The multi-faceted dynamics of HIV-1 transmission in Northern Alberta: A combined analysis of virus genetic and public health data.

Molecular epidemiology has become a key tool for tracking infectious disease epidemics. Here, the spread of the most prevalent HIV-1 subtypes in Northern Alberta, Canada, was characterized with a Bayesian phylogenetic approach using 1146 HIV-1 pol sequences collected between 2007 and 2013 for routine clinical management purposes. Available patient metadata were qualitatively interpreted and correlated with onwards transmission using Fisher exact tests and logistic regression. Most infections were from subtypes A (n=36), B (n=815) and C (n=211). Africa is the dominant origin location for subtypes A and C while the subtype B epidemic was seeded from the USA and Middle America and, from the early 1990s onwards, mostly by interprovincial spread. Subtypes A (77.8%) and C (74.0%) were usually heterosexually transmitted and circulate predominantly among Blacks (61.1% and 85% respectively). Subtype B was mostly found among Caucasians (48.6%) and First Nations (36.8%), and its modes of transmission were stratified by ethnic origin. Compared to subtypes A (5.6%) and C (3.8-10.0%), a larger portion of subtype B patients were found within putative provincial transmission networks (20.3-29.5%), and this almost doubled when focusing on nationwide transmission clusters (37.9-57.5%). No clear association between cluster membership and particular patient characteristics was found. This study reveals complex and multi-faceted transmission dynamics of the HIV-1 epidemic in this otherwise low HIV prevalence population in Northern Alberta, Canada. These findings can aid public health planning.

An electroluminescence device for printable electronics using coprecipitated ZnS:Mn nanocrystal ink.

Electroluminescence (EL) devices for printable electronics using coprecipitated ZnS:Mn nanocrystal (NC) ink are demonstrated. The EL properties of these devices were investigated along with the structural and optical properties of ZnS:Mn NCs with an emphasis on their dependence on crystal size. Transmission electron microscopy and x-ray diffraction studies revealed that the NCs, with a crystal size of 3-4 nm, are nearly monodisperse; the crystal size can be controlled by the Zn(2+) concentration in the starting solution for coprecipitation. The results of optical studies indicate the presence of quantum confinement effects; in addition, the NC surfaces are well passivated, regardless of the crystal size. Finally, an increase in the luminance of EL devices with a decrease in crystal size is observed, which suggests the excitation mechanism of ZnS:Mn NC EL devices.

Design of a single tube RT-PCR assay for the diagnosis of human infection with highly pathogenic influenza A(H5) viruses.

Concerns about emergence of a pandemic strain of influenza have been increasing. The strains of highly pathogenic influenza A(H5N1) currently circulating are considered among the most plausible candidates for giving rise to a pandemic strain. In this study the design and development of a RT-PCR assay specific for these highly pathogenic influenza A(H5) strains is presented. This is achieved in part by the design of a primer targeting the coding region for the protease cleavage site of the hemagglutinin, and another primer derived from a pan-hemagglutinin RT-PCR assay also presented in this study. It is shown that the HPAI A(H5) specific assay amplifies only the nucleic acids of highly pathogenic A(H5), with a high sensitivity.

A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses.

Mosquito-borne flaviviruses include several important agents of human disease and have provided striking examples of emerging infections. In this study we present the design and validation of a single tube RT-PCR assay using a pair of consensus primers for the detection of mosquito-borne flaviviruses. Sequencing of the amplicons permits the species identification. The assay was validated using RNA from the yellow fever virus vaccine strain and from representative strains of dengue viruses 1, 2, 3 and 4, West Nile virus, Kunjin virus (a clade of West Nile virus), and St. Louis encephalitis virus.

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay.

The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

Acute disseminated encephalomyelitis (ADEM) after allogeneic bone marrow transplantation for acute myeloid leukemia.

Acute disseminated encephalomyelitis (ADEM) is a rare inflammatory demyelinating disease of the central nervous system. We describe here a patient who developed ADEM after allogeneic bone marrow transplantation (BMT). A 48-year-old woman with acute myeloid leukemia (M2) underwent allogeneic BMT from her HLA-identical sister. Cyclosporin for prophylaxis of acute graft-versus-host disease (GVHD) was discontinued from day 15 because of its toxicity. She was relatively well after the resolution of cytomegalovirus reactivation and chronic GVHD. Nine months after BMT, she suddenly developed diplopia, dysarthria, and gait disturbance. Computed tomography of the brain at that time revealed no abnormal findings. Leukemia recurrence was not revealed. The neurological symptoms were very mild without further deterioration. Her clinical course was carefully watched without therapy. Two weeks after onset, fluid attenuated inversion recovery magnetic resonance imaging (MRI) revealed multifocal abnormal high-signal intensity mainly in the white matter of the cerebrum as well as in the cerebellum and brainstem. Cerebrospinal fluid examination showed no abnormal findings. No laboratory findings suggested the presence of infectious agents. The typical MRI findings and an acute monophasic clinical course of this patient led to a diagnosis of ADEM. Twelve weeks after onset, the symptoms had almost resolved. Follow-up MRI showed a substantial improvement of the previous lesions without any new lesions. The symptoms had completely resolved 5 months after onset. This is a rare case of ADEM developing after allogeneic BMT.

