bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulin levels and phagocytes in the cervical mucus plug at term of pregnancy
Authors: Hein Merete , Petersen Astrid C. , Helmig Rikke Bek , Uldbjerg Niels , Reinholdt Jesper
Published: July 2005

Background.  To characterize the potential for adaptive immune protection in cervical mucus plugs with respect to immunoglobulin isotypes and effector cells (phagocytes).

Methods.  Thirty‐one cervical mucus plugs were collected from healthy women in labor at term. The cervical mucus plugs were allocated either to analysis of immunoglobulins by enzyme‐linked immunosorbent assay (ELISA), gel chromatography and Western blotting (n = 20) or to microscopical, including immunocytochemical, analyses. The levels of immunoglobulin in the plugs were compared to the levels in 10 samples of ovulatory cervical mucus from nonpregnant women.

Results.  In the cervical mucus plugs, levels of immunoglobulin G (IgG) [median 3270 µg/mL (100–14 500)] and IgA [540 (22–2820)], but not IgM [30.5 (1.0–160)], were significantly elevated compared to cervical mucus from nonpregnant women (p < 0.02 for IgG and IgA). The IgG : IgA ratio in the plugs was also elevated (p < 0.02). The proportion of secretory immunoglobulin A (SIgA) relative to total IgA in the plugs ranged from 16 to 65% (n = 5). IgA and IgG were largely intact. Microscopically, the vagina‐proximal part of the cervical mucus plugs contained bacteria and was rich in cells, mainly phagocytes. Conversely, the uterine part contained few cells.

Conclusion.  The high immunoglobulin levels in combination with the presence of phagocytes suggest a potential for adaptive immune defense in the cervical mucus plug, which, together with innate immune factors, may act as an immunological gatekeeper protecting the fetomaternal unit against infection from the vagina.
Short‐term and long‐term results of resectoscopic myomectomy with and without pretreatment with GnRH analogs in premenopausal women
Authors: Campo Sebastiano , Campo Vincenzo , Gambadauro Pietro
Published: July 2005

Objective.  The aim of this study was to analyze the safety and efficacy of resectoscopic myomectomy and to evaluate the influence of GnRH analog pretreatment on short‐ and long‐term surgery outcome.

Methods.  Data were collected from 80 consecutive resectoscopic myomectomies performed on premenopausal women. Forty‐two patients (52·5%) had not received any preoperative medical treatment and in their case surgery was performed during the early proliferative phase (group A), whereas 38 patients (47·5%) had been treated with 2 months of intramuscular GnRH analog (triptorelin, Decapeptyl 3·75 mg) (group B). Myomas were classified according to the Wamsteker classification. The surgical time, recovery time, and complications were considered as short‐term results. A 24‐month follow‐up was performed in order to assess long‐term results, such as abnormal uterine bleeding recurrence, myomas recurrence, and need of repeat surgery.

Results.  Patients' mean age was 38·8 ± 5·39 years (range: 24–47) in group A and 38·97 ± 7·46 years (range: 20–49) in group B. Forty‐eight myomas were excised in group A (1·1 ± 0·53 myoma per patient; mean diameter: 28·72 ± 11·57 mm) and 42 in group B (1·09 ± 0·29; mean diameter: 29·73 ± 14·47 mm). Myoma type distribution was similar in both groups. Surgical time of the pretreated patients (group B) was significantly longer than that of untreated patients (group A 40 ± 18·06 versus group B 57·65 ± 29·61 min; P = 0·002). No significant difference was found between the two groups in the length of postoperative stay (group A 1·05 ± 0·22 days versus group B 1·15 ± 0·44). Only one complication occurred, represented by a uterine perforation. Histopathology revealed the presence of cellular myomas, without atypia, in three patients of each group. At follow‐up, abnormal uterine bleeding occurred in 36·3% of group A patients and in 26·6% of group B patients, but the difference showed no statistical significance (P = 0·57). Five patients of myoma recurrence were observed, three in group A and two in group B. Only two patients, one from each group, required repeat hysteroscopic surgery.

