Retrovirally Infected Research Articles

Antibody response to feline panleukopenia virus vaccination in cats with asymptomatic retrovirus infections: a pilot study.

Currently, there are only a few studies on how immunocompromised cats, such as cats infected with feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV), respond to vaccination. Therefore, this study measured feline panleukopenia virus (FPV) antibodies in retrovirus-infected cats within a period of 28 days after FPV vaccination, and compared the immune response to that of non-infected cats. Eight asymptomatic retrovirus-infected cats (four FeLV, four FIV), and non-infected age-matched control cats (n = 67) were vaccinated with a commercial FPV modified live virus (MLV). Pre- and post-vaccination antibody titres were measured by haemagglutination inhibition (HI) on days 0, 7 and 28. An HI titre ⩾1:40 was defined as protective. An adequate response to vaccination was defined as a four-fold titre increase or higher. Comparison of the immune response of retrovirus-infected and non-infected cats was performed. Pre-vaccination FPV antibody titres ⩾1:40 were present in 100% (n = 8/8; 95% confidence interval [CI] 62.8-100) of retrovirus-infected and in 77.6% (n = 52/67; 95% CI 66.2-86.0) of non-infected cats. An adequate response to vaccination (titre increase ⩾four-fold) was seen in 1/8 retrovirus-infected cats (12.5%; 95% CI 0.1-49.2) compared with 22/67 non-infected cats (32.8%; 95% CI 22.8-44.8). In cats with high pre-vaccination titres (⩾1:160), a four-fold titre increase or higher was observed in 1/8 retrovirus infected cats (12.5%; 95% CI 0.1-49.2) compared with 4/42 non-infected cats (9.5%; 95% CI 3.2-22.6). None of the eight retrovirus-infected cats developed illness or vaccination side effects after vaccination with MLV against FPV within the 28 days. There were no significant differences between groups: for pre-vaccination titres; for at least four-fold titre increases following vaccination in either all cats or the cats with high pre-vaccination titres; and concerning adverse effects. All retrovirus-infected asymptomatic cats had pre-vaccination FPV antibodies indicating protection against panleukopenia. Response of retrovirus-infected cats to vaccination was similar to the response of non-infected cats.

Hemophagocytic Syndrome in a Pancytopenic Simian Retrovirus–Infected Male Rhesus Macaque (Macaca mulatta)

Hemophagocytic syndrome (HPS) is a macrophage hyperactivation disorder triggered by disrupted T-cell macrophage cytokine interaction. HPS has been reported in humans, dogs, cats, and cattle, and it is infrequent and poorly characterized in animals. A 16-year-old male rhesus macaque was euthanized because of severe pancytopenia, including nonregenerative anemia (hematocrit = 5.5%), neutropenia (0.29 K/μl), and thrombocytopenia (21 K/μl). Bone marrow was hypocellular with normal maturation, myeloid hypoplasia, and few megakaryocytes. There were numerous morphologically normal macrophages (12% of nucleated cells), with 6% of nucleated cells being hemophagocytic macrophages in the bone marrow. Serology was negative, but polymerase chain reaction and immunohistochemistry were positive for simian retrovirus type 2. Blood and bone marrow findings were consistent with HPS. Cytopenias are common in simian retrovirus-infected macaques, but HPS has not been reported. An association between simian retrovirus infection and HPS is undetermined, but retrovirus-associated HPS has been observed in humans.

Coordinate Suppression of ERBB2 and ERBB3 by Enforced Expression of Micro-RNA miR-125a or miR-125b

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

RPA2 Is a Direct Downstream Target for ATR to Regulate the S-phase Checkpoint

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.

Retroviral Infection and Selection of Culture-Derived Platelets Allows Study of the Effect of Transgenes on Platelet Physiology Ex Vivo and on Thrombus Formation In Vivo

Background- We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets. We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings. After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy. The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.

CpG ODNs Treatments of HIV-1 Infected Patients May Cause the Decline of Transmission in High Risk Populations – A Review, Hypothesis and Implications

The Joint United Nations Program on HIV-1/AIDS (UNAIDS) announced its goal to stop HIV-1 transmission by antiviral (HAART) treatment of patients since at the end of 2003 the number of people living with HIV-1 was 38 million, 25 million in the sub-Saharan region of Africa. The present review deals with a new approach to simultaneously treat HIV-1/AIDS patients in HIV-1 endemic regions with CpG oligodeoxynucleotides (ODNs) and people at high risk of infection with a vaccine containing CpG ODNs combined with synthetic HIV-1 peptides by intranasal and intradermal applications.