A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

[Case of fulminant mixed cryoglobulinemia developing after atrial myxoma resection].

A 63-year-old man, with a 13-year history of asymptomatic proteinuria, was diagnosed with left atrial myxoma at the onset of heart failure. After resection of the tumor by hypothermal surgery, the patient developed fever, renal failure and skin rash. The diagnosis was type II mixed cryoglobulinemia accompanied by an IgMlambda clone with high titers of rheumatoid factor activity and polyclonal IgG. Treatment with high doses of steroids and plasmapheresis was ineffective, and the patient died of colon necrosis due to thrombotic occlusion in the supra-mesenteric arteries. Although the patient had suffered from sporadic Raynaud's phenomenon and purpura of the lower extremities from the age of 60 years, cryoglobulinemia was not suspected before surgery because of the atrial myxoma. Thus, we suggest that it is important to perform laboratory tests for cryoproteins before hypothermal surgery.

Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells.

There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears promising. Before initiating a clinical study using an ex vivo gene-transduced autologous cell vaccine-based immunogene therapy for RCC in Japan, in 1992 we initially planned a Japanese version of a clinical protocol in collaboration with a US group. In 1993, the original protocol was refined. We performed five preclinical qualification studies using RCC nephrectomy specimens from patients in 1997, and the results showed that preparation of RCC cells for autologous vaccines at the Clinical Cell Technology Facility, Research Hospital of the Institute of Medical Science, University of Tokyo, was feasible. Subsequently in August 1998, the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sport approved our clinical protocol. We have recruited two patients with stage IV RCC to our study so far. Here we report the background to the initiation of cancer gene therapy in Japan.

Clinical, serological and genetic study in patients with CREST syndrome.

OBJECTIVE: To assess the clinical, serological and genetic features of Japanese patients with CREST syndrome. PATIENTS AND METHODS: Clinical features, autoantibodies and human histocompatibility leukocyte antigen (HLA) typing were studied in thirty patients with CREST syndrome, including 29 females and one male, with a mean age of 59.0 years (ranging from 40 to 76 years). RESULTS: Interstitial pneumonia on chest X-ray and renal involvement were rare. Mitral regurgitation and tricuspid regurgitation were present in 56.7% and 76.7%, respectively. Sjören's syndrome (SS) and primary biliary cirrhosis (PBC) were highly associated, however the positivity of the marker antibodies to those syndromes, such as anti-SSA, anti-SSB, anti-mitochondrial (AMA) and anti-smooth muscle autoantibodies were less frequent than that of primary SS and PBC without the other autoimmune diseases. The histological findings of PBC were all early stages in Scheuer's classification. HLA-Cw6 were associated with CREST-PBC overlap syndrome (p<0.05). However the HLA antigen was not correlated with CREST syndrome, and the frequency of HLA-DR2 between CREST syndrome with or without PBC was significantly different (p<0.01). CONCLUSION: It was suggested that there was a genetic difference between CREST syndrome alone and CREST-PBC overlap syndrome and there were differences (the positivity of AMA and the severity of bile duct lesion) between PBC and CREST-PBC overlap syndrome.

p53-independent tumor growth and in vitro cell survival for F-MuLV-induced erythroleukemias.

Retroviral insertional activation of the Fli-1 proto-oncogene is the first genetic event associated with the induction of erythroleukemias by the Friend murine leukemia virus (F-MuLV). Mutations within p53, which are only detected in cell lines established from transplanted tumors, have been previously shown to be associated with the immortalization of erythroleukemic cells in culture. In this study, we have demonstrated that primary erythroleukemic cells grown in liquid culture undergo rapid apoptosis independent of the stabilization of wild-type p53 protein. Further confirmation that the programmed cell death observed for liquid-cultured F-MuLV-induced primary erythroleukemic cells is largely p53 independent was provided by experimentation with a transgenic mouse line containing multiple copies of the dominant negative mutant p53Pro-193 allele. Erythroleukemic cells taken from tumor-bearing transgenic mice expressing high levels of the mutant p53Pro-193 undergo programmed cell death in culture in a manner that is largely identical to that observed for tumor cells derived from nontransgenic littermates. Furthermore, the rate of development of F-MuLV-induced erythroleukemias for both p53Pro-193-transgenic and nontransgenic littermates are similar. Moreover, cytogenetic analysis indicates that primary erythroleukemia cells are diploid, whereas chromosomal aberrations were observed in all established cell lines. These results are consistent with the notion that mutations within the p53 tumor suppressor gene affect genomic stability, subsequently leading to changes in gene expression that are associated with the immortalization of erythroid progenitor cells.