Conclusions.  GnRH analog pretreatment, even if extremely beneficial in anemic patients, does not seem to improve short‐ and long‐term outcome following resectoscopic myomectomy. Surgical time of pretreated patients is significantly longer, maybe because of difficulty in cervical dilatation, although further study is needed in order to confirm this hypothesis.
A three‐armed randomized trial comparing open Burch colposuspension using sutures with laparoscopic colposuspension using sutures and laparoscopic colposuspension using mesh and staples in women with stress urinary incontinence
Authors: Ankardal Maud , Milsom Ian , Stjerndahl Jan‐Henrik , Engh Marie Ellström
Published: July 2005

Objective.  To compare open Burch colposuspension using sutures (OC) with laparoscopic colposuspension using sutures (LCS) and laparoscopic colposuspension using mesh and staples (LCM) in women with stress urinary incontinence.

Design.  Prospective randomized trial.

Setting.  Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Göteborg, Sweden.

Population.  Women with genuine stress urinary incontinence or mixed incontinence with a predominantly stress component attending the Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Göteborg, Sweden.

Methods.  The women were assessed objectively using a 48‐h frequency‐volume chart, a 48‐h pad test and a standardized stress test. Subjectively, the women were assessed by a questionnaire including influence on quality of life. The women were randomized to OC using sutures (n = 79), LCS (n = 53) or LCM (n = 79). Anaesthesia/operation time, blood loss, complications and other related surgical parameters were compared.

Main outcome measures.  Objective and subjective cure rate.

Results.  Objective cure rates 1 year after surgery were significantly higher in the OC and LCS groups compared to the LCM group analyzed by a standardized stress test. Subjective findings were in concordance with the objective results. Performing an OC was less time consuming than performing a LCS and resulted in more blood loss compared to the two laparoscopic techniques. Patients in the LCM group had a shorter duration of catheter use and hospital stay.

Conclusion.  The use of sutures, irrespective of whether the surgical approach was laparoscopic or open surgery, was superior to the laparoscopic mesh and staple technique.
Placental imbalance of Th1‐ and Th2‐type cytokines in preeclampsia
Authors: Dong Minyue , He Jing , Wang Zhengping , Xie Xing , Wang Hanzhi
Published: July 2005

Objectives.  To characterize the changes in the level of T helper 1 (Th1)‐ [interleukin (IL)‐2 and tumor necrosis factor (TNF)‐α] and Th2‐type cytokine (IL‐10) and the ratios of Th1/Th2 (IL‐2/IL‐10 and TNF‐α/IL‐10) in placentae from women with preeclampsia and women with gestational hypertension.

Methods.  Placental levels of IL‐2, TNF‐α, and IL‐10 were determined with radioimmunoassay and Th1/Th2 ratios (IL‐2/IL‐10 and TNF‐α/IL‐10) calculated in the placentae from 22 women with preeclampsia, 15 women with gestational hypertension, and 32 normal term pregnant women.

Results.  Although preeclampsia had the trend of the increase in the placental levels of IL‐2 and TNF‐α and the trend of the decrease in placental IL‐10, there were not significant difference in placental levels of IL‐2, IL‐10, and TNF‐α among preeclampsia, gestational hypertension, and normal pregnancy (P > 0.05 for all). Placental ratios of IL‐2/IL‐10 and TNF‐α/IL‐10 were significantly higher in preeclampsia than in normal pregnancy (P = 0.035 and P = 0.005, respectively). No differences of Th1/Th2 ratios were found between preeclampsia and gestational hypertension and between gestational hypertension and normal pregnancy (P > 0.05 for all).

Conclusions.  Alterations of placental balances of cytokines with Th1 predominance were demonstrated in preeclampsia. These associations may offer insights into the pathogenesis of preeclampsia.
Psychological sequelae of medical and surgical abortion at 10–13 weeks gestation
Authors: Ashok Premila W. , Hamoda Haitham , Flett Gillian M. M. , Kidd Avril , Fitzmaurice Ann , Templeton Allan
Published: July 2005

Background.  Although not much research comparing the emotional distress following medical and surgical abortion is available, few studies have compared psychological sequelae following both methods of abortion early in the first trimester of pregnancy. The aim of this review was to assess the psychological sequelae and emotional distress following medical and surgical abortion at 10–13 weeks gestation.

