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Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1). more...

The planning and execution of avalanche rescue missions to search for totally buried avalanche victims are mostly based on personal experience and preference, as evidence-based information from literature is almost completely missing. Hence, the aim of this study was to identify major factors determining the survival probability of totally buried victims during avalanche rescue missions carried out by organized rescue teams (Austrian Mountain Rescue Service, Tyrol). During the 12-year period studied, 109 totally buried persons (56 off-piste, 53 backcountry), were rescued or recovered; 18.3% survived to hospital discharge. Median depth of burial was 1.25 m; median duration of burial was 85 min. The majority (61.6%) of the rescue missions were conducted under considerably dangerous avalanche conditions. The probability of survival was highest when located visually and lowest for those located by avalanche transceiver; survival did not significantly differ between those found by rescue dogs and those located with avalanche probes. Multivariate analysis revealed short duration of burial and off-piste terrain to be the two independent predictors of survival. Whenever companion rescue fails, snow burial in an avalanche is associated with extraordinarily high mortality. Searching the avalanche debris with probe lines seems to be equally effective as compared to searching with rescue dogs. The potential hazard for rescuers during avalanche rescue missions comes mainly from self-triggered avalanches, hence thorough mission planning and critical risk-benefit assessment are of utmost importance for risk reduction. more...

Purpose: To investigate the membrane physiology of Müller glial cells from retinae of DBA/2J mice (which develop ocular hypertension) and of C57BL/6 control mice of different ages., Methods: Retinae were obtained at the ages of 3, 6, and 12 months from DBA/2J mice and from C57BL/6 controls. Immunohistochemistry was performed using antibodies against glial fibrillary acidic protein (GFAP). Whole-cell membrane currents, membrane potentials and capacitances were recorded from freshly isolated Müller cells., Results: Strong immunostaining for GFAP was found in Müller cells of 12-month-old DBA/2J mice, whereas only astrocytes were immunopositive in C57BL/6 retinae. No significant alterations of membrane currents or potentials of Müller cells from DBA/2J mice as compared to controls were observed at any age; however, the membrane capacitance was increased in Müller cells from DBA/2J mice at the age of 6 months., Conclusions: Although Müller cells of DBA/2J mice display some symptoms of reactive gliosis, the lack of significant alterations of the membrane physiology confirm previous data demonstrating that these cells undergo a nonproliferative gliosis. more...

The promise of cancer immunotherapy is long-term disease control with high specificity and low toxicity. However, many cancers fail immune interventions, and secretion of immunosuppressive factors, defective antigen presentation, and expression of death ligands or serpins are regarded as main escape mechanisms. Here, we study whether deregulation of growth and survival factor signaling, which is encountered in most human cancers, provides another level of protection against immunologic tumor eradication. We show in two models that activated cell autonomous protein kinase B (PKB)/AKT signaling mediates resistance against tumor suppression by antigen-specific CTLs in vitro and adoptively transferred cellular immune effectors in vivo. PKB/AKT-dependent immunoresistance of established tumors is reversed by genetic suppression of endogenous Mcl-1, an antiapoptotic member of the Bcl-2 family. Mechanistically, deregulated PKB/AKT stabilizes Mcl-1 expression in a mammalian target of rapamycin (mTOR)-dependent pathway. Treatment with the mTOR inhibitor rapamycin effectively sensitizes established cancers to adoptive immunotherapy in vivo. In conclusion, cancer cell-intrinsic PKB/AKT signaling regulates the susceptibility to immune-mediated cytotoxicity. Combined targeting of signal transduction pathways may be critical for improvement of cancer immunotherapies. more...

Purpose: To investigate the intraocular effect of rhodamine 6G (R6G) on retinal structures and function in an in vivo rat model and to develop an in vivo method for accurate evaluation of new dyes for intraocular surgery., Methods: R6G in physiologic saline solution (PSS) was injected into the vitreous of adult Brown Norway rats at concentrations of 0.0002%, 0.002%, 0.02%, 0.2%, and 0.5%. Control animals received only PSS. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts, light microscopy 7 days later, photopic electroretinography (ERG), and measurement of scotopic sensitivity and recovery of dark adaptation 48 hours and 7 days after intravitreous injection., Results: R6G at concentrations of 0.2% and 0.5% led to a dose-dependent loss of RGC. The most significant loss occurred at 0.5%. Lower concentrations (0.0002%, 0.002%, and 0.02%) produced no statistically significant retinal ganglion cell loss. Analysis of the eyes by light microscopy showed no structural changes in the central retina, although injections of 0.5% R6G were followed by impressive degenerative changes adjacent to the injection sites. ERGs showed no effects of the highest R6G concentration on rods, kinetics of rhodopsin recovery after bleaching, or cone-driven responses., Conclusions: R6G can be safely injected in doses of up to 0.02% in rats, but has a toxic effect on retinal ganglion cells at higher concentrations. Accumulation of R6G may be a problem at higher concentrations, particularly at the injection site. more...