Dilated Cardiomyopathy in Retrovirally Infected Mice: A Novel Model for Silent Viral DCM?

Dilated cardiomyopathy (DCM) is a clinically relevant disease that can occur independently or secondary to other diseases such as HIV infection and AIDS. To study this latter process, we used a model in which mice are infected with the LP-BM5 murine AIDS (MAIDS) retrovirus. Cardiac function of control and infected mice was determined through the in vivo analysis of left ventricular pressure-volume loops. Furthermore, the role of myocarditis was investigated through immunohistochemistry for T-cell, B-cell, and macrophage cardiac infiltrates and Northern blot analysis for tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS). End-systolic and end-diastolic volumes were significantly increased and ventricular stiffness was significantly decreased in infected mice, consistent with DCM; however, no staining for inflammatory cellular infiltrates or TNF-alpha and iNOS was seen. These data support the conclusion that the LP-BM5 HIV model virus causes DCM in the absence of chronic cardiac inflammation. These findings support MAIDS retroviral infection as a new model of idiopathic DCM in which myo-carditis does not occur.

Side-stream cigarette smoke accentuates immunomodulation during murine AIDS

Side-stream cigarette smoke has become a hotly debated social, political, and scientific health and safety issue for nonsmokers. The harmful influences of side-stream cigarette smoke on human health are its adverse effects on the immune system, especially when already compromised by other agents. Acquired immune deficiency syndrome (AIDS) is a clinical disorder caused by human immunodeficiency virus (HIV). To facilitate studies, murine AIDS was induced in C57BL/6 mice by LP-BM5 murine leukemia virus infection, which mimics human AIDS. After 2 weeks of retroviral infection, the mice were exposed to side-stream cigarette smoke for 30 min, 5 days/week for 12 weeks using a side-stream cigarette smoke exposure system. Murine retrovirus infection reduced the in vitro proliferation of T lymphocytes stimulated by concanavalin A, increased the release of pro-inflammatory cytokine interleukin-6 (IL-6) tumor necrosis factor-alpha (TNF-α), increased the hepatic lipid peroxidation and decreased the α-tocopherol levels in liver, lung and heart. Concomitant side-stream cigarette smoke exposure for 12 weeks further inhibited the proliferation of T cells, increased the release of TNF-α, IL-6 cytokines and enhanced the hepatic lipid peroxidation from retrovirus infected mice. The loss of α-tocopherol was also further enhanced by side-stream cigarette smoke exposure during retrovirus infection. Our conclusions are that side-stream cigarette smoke induced increasing oxidative stress, reducing nutrient concentrations and suppressing immune function could make mice with murine AIDS more susceptible to opportunistic infections, potentially accelerating murine AIDS progression. Thus, the reduction of side-stream cigarette smoke exposure is an important health issue in AIDS patients to improve the quality and quantity of their lives.

T-Cell Receptor-Derived Peptides in Immunoregulation and Therapy of Retrovirally Induced Immunosuppression

Retrovirally infected humans and mice showed progressive acquired immunodeficiency accompanied by the production of elevated levels of autoantibodies directed against T-cell receptor variable-domain epitopes. Epitope mapping analyses indicated that a major determinant recognized was defined by a 16-mer peptide containing the entire CDR1 segment and part of the FR2 region of human Vbeta8, and that both species showed reactivity to the same sequence. Either prophylactic or therapeutic administration of this peptide to retrovirus-infected C57/BL/6 mice normalized the balance of T(H)1- and T(H)2-type helper activity and restored the resistance to infection by the opportunistic parasite Cryptosporidium. Administration of the peptide did not generate significantly increased levels of autoantibody, but had a profound effect on T-cell activity as well as other aspects of inflammation, including NK-cell activity. A 16-mer derived from the Jbeta sequence showed similar functional effects on T cells from retrovirus-infected mice. Direct binding of the VbetaCDR1 peptide to recombinant TCR Valpha/Vbeta constructs, as well as to IgM natural autoantibodies, suggests that the cell surface receptor for the peptide is the alpha/beta TCR on T cells and surface IgM in B cells. The Vbeta CDR1 peptide stimulated division of murine splenocytes in vitro, stimulated the production of the T(H)1 cytokine IL-2, and synergized with the T-cell mitogen concanavalin A in proliferation and IL-2 production. These studies indicate that administration of peptides derived from T-cell receptor variable domains to animals immunosuppressed as a result of retroviral infection has a profound immunomodulatory effect enhancing overall T-cell functional capacity, particularly with respect to the cytokine production characteristic of T(H)1-type cells. Our studies are interpreted in the context of other recent investigations of immunomodulatory peptides.