Methods.  Partially randomized patient preference trial in a Scottish Teaching Hospital was conducted. The hospital anxiety and depression scales were used to assess emotional distress. Anxiety levels were also assessed using visual analog scales while semantic differential rating scales were used to measure self‐esteem. A total of 368 women were randomized, while 77 entered the preference cohort.

Results.  There were no significant differences in hospital anxiety and depression scales scores for anxiety or depression between the groups. Visual analog scales showed higher anxiety levels in women randomized to surgery prior to abortion (P < 0.0001), while women randomized to surgical treatment were less anxious after abortion (P < 0.0001). Semantic differential rating scores showed a fall in self‐esteem in the randomized medical group compared to those undergoing surgery (P = 0.02).

Conclusions.  Medical abortion at 10–13 weeks is effective and does not increase psychological morbidity compared to surgical vacuum aspiration and hence should be made available to all women undergoing abortion at these gestations.
Sensitivity to activated protein C in patients with deep vein thrombosis during early puerperium period
Authors: Ohashi Ryota , Sugimura Motoi , Kawamura Takakazu , Tamura Naoaki , Kanayama Naohiro
Published: July 2005

Background.  In this study, we used an endogenous thrombin potential (ETP)‐based assay to examine the response to activated protein C (APC) during early puerperium of 6 Japanese patients of deep vein thrombosis (DVT) and pulmonary embolism (PE) without a history of familial thrombophilia or antiphospholipid syndrome.

Methods.  The patient group comprised 6 Japanese women diagnosed for DVT or PE at Hamamatsu University School of Medicine, between July 1997 and April 2000. The control group comprised 30 healthy Japanese women in early puerperium. APC‐sensitivity ratio (APC‐sr), complete blood count (CBC), and other coagulant variables (thrombin‐antithrombin‐complex (TAT) and fibrin/fibrinogen‐degration product (FDP)‐D‐dimer levels) were determined.

Results.  Mean APC‐sr was 2.0 ± 0.3 in the control group and significantly higher (3.3 ± 1.0) in the patient group (P < 0.05). The FDP‐D‐dimer level was significantly higher in the patient group than in the control group, but there was no significant difference in the TAT complex level.

Conclusions.  We conclude that sensitivity to APC is reduced in patients with DVT or PE during early puerperium. Although further clinical studies are needed to confirm, it appears that sensitivity to APC detected by ETP‐based assay could become a useful marker to estimate the occurrence of DVT or PE.
Involvement of Actin Dephosphorylation in Germination of Physarum Sclerotium
Authors: FURUHASHI KIYOSHI
Published: July 2005

ABSTRACT. The plasmodium of Physarum polycephalum grows without cytokinesis and shows an active cytoplasmic streaming under wet and nutritious conditions. It can undergo reversible differentiation into several types of dormancy to survive in adverse environments. Temperature change or osmotic stress leads to cytoplasmic division of the plasmodium into cells containing one or more nuclei: these form a macrocyst, the spherule. Desiccation also induces cell division of the plasmodium followed by formation of a sclerotium, a dormant body resistant to dry stress. More than half of the actin in a sclerotium is phosphorylated at a single site, threonine 203, resulting in loss of its ability to polymerize into actin filaments. In the present study, actin phosphorylation was found in the sclerotium but not in either the plasmodium or in the spherule. This result suggests that phosphorylation of sclerotium actin may be related to the mechanism associated with desiccation resistance rather than morphological changes through cell compartmentalization in the macrocyst formation. Moreover, dephosphorylation of the phosphorylated form of sclerotium actin began within 5 min after addition of water. Dephosphorylation was not affected by sucrose and sorbitol sugars, but was inhibited by ammonium bicarbonate, ammonium phosphate, sodium phosphate, NaC1, and KC1 in a dose‐dependent manner. On the other hand, in germination of the sclerotium there was measurable sensitivity to both carbohydrates and salts. Actin dephosphorylation seems to be one of the important processes in the early phase of sclerotium germination.
Comparative Effects of Curcumin and an Analogue of Curcumin in Carbon Tetrachloride‐Induced Hepatotoxicity in Rats
Authors: Kamalakkannan Narasimhanaidu , Rukkumani Rajagopalan , Varma Penumathsa Suresh , Viswanathan Periyasamy , Rajasekharan Kallikat Narayanan , Menon Venugopal Padmanabhan
Published: July 2005

