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Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.

Cdc55, a regulatory B subunit of the protein phosphatase 2A (PP2A) complex, plays various functions during mitosis. Sequestration of Cdc55 from the nucleus by Zds1 and Zds2 is important for robust activation of mitotic Cdk1 and mitotic progression in budding yeast. However, Zds1-family proteins are found only in fungi but not in higher eukaryotes. In animal cells, highly conserved ENSA/ARPP-19 family proteins bind and inhibit PP2A–B55 activity for mitotic entry. In this study, we compared the relative contribution of Zds1/Zds2 and ENSA-family proteins Igo1/Igo2 on Cdc55 functions in budding yeast mitosis. We confirmed that Igo1/Igo2 can inhibit Cdc55 in early mitosis, but their contribution to Cdc55 regulation is relatively minor compared with the role of Zds1/Zds2. In contrast to Zds1, which primarily localized to the sites of cell polarity and in the cytoplasm, Igo1 is localized in the nucleus, suggesting that Igo1/Igo2 inhibit Cdc55 in a manner distinct from Zds1/Zds2.Our analysis confirmed an evolutionarily conserved function of ENSA-family proteins in inhibiting PP2A-Cdc55, and we propose that Zds1-dependent sequestration of PP2A-Cdc55 from the nucleus is uniquely evolved to facilitate closed mitosis in fungal species.

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea.

ABSTRACTTransient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8+lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8+cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8+cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

ABSTRACTMolecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrinamacaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P= 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4+T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.

ABSTRACTTo better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir {9-[2-(R)-(phosphonomethoxy)propyl]adenine}while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS.

Phorbol myristate acetate (PMA) treatment of an EL‐4 thymoma cell line (EL‐4farrar) induced secretion of a factor that inhibited intracellular killing of Leishmania majoramastigotes by activated macrophages. Analysis of the cytokines produced by EL‐4 cells after PMA stimulation identified interleukin‐2 (IL‐2, 2500 U/ml), IL‐4 (1280 U/ml), interferon‐γ(IFN‐γ; 100 U/ml), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor β(TGF‐β) was detected. Each of the cytokines present in EL‐4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN‐γand GM‐CSF both activated macrophages to kill Leishmania; I L‐2 and IL‐4 had no activity for induction of this anti‐microbial effector function. IL‐2 and IL‐4 were tested for their capacity to inhibit lymphokine‐ or IFN‐γ‐induced destruction of L. majorby macrophages: IL‐4 was ineffective, but IL‐2 markedly suppressed the activation of macrophages for intracellular killing. Addition of ≥ 10 U/ml of IL‐2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL‐4 fluids with anti‐IL‐2 antibody resulted in >80% reduction in suppression of intracellular killing. The suppressive effects of IL‐2 were not direct, but mediated by TGF‐β. IL‐2 induced resident peritoneal macrophages to secrete >5000 pg/ml TGF‐β1, a quantity that is > 500‐fold higher than constitutive background levels (20–40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF‐βwas not dependent increases in TGF‐β1 mRNA. Treatment of cultures with EL‐4 fluids or recombinant IL‐2 in the presence of antibody to TGF‐β1 blocked the suppressive activity of both. Thus, IL‐2 was the major suppressor factor in EL‐4 fluids, and it acted indirectly through the induction and autocrine action of TGF‐β. J. Leukoc. Biol.55: 81–90; 1994.

ABSTRACTWhole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4+target cells and mediated virus-induced, CD4-dependent “fusion from without” comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN- alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN- alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN- alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

The amino acid sequence of a biologically active polypeptide isolated from calf thymus, termed thymosin alpha1, has been determined. Thymosin alpha1 is a heat stable, highly acidic molecule composed of 28 amino acid residues. This peptide is one of several present in thymosin fraction 5 that may participate in the regulation, differentiation and function of thymus-dependent lymphocytes (T cells). A nomenclature for the family of polypeptides present in thymosin fraction 5 is suggested.

ABSTRACTWe tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4+T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4+T-cell depletion, consistent with mechanisms requiring productive infection.

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4(+) T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4(+) T-cell depletion, consistent with mechanisms requiring productive infection.