Retroviral infection of the FGF2 gene into MCF-7 cells induces branching morphogenesis, retards cell growth and suppresses tumorigenicity in nude mice

FGF2 (basic fibroblast growth factor) is a multifunctional growth factor and exhibits diverse function in different cell types. In breast, loss of FGF2 expression is associated with malignant progression. In order to understand the role of FGF2 in maintenance of normal breast structures and control of cell growth, we restored FGF2 expression in the breast cancer cell line MCF-7. The FGF2 retrovirally infected MCF-7 cells (MCF-7.F2.5) not only expressed FGF2 in cytoplasm and nuclei, but also released FGF2 into culture medium both on plastic and in Matrigel conditions. The MCF-7.F2.5 cells formed branches in Matrigel and this effect was abolished by the addition of a neutralising anti-FGF2 antibody or function blocking antibodies to α2, α3 and β1 integrins. Furthermore, MCF-7.F2.5 cells lost their ability for anchorage-independent growth in soft agar. When MCF-7 and MCF-7.F2.5 cells were injected into nude mice, there was a 1.6- to 3.2-fold reduction of tumour volume with MCF-7.F2.5 cells in comparison with the parental MCF-7 cells. MCF-7.F2.5 cells also demonstrated a reduction in oestrogen receptor-α (ERα) both in vitro and in vivo. Our results suggest that introduction of the FGF2 gene into MCF-7 cells altered the malignant tumour cells towards a more benign phenotype in vitro and in vivo.

Retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with viral particles and are devoid of viral vector RNA.

Obtaining high expression levels of a therapeutic gene in target cells could be achieved by integrating multiple copies of a recombinant retrovirus. However, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copies, suggesting that maximum retroviral transduction was achieved at relatively low MOIs. The same results were obtained when purified virus, free of most medium components, was used. Retroviral infection was shown to be inhibited by supernatants of other viral producer cell lines, and this inhibition could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparations showed that the amount of retroviral vector RNA present was similar to the amount expected on the basis of virus titers. Our data suggest that retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with retroviruses and do not contain viral vector RNA. We postulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more effective packaging cell lines.

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b5 anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb5 anchor (1–266-b5) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb5 anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.

Effects of Selegiline in a retroviral rat model for neurodegenerative disease.

Upon inoculation into neonatal rats, murine leukemia virus (MuLV) NT40 causes a non-inflammatory degeneration of the central nervous system. While microglia cells appear to be the major target cells within the brain parenchyma for neurovirulent MuLV, degenerating neurons do not express retroviral gene products. In order to protect rats from neuronal damage we treated retrovirally infected rats once with monoamine oxidase (MAO) B inhibitor Selegiline which--under different conditions--exerts neuroprotective effects. Unexpectedly, when administered at 17 days post-infection (d.p.i.) a single intraperitoneal dose of Selegilin (1 mg/kg bodyweight) significantly shortened the incubation period for neurological disease. In contrast, Selegiline given in a lower dosage (0.05 mg/kg bodyweight) and/or at a different time point (13 d.p.i.) at the low (0.05 mg/kg bodyweight) and the high dose (1.0 mg/kg bodyweight) had no effect on the outcome of neurological disease. Animals treated with Selegiline (1.0 mg/kg bodyweight at 17 d.p.i.) contained higher amounts of viral loads in the CNS, higher numbers of brain cells expressing major histocompatibility complex class II molecules, and exhibited inhibition of MAO-B in comparison to untreated yet infected (control) animals. Supposedly, Selegiline activated the major target cell population of the CNS for MuLV-NT40, microglia, with the consequence of enhanced susceptibility for retroviral infection and triggered endogenous mechanism(s) involved in the pathogenesis of retroviral neurodegeneration.