Abstract: We have evaluated the comparative effect of curcumin (diferuloyl methane) and its analogue [bis‐1,7‐(2‐hydroxyphenyl)‐hepta‐1,6‐diene‐3,5‐dione] (BDMC‐A) on carbon tetrachloride‐induced hepatotoxicity in rats. Administration of carbon tetrachloride (3 ml/kg/week) for three months significantly (P<0.05) increased the levels of marker enzymes such as aspartate transaminase (AST), alkaline phosphatase (ALP) and γ‐glutamyl transferase (GGT). The levels of plasma thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides were also significantly (P<0.05) increased. We have observed a significant (P<0.05) decrease in the levels of plasma reduced glutathione (GSH), vitamin C and vitamin E. There was a significant (P<0.05) increase in the levels of TBARS and hydroperoxides in liver and kidney and a significant (P<0.05) decrease in the activities of enzymic antioxidants‐ superoxide dismutase (SOD), catalase and GSH peroxidase along with GSH in CCl4‐treated rats. Oral administration of curcumin and BDMC‐A to CCl4‐induced rats for a period of three months significantly (P<0.05) decreased the levels of marker enzymes, plasma TBARS and hydroperoxides and increased the levels of plasma and tissue antioxidants. Histopathological studies of liver also showed protective effect of curcumin and BDMC‐A. We have observed thickening of blood vessels and microvesicular fatty changes around the portal triad in CCl4‐treated rat liver. Treatment with curcumin showed only mild sinusoidal dilatation while with BDMC‐A there was only mild portal inflammation. The effect exerted by BDMC‐A was found to be more promising than curcumin.
Intramuscular Injection of Hypertonic Saline: In vitro and in vivo Muscle Tissue Toxicity and Spinal Neurone c‐fos Expression
Authors: Svendsen Ove , Edwards C. Nick , Lauritzen Brian , Rasmussen Allan D.
Published: July 2005

Abstract: Intramuscular injection of hypertonic saline (4–6% NaCl) is widely used to induce muscle pain in volunteers. The quality of the pain is comparable to clinical muscle pain with localised and referred pain. The objective was to evaluate the muscle toxicity of hypertonic saline by characterisation of 1) cytotoxicity in vitro, 2) local muscle toxicity in rabbits and 3) number of spinal dorsal horn neurones expressing c‐fos after intramuscular injection in pigs as an indicator of nociception. Rat myocyte cultures and erythrocyte suspensions were treated with hypertonic NaCl solutions. The creatine kinase activity remaining in the myocytes and haemolysis were measured. Groups of six rabbits were given an intramuscular injection of 0.5 ml of 0.9, 3 or 6% NaCl. Three days later, creatine kinase activity was determined in injection site muscle tissue and normal contralateral muscle. The amount of injection site muscle tissue totally depleted of creatine kinase was calculated. Groups of two pigs were given an intramuscular injection of 3.0 ml of 6% NaCl. The spinal cord was sampled 1, 2 or 3 hr later and processed for stereological quantification of the number of dorsal horn neurones expressing c‐fos. Saline was not toxic in vitro at 0.9–6%, but toxic to erythrocytes at 7% or higher and rat myocytes at 15% or higher. No muscle toxicity was seen in rabbits. The number of dorsal horn neurones expressing c‐fos was not above basal level. In conclusion, 6% saline caused no in vitro or in vivo toxicity in sensitive models. Consequently, the pain caused by intramuscular injection of hypertonic saline is most likely not related to tissue damage. Consistently, intramuscular injection of 6% NaCl did not activate dorsal horn neurones in pigs to express c‐fos beyond basal level.
Chimeric Influenza Virus Hemagglutinin Proteins Containing Large Domains of the
Bacillus anthracis
Protective Antigen: Protein Characterization, Incorporation into Infectious Influenza Viruses, and Antigenicity
Authors: Li Zhu-Nan , Mueller Scott N. , Ye Ling , Bu Zhigao , Yang Chinglai , Ahmed Rafi , Steinhauer David A.
Published: July 2005

