Anssi Lagerstedt

Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines

SummaryInformation concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6–9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12–48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.

The effects of lifelong ubiquinone Q10 supplementation on the Q9 and Q10 tissue concentrations and life span of male rats and mice

The effect of lifelong oral supplementation with ubiquinone Q10 (10 mg/kg/day) was examined in Sprague‐Dawley rats and C57/B17 mice. There were no significant differences in survival or life‐span found in either rats or mice. Histopathologic examination of different rat tissues showed no differences between the groups. In Q10 supplemented rats, plasma and liver Q10 levels were 2.6 to 8.4 times higher at all age points than in control rats. Interestingly, in supplemented rats the Q9 levels also were significantly higher (p<0.05) in plasma and liver at ages 18 and 24 months. Neither Q9 nor Q10 levels were affected by supplementation in kidney, heart, or brain tissues. In spite of the significant changes in plasma and liver ubiquinone concentrations, lifelong Q10 supplementation did not prolong or shorten the lifespan of either rats or mice.

Effect of PUVA on immunologic and virologic findings in HIV-infected patients

Psoralen and UVA radiation inactivate human immunodeficiency virus (HIV) in vitro whereas UVB and UVC radiation under experimental conditions transactivate HIV. We studied the effect of systemic PUVA treatment on immunologic and virologic findings in five HIV-infected patients. Systemic PUVA was given in two-4-week periods, 2 months apart. The total irradiation ranged from 30 to 262 joules/cm2. All skin lesions, including therapy-resistant psoriasis vulgaris, seborrheic dermatitis, folliculitis, and chronic urticaria, cleared during the first weeks of PUVA. A slight increase in the CD4 lymphocyte numbers was seen in two patients. Serum beta 2-microglobulin values and urine neopterin values remained steady, and the elevated serum immunoglobulin values became normal in all patients. The PUVA treatment did not induce appearance of HIV antigen in serum and HIV isolation was repeatedly negative in all patients whose cultures were initially negative. Lymphocyte recall responses to purified protein derivative (tuberculin) became positive in three and to HIV-specific antigens in two patients. These responses, however, were transient. All patients except one, who was positive for HIV antigen at entry, have remained well 1 year after PUVA therapy.

Immunological variation and immunohistochemical localization of HIV-1 Nef demonstrated with monoclonal antibodies.

ObjectiveTo study the immunological and immunohistochemicai nature of HIV-1 Nef. DesignMonoclonal anti-Nef antibodies were generated and used to identify antigenic epitopes in Nef, to study immunological cross-reactivity between Nef from different isolates and to reveal the subcelluiar localization of Nef. MethodsMonoclonal antibodies against recombinant HIV-1 Nef protein (BRU isolate) were generated in BALB/c mice. The epitope mapping was carried out with the use of overlapping 15–20mer lipopeptides linked to a lipid group at the amino-terminus. Immunoperoxidase method was used for histochemical studies. ResultsTen stable antibody-producing clones, mainly of the immunoglobulin (Ig) G1 subtype, with strong Western blot and enzyme-linked immunosorbent assay reactivity toward the recombinant Nef protein, were obtained. The epitopes recognized were located on amino-acid sequences 21–41, 31–50, 51–71, 61–80, 151–170, 161–180, and 171–190. All 10 monoclonal antibodies also reacted with the native Nef of HIV-1BRU, and eight reacted with native HIV-1IIIB. Most antibodies also reacted with Nef from more divergent HIV-1 strains. In Western blotting, two forms of Nef (24 and 27 kDa) were observed with most isolates studied. Immunohistochemical staining of HIV-1 -infected H9 or MT-4 lymphoid cells demonstrated that Nef was expressed mainly in the Golgi complex and at the nuclear membrane, but occasionally also in the nucleus. The nuclear localization of Nef was especially frequent in the HIV-1-infected MT-4 cells. ConclusionsOur findings suggest that Nef is expressed in two isomorphic forms, and that it may also act as a nuclear protein and thus have a direct regulatory function at the RNA/DNA level.

Interleukin-10 gene expression induced by HIV-1 Tat and Rev in the cells of HIV-1 infected individuals.

The role of cytokines in the regulation and function of the immune system is of great importance. In human immunodeficiency virus (HIV) infection, with progressive deterioration of cell-mediated immune response, cytokines are dysregulated. We have therefore investigated cytokine mRNA expression in type-1 and type-2 helper T cells of HIV-seropositive (HIV+) individuals, stimulated with mitogen (leukoagglutinin) and HIV-1 Tat and Rev peptides, previously found to induce proliferative T-cell responses in these individuals. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect interleukin 2 (IL-2), interferon gamma (IFN-gamma), IL-4, and IL-10 mRNAs. There was no difference in the mRNA expression of these cytokines when the cells of HIV-infected or noninfected individuals were polyclonally stimulated with the mitogen, as all cytokine mRNAs were detected in both groups. Baseline cytokine expression of unstimulated cells was, however, different in these two groups: the cells of HIV+ persons did not show comparable expression of mRNAs to HIV-seronegative (HIV+) individuals. When the cells of HIV+ individuals were stimulated with the peptides, 70% of the cases showed IL-10 mRNA expression, 20% IFN-gamma, and 10% IL-2, with no detection of IL-4 mRNA in any of the cases. Our results thus show that HIV-specific T-cell antigens induce production of IL-10 in HIV-infected individuals. The increase in IL-10 demonstrated here may have a role in hyperactivation of B cells, as well as in immunosuppression of T cells often seen in HIV-infected individuals.