Physical inactivity of murine retrovirus infected C57BL/6 mice is prevented by melatonin and dehydroepiandrosterone.

LP-BM5 retrovirus infection and hormone synthesis modulate many physiological systems that could affect physical activity. Infection induced oxidative damage and immunodeficiency of female mice which hormone supplement prevented. Therefore, the effects of retrovirus infection and hormone supplementation were assessed on physical activity using a computerized video system. Retroviral infection increased activity when stationary while lowering average speed and resting time. Hormone supplementation partially modified changes due to murine AIDS. Dehydroepiandrosterone (DHEA) or melatonin (MLT) supplementation restored the average speed; ambulatory time and distance traveled of retrovirus infected mice. MLT as well as the combination of DHEA + MLT increased body movement, but decreased average speed and distance traveled. Thus, retrovirus infection had significant effects on physical activity. Further studies into the relationship between the DHEA and/or MLT and physical activity will assert the contribution of these hormones to the treatment of murine AIDS.

Dehydroepiandrosterone (DHEA) sulfate prevents reduction in tissue vitamin E and increased lipid peroxidation due to murine retrovirus infection of aged mice.

Dietary effects of dehydroepiandrosterone sulfate (DHEAS) supplementation on tissue antioxidants and lipids were investigated in retrovirus infected mice. DHEA is a powerful antioxidant and immunomodulator whose production declines with age. For this study, twenty-four female, 15-month-old C57BL/6 mice were left uninfected while twenty-four were infected with LP-BM5 murine leukemia virus, causing murine AIDS. The retroviral infection caused immune dysfunction and loss of hepatic and cardiac vitamins E and A, resulting in increased lipid peroxides. Treatment with DHEAS at 0.01 or 0.005% in drinking water for 10 weeks post-infection significantly (P < 0.05) lowered lipid peroxidation in both heart and liver tissues. Treatment with DHEAS also largely prevented loss of the antioxidants, such as vitamin E and A, and prevented loss of phospholipid in the hearts and livers of the old uninfected as well as infected mice. This study suggests that DHEAS supplementation reduces damage associated with elevated oxidation due to aging and retrovirus infection.

Cytokine dysregulation and increased oxidation is prevented by dehydroepiandrosterone in mice infected with murine leukemia retrovirus.

The effects of murine leukemia retrovirus infection on production of cytokines was investigated in mice fed different doses of dehydroepiandrosterone (DHEA). Young C57BL/6 female mice were injected with LP-BM5 murine retrovirus or were kept as uninfected controls. Two weeks later, each group was divided into subgroups: fed unsupplemented AIN 93 diet as the control, or diets supplemented with 0.02% DHEA (0.9 mg/mouse/day) or 0.06% DHEA (2.7 mg/mouse/day). The uninfected mice supplemented with 0.06% DHEA showed a significant (P < 0.05) increase in interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) production, and hepatic vitamin E levels. Retroviral infection induced severe oxidative stress that was reduced by DHEAS supplementation in retrovirally infected mice. DHEA supplementation prevented the retrovirus-induced loss of cytokines (IL-2 and IFN-gamma) secretion by mitogen stimulated spleen cells. DHEA also suppressed the production of cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) by T helper 2 (Th2) cells which were otherwise stimulated by retrovirus infection. Thus, immune dysfunction and increased oxidation induced by murine retrovirus infection were largely prevented by DHEA.