Large polypeptides of the
Bacillus anthracis
protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1′, the region responsible for binding to the other toxin components, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD), were inserted at the C-terminal flank of the HA signal peptide and incorporated into the HA1 subunit of HA. The chimeric proteins, designated as LEF/HA (90 amino acid insertion) and RBD/HA (140 amino acid insertion), were initially analyzed following expression using recombinant vaccinia viruses. Both chimeric proteins were shown to display functional phenotypes similar to that of the wild-type HA. They transport to the cell surface, can be cleaved into the HA1 and HA2 subunits by trypsin to activate membrane fusion potential, are able to undergo the low-pH-induced conformational changes required for fusion, and are capable of inducing the fusion process. We were also able to generate recombinant influenza viruses containing the chimeric RBD/HA and LEF/HA genes, and the inserted PA domains were maintained in the HA gene segments following several passages in MDCK cells or embryonated chicken eggs. Furthermore, DNA immunization of mice with plasmids that express the chimeric RBD/HA and LEF/HA proteins, and the recombinant viruses containing them, induced antibody responses against both the HA and PA components of the protein. These approaches may provide useful tools for vaccines against anthrax and other diseases.
Interferon Regulatory Factor 7 Is Negatively Regulated by the Epstein-Barr Virus Immediate-Early Gene,BZLF-1
Authors: Hahn Angela M. , Huye Leslie E. , Ning Shunbin , Webster-Cyriaque Jennifer , Pagano Joseph S.
Published: July 2005

Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-α/β) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-α4, IFN-β, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-α4, IFN-β, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-α4, IFN-β, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.
Nef Induces Multiple Genes Involved in Cholesterol Synthesis and Uptake in Human Immunodeficiency Virus Type 1-Infected T Cells
Authors: van ′t Wout Angélique B. , Swain J. Victor , Schindler Michael , Rao Ushnal , Pathmajeyan Melissa S. , Mullins James I. , Kirchhoff Frank
Published: July 2005

Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4
+
T cells. Consistent with our microarray data,
14
C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.
Degeneration of Neuronal Processes after Infection with Pathogenic, but Not Attenuated, Rabies Viruses
Authors: Li Xia-Qing , Sarmento Luciana , Fu Zhen F.
Published: July 2005

The structural alterations of neuronal processes in mice were investigated after the mice were infected with rabies virus (RV). Silver staining of infected brain sections showed severe destruction and disorganization of neuronal processes in mice infected with pathogenic RV but not with attenuated RV. However, neuronal bodies showed very little pathological changes. Electron microscopy revealed the disappearance of intracellular organelles, as well as the disappearance of synaptic structures and vesicles. Infection of primary neurons with pathogenic, but not attenuated, RV resulted in the destruction of neuronal processes and disappearance of microtubule-associated protein 2 and neurofilament immunoreactivity, which suggests that pathogenic RV causes degeneration of neuronal processes possibly by interrupting cytoskeletal integrity.
Reduced Prevalence of Epstein-Barr Virus-Related Lymphocryptovirus Infection in Sera from a New World Primate
Authors: Fogg Mark H. , Carville Angela , Cameron Jennifer , Quink Carol , Wang Fred
Published: July 2005

The recent discovery of an Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) naturally infecting common marmosets demonstrated that gamma-1 herpesviruses are not limited to human and Old World nonhuman primate hosts. We developed serologic assays to detect serum antibodies against lytic- and latent-infection marmoset LCV antigens in order to perform the first seroepidemiologic study of LCV infection in New World primates. In three different domestic colonies and in animals recently captured from the wild, we found that the seroprevalence of marmoset LCV infection was not as ubiquitous as with EBV or Old World LCV. These biologic differences in LCV infection of New World versus human and Old World primate hosts correlate with the evolution of the LCV viral gene repertoire.
A New Common Integration Site, Int7, for the Mouse Mammary Tumor Virus in Mouse Mammary Tumors Identifies a Gene Whose Product Has Furin-Like and Thrombospondin-Like Sequences
Authors: Lowther William , Wiley Korah , Smith Gilbert H. , Callahan Robert
Published: July 2005