Multiple features of advanced melanoma recapitulated in tumorigenic variants of early stage (radial growth phase) human melanoma cell lines: evidence for a dominant phenotype.

The vast majority of primary human cutaneous melanomas undergo a slow and gradual progression from a clinically indolent, curable radial growth phase (RGP) to a malignant vertical growth phase. We sought to develop a way of isolating genetically related malignant variants from a benign RGP human melanoma, called WM35. The parent and variants were then used as a model system to examine to what extent the expression of clinically and biologically relevant phenotypic features characteristic of advanced melanomas are associated with (and thus perhaps causative of) such a malignant conversion. Such a model system could also be used as a means of eventually identifying genetic alterations and cellular changes involved in the malignant switch in melanoma progression. To develop such a model, we subjected WM35 cells to retroviral insertional mutagenesis, which was followed by selection for progressive growth of solid tumors in nude mice. Highly aggressive and phenotypically stable tumorigenic variants were derived which contained at least four integrated proviruses. In contrast to the parental WM35 cells, these cell lines expressed several phenotypic features characteristic of naturally derived, advanced-stage malignant melanoma cells. Thus, in addition to tumor-forming ability in nude mice, the variants were growth factor and anchorage independent, overexpressed the MUC18 adhesion molecule, and lost responsiveness to the growth-inhibitory effect of several cytokines, including interleukin 6, transforming growth factor beta, interleukin 1beta, and tumor necrosis factor-alpha. Tumorigenicity and "multicytokine resistance" were dominant traits since in somatic cell hybrids between the parental cells and a tumorigenic subline no suppressive effect of the former cell population was observed. These findings suggest that one or more dominantly acting genetic alterations might be involved in this progression of RGP melanoma cells. The identity of such alterations remains to be determined.

Retroviral insertional mutagenesis as a strategy for the identification of genes associated with cis-diamminedichloroplatinum(II) resistance.

Expression of resistance to cis-diamminedichloroplatinum(II) (CDDP), one of the most effective chemotherapeutic drugs used to treat a variety of malignancies, remains a serious obstacle for improving cancer treatment. To study possible genetic mechanisms underlying the development of CDDP resistance, we have adopted the approach of retroviral insertional mutagenesis. An early-stage CDDP-sensitive human melanoma cell line, WM35, was infected with a defective amphotropic murine retrovirus (murine stem cell virus), and the pooled cells were subsequently selected for CDDP-resistant variants. Nine CDDP-resistant clones independently derived from murine stem cell virus-infected WM35 cells were analyzed and it was found that five of these clones acquired an identical retroviral integration site, designated as CDDP resistance locus 1 (CRL-1), as revealed by isolation of retroviral flanking sequences. Furthermore, using the flanking sequence as probe, we have detected a 3.5-4.0-kilobase message, the expression of which is strongly increased in clones carrying a rearranged CRL-1 locus. These results strongly suggest that overexpression of CRL-1 confers resistance to CDDP in these clones. In addition, the present study indicates that retroviral insertional mutagenesis represents a potential strategy to identify genes responsible for CDDP resistance and possibly other chemotherapeutic drugs as well.

Neurotensin binding to dopamine.

Rotating disk electrode voltammetry at glassy carbon electrodes and ultraviolet/visible spectroscopy were used to demonstrate that dopamine binds to neurotensin with a dissociation constant of 7.5 x 10(-8). By measuring the binding constants of various neurotensin analogs, it was found that the -Arg8-Arg9-portion of the neurotensin sequence is critical for binding dopamine. Neurotensin also formed a complex with 4-ethylcatechol, 4-methylcatechol, 3-methoxytyramine, and norepinephrine. Although changes in the side chain did not alter the binding constant, methoxylation of the catechol moiety significantly increased the dissociation constant. These data along with additional studies of dopamine interactions with arginine derivatives suggest that the guanidino groups of arginine and the catechol hydroxyls of dopamine are responsible for mediating the observed binding. It is hypothesized that the capacity of neurotensin to bind directly to dopamine may be partly responsible for its previously observed antagonism of dopamine-induced locomotor activity.