ALTERATION OF THE CD34 +Tf-1β CELL LINE PROFILE IN RESPONSE TO LONG-TERM EXPOSURE TO IL-15

Interleukin 15 (IL-15) is a cytokine with many functional characteristics that are similar to IL-2. Most of the functional activities that IL-2 and IL-15 support have been evaluated in short-term assays. It was our intention, then, to determine the long-term effects of IL-15 in comparison to IL-2. These studies were performed using the growth factor-dependent myelomonocytic cell line, Tf-1, which has been well characterized with regard to morphology, CD marker expression, responses to certain growth factors and cytokines (GM-CSF, IL-4, erythropoietin), and can differentiate-through the myeloid and erythroid lineages. In order to study IL-2 and IL-15 responses, Tf-1 cells were retrovirally infected with the IL-2Rβ chain gene as a means to confer IL-2 responsiveness to this cell type. The results of this study demonstrate that retroviral infection of Tf-1 successfully generated a stable IL-2 responsive cell line, Tf-1β, without interfering with the original characteristics of the Tf-1 cell. Tf-1β cells respond functionally to both IL-2 and IL-15. When Tf-1β cells are grown for 8 weeks in Il-2 (Tf-1β2), rather than GM-CSF, the original morphology, CD marker expression, esterase activity and proliferative response is unaltered in comparison to that of the original Tf-1β line maintained in GM-CSF. However, long-term growth of Tf-1β in IL-15 (Tf-1β15) results in morphological alterations, downregulation of CD33, CD38, and HLA-DR, and a decreased response to IL-15 in comparison to Tf-1β2. These studies support the concept that retroviral infection, even when it confers new functions upon a cell, does not necessarily alter all other functions, as assessed by evaluation of its phenotypic profile. Furthermore, the production of the Tf-1β2 and Tf-1β15 sublines demonstrates that IL-2 and IL-15 can support long-term growth. However, this long term growth in IL-15 leads to subtle alterations in the cell profile that are not seen with IL-2, suggesting that distinctions in IL-2 and IL-15 function do exist. Further study of the Tf-1β15 cell line will be useful to clarify these functional distinctions between IL-2 and IL-15.

Inclusion body myositis in HIV-1 and HTLV-1 infected patients

Sporadic inclusion body myositis (IBM) is the most common inflammatory myopathy affecting patients over the age of 50 years. Dysimmune and degenerative aetiologies have been postulated, but viral infections have not been associated with the disease. Two HIV-I (human immunodeficiency virus type 1) infected men and one woman infected with HTLV-1 (human T cell leukaemia virus type 1) developed progressive proximal muscle weakness unrelated to antiretroviral therapy. Their muscle biopsies were studied by light and electron microscopy, by immunocytochemistry to determine the expression of major histocompatibility complex (MHC) molecules and identify the type of infiltrating cells and T cell receptor (TCR) subunits, and by reverse transcription-polymerase chain reaction (RT-PCR) and single or double immunocytochemistry to search for retrovirally infected endomysial cells. The clinical features were consistent with sporadic IBM. The muscle biopsies showed primary endomysial inflammation, red-rimmed vacuoles, amyloid deposits, eosinophilic inclusions, and small round fibres in groups, all diagnostic of IBM. The muscle fibres expressed MHC class-1 antigens and were invaded primarily by CD8+ T-lymphocytes preferentially bearing TCR V beta 5.1 and V beta 13 chains. The HIV-1 or HTLV-1 antigens were detected only on endomysial macrophages on or around muscle fibres, but not within the muscle fibres. We conclude that IBM occurs in HIV-1 and HTLV-1 infected individuals and has a clinical, histological and immunological pattern identical to sporadic IBM in the non-retrovirally infected patients. Retroviruses do not directly infect the muscle, but persistent retroviral infections may provide superantigenic stimulation and trigger an endomysial inflammatory response identical to that occurring in sporadic IBM.

Immunomodulation by pycnogenol® in retrovirus-infected or ethanol-fed mice

Pycnogenol ® is a commercial mixture of bioflavonoids that exhibits antioxidative activity. The effects of dietary pycnogenol on immune dysfunction in normal mice as well as those fed ethanol or infected with the LP-BM5 murine retrovirus were determined. The ethanol consumption and retrovirus infection cause abnormalities in the function and/or structure of a broad array of cells involved in humoral and cellular immunity. Pycnogenol enhanced in vitro IL-2 production by mitogen-stimulated splenocytes if its production was suppressed in ethanolfed or retrovirus-infected mice. Mitogenesis of splenocytes did not show a significant change in mice treated with pycnogenol. It reduced the elevated levels of interleukin-6 produced in vitro by cells from retrovirus infected mice and IL-10 secreted by spleen cells from mice consuming ethanol. Natural killer cell cytotoxicity was increased with pycnogenol treatment.

Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells.

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neor gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neor gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.