A novel common integration site for the mouse mammary tumor virus (MMTV) was identified (designated Int7) in five independently arising mouse mammary tumors. The insertion sites all cluster within a 1-kb region that is 2 to 3 kb 5′ of the transcription initiation site of a gene, 2610028F08RIK, whose gene product contains furin-like and thrombospondin-like sequences. Expression of Int7 is normally very low or silent during various stages of mammary gland development, but MMTV integration at this site results in the activation of high steady-state levels of expression of the gene. These five tumors were also found to have two or three additional viral insertions, which in each case occurred flanking a member of either the Wnt and/or FGF gene family. Reverse transcriptase PCR results demonstrated that each of the viral insertions led to elevated expression of the presumed target flanking genes.
Important Roles for Gamma Interferon and NKG2D in γδ T-Cell-Induced Demyelination in T-Cell Receptor β-Deficient Mice Infected with a Coronavirus
Authors: Dandekar Ajai A. , O'Malley Katherine , Perlman Stanley
Published: July 2005

γδ T cells mediate demyelination in athymic (nude) mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM. Now, we show that these cells also mediate the same process in mice lacking αβ T cells (T-cell receptor β-deficient [TCRβ−/−] mice) and demyelination is gamma interferon (IFN-γ) dependent. Most strikingly, our results also show a major role for NKG2D, expressed on γδ T cells, in the demyelinating process with in vivo blockade of NKG2D interactions resulting in a 60% reduction in demyelination. NKG2D may serve as a primary recognition receptor or as a costimulatory molecule. We show that NKG2D+γδ T cells in the JHM-infected central nervous system express the adaptor molecule DAP12 and an NKG2D isoform (NKG2D short), both required for NKG2D to serve as a primary receptor. These results are consistent with models in which γδ T cells mediate demyelination using the same effector cytokine, IFN-γ, as CD8 T cells and do so without a requirement for signaling through the TCR.
Variant-Specific Tropism of Human Herpesvirus 6 in Human Astrocytes
Authors: Donati Donatella , Martinelli Elena , Cassiani-Ingoni Riccardo , Ahlqvist Jenny , Hou Jean , Major Eugene O. , Jacobson Steve
Published: July 2005

Though first described as a lymphotropic virus, human herpesvirus 6 (HHV-6) is highly neuropathogenic. Two viral variants are known: HHV-6A and HHV-6B. Both variants can infect glial cells and have been differentially associated with central nervous system diseases, suggesting an HHV-6 variant-specific tropism for glial cell subtypes. We have performed infections with both viral variants in human progenitor-derived astrocytes (HPDA) and monitored infected cell cultures for cytopathic effect (CPE), intra- and extracellular viral DNA load, the presence of viral particles by electronic microscopy, mRNA transcription, and viral protein expression. HHV-6A established a productive infection with CPE, visible intracellular virions, and high virus DNA loads. HHV-6B-infected HPDA showed no morphological changes, intracellular viral particles, and decreasing intra- and extracellular viral DNA over time. After long-term passage, HHV-6B-infected HPDA had stable but low levels of intracellular viral DNA load with no detectable viral mRNA. Our results demonstrate that HHV-6A and HHV-6B have differential tropisms and patterns of infection for HPDA in vitro, where HHV-6A results in a productive lytic infection. In contrast, HHV-6B was associated with a nonproductive infection. These findings suggest that HHV-6 variants might be responsible for specific infection patterns in glial cells in vivo. Astrocytes may be an important reservoir for this virus in which differential tropism of HHV-6A and HHV-6B may be associated with different disease outcomes.
Human T-Lymphotropic Virus Type 1 Mitochondrion-Localizing Protein p13
II
Sensitizes Jurkat T Cells to Ras-Mediated Apoptosis
Authors: Hiraragi Hajime , Michael Bindhu , Nair Amrithraj , Silic-Benussi Micol , Ciminale Vincenzo , Lairmore Michael
Published: July 2005

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13
II
. We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13
II
localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13
II
, using both transient and stable expression vectors. Our data indicate that p13
II
-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13
II
-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13
II
-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13
II
's influence on lymphocyte survival. Our data are the first to demonstrate that p13
II
alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.
Apical Entry and Release of Severe Acute Respiratory Syndrome-Associated Coronavirus in Polarized Calu-3 Lung Epithelial Cells
Authors: Tseng Chien-Te K. , Tseng Jennifer , Perrone Lucy , Worthy Melissa , Popov Vsevolod , Peters Clarence J.
Published: July 2005

Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (CoV) known as SARS-CoV, is a contagious and life-threatening respiratory illness with pneumocytes as its main target. A full understanding of how SARS-CoV would interact with lung epithelial cells will be vital for advancing our knowledge of SARS pathogenesis. However, an in vitro model of SARS-CoV infection using relevant lung epithelial cells is not yet available, making it difficult to dissect the pathogenesis of SARS-CoV in the lungs. Here, we report that SARS-CoV can productively infect human bronchial epithelial Calu-3 cells, causing cytopathic effects, a process reflective of its natural course of infection in the lungs. Indirect immunofluorescence studies revealed a preferential expression of angiotensin-converting enzyme 2 (ACE-2), the functional receptor of SARS-CoV, on the apical surface. Importantly, both ACE-2 and viral antigen appeared to preferentially colocalize at the apical domain of infected cells. In highly polarized Calu-3 cells grown on the membrane inserts, we found that cells exposed to virus through the apical rather than the basolateral surface showed high levels of viral replication. Progeny virus was released into the apical chamber at titers up to 5 logs higher than those recovered from the basolateral chambers of polarized cultures. Taken together, these results indicate that SARS-CoV almost exclusively entered and was released from the apical domain of polarized Calu-3 cells, which might provide important insight into the mechanism of transmission and pathogenesis of SARS-CoV.
Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus
Authors: Bukreyev Alexander , Belyakov Igor M. , Prince Gregory A. , Yim Kevin C. , Harris Katie K. , Berzofsky Jay A. , Collins Peter L.
Published: July 2005

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8+cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8+CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.
CD4+T-Cell Reconstitution Reduces Cytomegalovirus in the Immunocompromised Brain
Authors: Reuter Jon D. , Wilson Jean H. , Idoko Kimberly E , van den Pol Anthony N.
Published: July 2005

Cytomegalovirus (CMV) infection is the most common opportunistic infection of the central nervous system in patients with human immunodeficiency virus or AIDS or on immunosuppressive drug therapy. Despite medical management, infection may be refractory to treatment and continues to cause significant morbidity and mortality. We investigated adoptive transfer as an approach to treat and prevent neurotropic CMV infection in an adult immunodeficient mouse model. SCID mice were challenged with intracranial murine CMV (MCMV) and reconstituted with MCMV- or vesicular stomatitis virus (VSV)-sensitized splenocytes, T cells, or T-cell subsets. T cells labeled with vital dye or that constitutively generated green fluorescent protein (GFP) were identified in the brain as early as 3 days following peripheral transfer. Regardless of specificity, activated T cells localized to regions of the brain containing CMV, however, only those specific for CMV were effective at clearing virus. Reconstitution with unsorted MCMV-immune splenocytes, enriched T-cell fractions, or CD4+cells significantly reduced virus levels in the brain within 7 days and also prevented clinical disease, in significant contrast with mice given VSV-immune unsorted splenocytes, MCMV-immune CD8+T cells, and SCID control mice. Results suggest CMV-immune T cells (particularly CD4+) rapidly cross the blood-brain barrier, congregate at sites of specific CMV infection, and functionally eliminate acute CMV within the brain. In addition, when CMV-immune splenocytes were administered prior to a peripheral CMV challenge, CMV entry into the immunocompromised brain was prevented. Systemic adoptive transfer may be a rapid and effective approach to preventing CMV entrance into the brain and for reducing neurotropic infection.
Evidence that Low-Level Viremias during Effective Highly Active Antiretroviral Therapy Result from Two Processes: Expression of Archival Virus and Replication of Virus
Authors: Tobin Nicole H. , Learn Gerald H. , Holte Sarah E. , Wang Yang , Melvin Ann J. , McKernan Jennifer L. , Pawluk Diane M. , Mohan Kathleen M. , Lewis Paul F. , Mullins James I. , Frenkel Lisa M.
Published: July 2005

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.
Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus
Authors: Panavas Tadas , Nagy Peter D.
Published: July 2005