Humoral and cellular immune responses to HIV-1 nef in mice DNA-immunised with non-replicating or self-replicating expression vectors.

OBJECTIVE HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Wound Healing and Soft Tissue Effects of CO2, Contact Nd: YAG and Combined CO2-Nd: YAG Laser Beams on Rabbit Trachea

Rabbit trachea was used as an experimental model to study tissue effects and healing of full-thickness tracheal lesions produced by CO2, contact Nd: YAG and combined, coaxial CO2-Nd: YAG (Combo) laser beams. Two power settings (10 W and 16 W) were used with CO2 and contact Nd: YAG lasers. Three different CO2/Nd:YAG power ratios (1:1, 1:2 and 1:4) and power settings (12 W 15 W and 16 W) were used with the Combolaser. Histological specimens for light and transmission electron microscopy were prepared immediately and 1, 3, 5, 7, 14 and 21 days postoperatively. The wound with the most precise and fastest healing was produced by contact Nd: YAG laser. CO2 laser produced a moderate amount of charring and the largest amount of coagulated tissue with a slightly prolonged healing period. In the acute phase, tissue defects produced by the Combolaser with power ratios 1:1 and 1:2 resembled the CO2 laser lesions but with slightly less charring. The power ratio 1:4 diminished the cutting properties of the beam considerably. During the healing period the Combolaser produced the most intensive inflammation and granulation tissue formation resulting in delayed regeneration of the lesion. In transmission electron micrographs the most severe damage to chondrocytes was seen after using the Combolaser. These findings indicate that the Combolaser produces deeper tissue damage than CO2 or contact Nd:YAG laser. However, the Combolaser appears to be suitable for tracheobronchial operations, owing to its good simultaneous cutting and haemostatic properties.

Intravenous Calcitriol Therapy Restores Reduced Antigen-Induced T-Lymphocyte Response in 1,25-(OH)2D3-Deficient Hemodialysis Patients

Ten hemodialysis patients were treated with intravenous calcitriol (1-1.5 micrograms 3 times per week) for 3 months and parameters reflecting cell-mediated immunity were measured before and at the end of treatment. Peripheral blood CD4+ cells increased from 33.1 +/- 14.2 to 43.8 +/- 5.8% (p < 0.05) causing a comparable increase in CD3+ cells (67.3 +/- 14.3 vs. 77.1 +/- 7.9%, p < 0.05), whereas CD8+ cells did not change significantly (22.2 +/- 5.4 vs. 25.5 +/- 3.0%). Mitogen-induced lymphocyte stimulation responses were normal even before treatment and did not change. Antigen-induced T-cell responses were very heterogeneous before calcitriol therapy; those 5 with initially unmeasurably low serum 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) had a weaker response than the other patients (3,873 +/- 1,528 vs. 22,948 +/- 13,684 cpm, p < 0.05). After calcitriol treatment the patients with pretherapy unmeasurably low serum 1,25-(OH)2D3 had a comparable response to other patients (16,220 +/- 9,674 vs. 22,064 +/- 10,331 cpm). Our study shows that calcitriol therapy restores the depressed antigen-induced T-cell response of the hemodialysis patients most deficient in 1,25-(OH)2D3.

Immediate Histological Changes in Soft Palate after Uvulopalatopharyngoplasty with CO2, Contact Nd : YAG or Combined CO2 and Nd : YAG Laser Beams

CO2, contact Nd : YAG and Combolaser (combined, simultaneous and coaxial CO2 + Nd : YAG laser beam) were used for uvulopalatopharyngoplasty (UPPP). It has been proposed that the combined beam geometry diminishes thermal damage to surrounding tissues when compared with single laser radiation. To study the extent of thermal tissue damage produced by the lasers, tissue samples for light (LM) and transmission electron microscopy (TEM) were taken from the surface of the resected area and 2 and 4 mm below the resection plane. The depth of tissue damage and coagulation was measured. The results showed no consistent differences in the inflammatory reactions or the amount and depth of tissue coagulation observed in samples taken immediately after the operation. The CO2 laser typically produced a carbonized and coagulated wound edge. Combolaser and contact Nd : YAG lasers generated slightly less charring but otherwise resembled each other with coagulated and vacuolized resecate margins. These results indicate that ...

Effects of erythropoietin treatment on cell-mediated immune responses in predialysis patients

Objective Material and methods The effects of erythropoietin (EPO) treatment on the immune functions of dialysis patients have been shown to be controversial and there are only limited data concerning predialysis patients. Results Twenty-four predialysis patients with renal anemia were assigned to subcutaneous EPO treatment, and those in need (n=19) were additionally treated with i.v. iron every other week. We analyzed the effect of the start of EPO treatment on (i) lymphocyte and lymphocyte subclass counts, (ii) lymphocyte stimulation functions and (iii) persisting IgG-class antibody levels to the viral antigens of Epstein–Barr virus and cytomegalovirus. Conclusions Our main findings were a decrease in the absolute lymphocyte count, combined with decreases in all the main lymphocyte subclass counts. The absolute number of cells with activation and memory markers remained constant, and therefore their proportion slightly increased. The proliferation responses to phytohemagglutinin, tuberculin and tetanus declined significantly, while the amount of IgG-class viral antibodies remained unchanged, meaning that the humoral side of immunity was not affected by the start of the EPO treatment. Similarly, the proliferation response to pokeweed mitogen, a B-cell mitogen, was unchanged. EPO treatment has a suppressive effect on cellular immune functions of predialysis patients. This suppression does not correlate with erythropoiesis, kidney function or iron status.