper T cells, although infection of these cells by the virus, thus expressing all the viral gene products including tat, is known to downregulate CD4 antigen expression.

Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate.

A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme. The purpose of this purification was to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h-1 mg-1) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, the largest found to be 26 nucleotides in length in relation to DNA size markers. However, the oligoribonucleotides associated with the enzyme are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP for 4-10 h at 3 degrees C, a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [gamma-32P]ATP, and UV-irradiated DNA-cellulose contained exogenous [gamma-32P]ATP. [gamma-32P]ATP eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions as those for ATP. Higher (X5) concentrations of ADP and adenosine 5'-(beta, gamma-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. Purification and characterization.

Lumazine protein, a novel protein containing 6,7-dimethyl-8-ribityllumazine as a bound prosthetic group, is one of the several major proteins produced by the bioluminescent bacteria, Photobacterium phosphoreum. purification to complete homogeneity from cell extracts is achieved in six steps. Lumazine protein is a near spherical, monomeric protein of average molecular weight 20,000; in amino acid composition it is acidic with two isoelectric isomers, pI 4.9 and 5.0, and is hydrophilic (974 cal/residue) with single methionine and tryptophan residues and two accessible cysteines. It contains no carbohydrate. Reaction of the cysteines with dithionitrobenzoic acid results in quenching of the bound lumazine fluorescence but is otherwise reversible. Estimates of protein by dry weight results in a mole ratio of one bound lumazine group per protein.

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum.

The highly fluorescent prosthetic group of the blue fluorescence protein purified from the bioluminescent bacterium Photobacterium phosphoreum has been dissociated and separated from its apoprotein by affinity chromatography on Cibacron Blue-Sepharose. It has been identified as 6,7-dimethyl-8-(1'-D-ribityl)lumazine by several methods of characterization, all of which gave results identical to those for an authentic sample. In neutral solution, absorption maxima are at 407, 275 (shoulder), and 256 nm, with a single fluorescence maximum at 491 nm. The proton magnetic resonance spectrum exhibits a singlet at 2.66 ppm corresponding to the methyl substituted at the 6 position of lumazine and a multiplet centered at 3.85 ppm corresponding to the C-2'-5' protons of the ribityl group. A Raman spectrum was obtained by the technique of coherent anti-Stokes Raman scattering and the R(F) values by paper chromatography were determined in four solvent systems. The isolated compound was readily transformed into riboflavin by riboflavin synthetase. Fifty grams (dry weight) of P. phosphoreum contains at least 20 mg of this lumazine derivative, an amount comparable to that found in other microorganisms classified as riboflavin overproducers. The overproduction of this lumazine in this case apparently has to do with its function in the generation of bioluminescence.

The chromophore topography and binding environment of perididin.chlorophyll a.protein complexes from marine dinoflagellate algae.

1. The peridinin.chlorophyll a.protein complex from Amphidinium carterae (Plymouth 450) shows spectroscopic characteristic (absorption, CD, fluorescence polarization, lifetime and energy transfer) essentially identical with peridinin.chlorophyll a.protein complexes from Glenodinium sp., Gonyaulax polyedra and Amphidinium rhyncocephaleum. 2. The apoprotein of peridinin.chlorophyll a.protein complexes is globular, with an isotropic rotational relaxation time (e.g. 33 ns for the A. caterae peridinin.chlorophyll a.protein), as deduced from the dynamic depolarization data. 3. The chromophores (4 peridinins and 1 chlorophyll a for peridinin.chlorophyll a.protein complexes from Glenodinium sp., G. polyedra and A. rhyncocephaleum and 9 and 2, respectively, for peridinin.chlorophyll a.protein of A. carterae) are accommodated in a hydrophobic crevice and not exposed to the solvent. The surface of the protein is highly hydrophilic. 4. No evidence for chlorophyll-chlorophyll interactions in the A. carterae peridinin.chlorophyll a.protein was obtained. This implies that binding crevices for two chlorophylls and half of peridinins (four to five) are located at some distance from each other. 5. The peridinin.chlorophyll a.protein complexes function as the photosynthetic antenna pigment. In addition, peridinins effectively protect chlorophyll a from photodecomposition.

Molecular topology of the photosynthetic light-harvesting pigment complex, peridinin-chlorophyll a-protein, from marine dinoflagellates.

The photosynthetic light-harvesting complex, peridinin-chlorophyll a-protein, was isolated from several marine dinoflagellates including Glenodinium sp. by Sephadex and ion-exchange chromatography. The carotenoid (peridinin)-chlorophyll a ratio in the complex is estimated to be 4:1. The fluorescence excitation spectrum of the complex indicates that energy absorbed by the carotenoid is transferred to the chlorophyll a molecule with 100% efficiency. Fluorescence lifetime measurements indicate that the energy transfer is much faster than fluorescence emission from chlorophyll a. The four peridinin molecules within the complex appear to form two allowed exciton bands which split the main absorption band of the carotenoid into two circular dichronic bands (with negative ellipticity band at 538 nm and positive band at 463 nm in the case of peridinin-chlorophyl a-protein complex from Glenodinium sp.). The fluorescence polarization of chlorophyll a in the complex at 200 K is about 0.1 in both circular dichroic excitation bands of the carotenoid chromophore. From these circular dichroic and fluorescence polarization data, a possible molecular arrangement of the four peridinin and chlorophyll molecules has been deduced for the complex. The structure of the complex deduced is also consistent with the magnitude of the exciton spliting (ca. greater than 3000 cm-1) at the intermolecular distance in the dimer pair of peridinin (ca. 12 A). This structural feature accounts for the efficient light-harvesting process of dinoflagellates as the exciton interaction lengthens the lifetime of peridinin (radiative) and the complex topology increases the energy transfer probability. The complex is, therefore, a useful molecular model for elucidating the mechanism and efficiency of solar energy conversion in vivo as well as in vitro.