Replication of RNA viruses is regulated by
cis
-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro
trans
-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in
trans
by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.
Diversity, Divergence, and Evolution of Cell-Free Human Immunodeficiency Virus Type 1 in Vaginal Secretions and Blood of Chronically Infected Women: Associations with Immune Status
Authors: Sullivan Sharon T. , Mandava Usha , Evans-Strickfaden Tammy , Lennox Jeffrey L. , Ellerbrock Tedd V. , Hart Clyde E.
Published: July 2005

Most human immunodeficiency virus type 1 (HIV-1) infections are believed to be the result of exposure to the virus in genital secretions. However, prevention and therapeutic strategies are usually based on characterizations of HIV-1 in blood. To understand better the dynamics between HIV-1 quasispecies in the genital tract and blood, we performed heteroduplex assays on amplified
env
products from cell-free viral RNA in paired vaginal secretion (VS) and blood plasma (BP) samples of 14 women followed for 1.5 to 3.5 years. Diversity and divergence were less in VS than in BP (
P
= 0.03 and
P
< 0.01, respectively), and divergence at both sites was correlated with blood CD4
+
cell levels (VS,
P
= 0.05; BP,
P
= 0.01). Evolution of quasispecies was observed in 58% of the women; the loss or gain of quasispecies in VS or BP was always accompanied by such changes at the other site. In addition, sustained compartmentalization of quasispecies in VS was found for four women, even as CD4
+
cell levels decreased to low levels (<50 cells/μl). Quasispecies changes over time were associated with fluctuations in CD4
+
cell levels; concordant increases or decreases in VS and BP divergence had greater CD4
+
cell level changes than intervals with discordant changes (
P
= 0.05), and women with evolving quasispecies had greater decreases in CD4
+
cell levels compared to that for women who maintained the same quasispecies (
P
< 0.05). Thus, diversity, divergence, and evolution of cell-free HIV-1 in VS can be different from that in BP, and dynamics between their respective quasispecies are associated with changes in CD4
+
cell levels.
Human Immunodeficiency Virus Reactivation by Phorbol Esters or T-Cell Receptor Ligation Requires both PKCα and PKCθ
Authors: Trushin Sergey A. , Bren Gary D. , Asin Susana , Pennington Kevin N. , Paya Carlos V. , Badley Andrew D.
Published: July 2005

Latently human immunodeficiency virus (HIV)-infected memory CD4+T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCθ, PKCθA148E, and PKCα, PKCαA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+T cells and that both PKCθA148E and PKCαA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCα and PKCθ with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCα or PKCθ. Since suppression of PKCα and PKCθ together has no greater inhibitory effect on HIV reactivation than inhibition of PKCα alone, our data confirm that PKCα and PKCθ act in sequence. The requirement for PKCα and PKCθ for prostratin-induced HIV reactivation and the ability of selective PKCα or PKCθ agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.
A Peptide Derived from RNA Recognition Motif 2 of Human La Protein Binds to Hepatitis C Virus Internal Ribosome Entry Site, Prevents Ribosomal Assembly, and Inhibits Internal Initiation of Translation
Authors: Pudi Renuka , Ramamurthy Sudhamani S. , Das Saumitra
Published: July 2005

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.
Characterization of Immune Responses Induced by Intramuscular Vaccination with DNA Vaccines Encoding Measles Virus Hemagglutinin and/or Fusion Proteins
Authors: Song Man Ki , Vindurampulle Christofer J. , Capozzo Alejandra V. E. , Ulmer Jeffrey , Polo John M. , Pasetti Marcela F. , Barry Eileen M. , Levine Myron M.
Published: July 2005

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone. To address this question, we generated Sindbis virus-based pSINCP DNA vaccines that encode either MV-H or MV-F alone or bicistronic or fusion system vectors that encode both MV-H and MV-F (to mimic MV infection where both MV-H and MV-F proteins are expressed by the same mammalian cell). Mice immunized with DNA vaccine encoding MV-H alone developed significantly greater PRN titers than mice immunized with bicistronic constructs. Interestingly, the presence of MV-F in the bicistronic constructs stimulated serum MV-specific immunoglobulin G of reduced avidity. By contrast, mice immunized with bicistronic constructs induced equivalent or higher levels of MV-specific gamma interferon responses than mice immunized with DNA vaccine encoding MV-H alone. These data will help guide the design of DNA-based MV vaccines to be used early in life in a heterologous prime-boost strategy.