Adoptive cellular immunotherapy inducing a graft-versus-tumor (GVT) effect is the therapeutic mainstay of allogeneic hematopoietic stem cell transplantation (ASCT) for high-risk leukemias. Autologous immunotherapies using vaccines or adoptive transfer of ex vivo-manipulated lymphocytes are clinically explored in patients with various cancer entities. Main reason for failure of ASCT and cancer immunotherapy is progression of the underlying malignancy, which is more prevalent in patients with advanced disease. Elucidating the molecular mechanisms contributing to immune escape will help to develop strategies for the improvement of immunologic cancer treatment. To this end, we have undertaken functional screening and expression cloning of factors mediating resistance to antigen-specific cytotoxic T lymphocytes (CTLs). We have identified Cdc42, a GTPase regulating actin dynamics and growth factor signaling that is highly expressed in invasive cancers, as determinator of cancer cell susceptibility to antigen-specific CTLs in vitro and adoptively transferred immune effectors in vivo. Cdc42 prevents CTL-induced apoptosis via mitogen-activated protein kinase (MAPK) signaling and posttranscriptional stabilization of Bcl-2. Pharmacologic inhibition of MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) overcomes Cdc42-mediated immunoresistance and activation of Bcl-2 in vivo. In conclusion, Cdc42 signaling contributes to immune escape of cancer. Targeting Cdc42 may improve the efficacy of cancer immunotherapies. more...

Background: The DBA/2J mouse spontaneously develops ocular hypertension and time-dependent progressive retinal ganglion cell (RGC) loss. This study examines changes in amino acid levels in the vitreous, and changes in the expression of retinal glutamate transporters and receptors that occur during the progression of this pathology., Methods: Retinas were obtained from DBA/2J mice at ages 3, 6 and 11 months. C57BL/6 mice were used as age-matched controls. Vitreal amino acid content was measured with HPLC. Western blotting and immunohistochemistry were performed using specific antibodies against the glutamate transporters (GLAST, GLT-1v, EAAC-1) and glutamate receptors, particularly NMDA (NR1, NR2A, NR2B) and AMPA (GluR1, GluR2/3, GluR4) receptors., Results: HPLC showed retinal concentrations of glutamate, glutamine, glycine, alanine, lysine, serine, and arginine to be significantly higher in DBA/2J mice at 11 months of age compared to age-matched controls. Western Blots revealed a moderate decrease of GLAST and GLT-1v expression in DBA/2J mice at 6 and 11 months as compared to age-matched controls while there was no change in EAAC1. Immunohistochemically, no changes in expression of NMDA and AMPA receptors were seen., Conclusion: Alterations of amino acid content and enhanced glutamate neurotransmission might be involved in the pathogenesis of retinal neurodegeneration in the DBA/ 2J mouse model of ocular hypertension. Moreover, these mice provide an animal model for studying excitotoxic retinal damage. more...

An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity. more...

The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection. more...

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression. more...

Purpose: To investigate the effect of intravitreal injections of new vital dyes on the retina, the retinal pigment epithelium (RPE) and the choroid in an in vivo rat model., Methods: Rats were injected intravitreally with four dyes: light-green SF yellowish (LGSF), copper(II)phthalocyanine-tetrasulfonic acid (E68), bromphenol blue (BPB), and Chicago blue (CB) dissolved in physiologic saline solution (PSS) at concentrations of 0.5% and 0.02%. PSS served as the control. Additional animals were treated with single injections of 0.5%, 0.02%, 0.002%, and 0.0002% ICG or 0.002% E68 into one eye. Adverse effects on anterior and posterior segments were evaluated by slit lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by histology and retinal ganglion cell (RGC) quantification 7 days after dye administration., Results: Eyes treated with 0.5% E68, 0.5% ICG, or 0.5% CB showed discrete staining of both cornea and lens not seen at lower concentrations or with other dyes. Histology revealed dose-dependent reactions after E68 administration. ICG 0.5% induced significant thinning of inner retinal layers compared with PSS. ICG 0.02% caused focal degenerative changes of the outer retina in three of seven eyes, whereas 0.002% and 0.0002% ICG did not. CB led to heterogeneous morphologic alterations. BPB- or LGSF-treated eyes showed normal retinal morphology. ICG at all tested concentrations induced significant RGC loss, as did E68 at 0.5% but not at lower concentrations., Conclusions: BPB or LGSF produced no significantly detectable toxic effects on the retina in vivo. The safety of these new dyes must be established in other models and/or in preclinical studies before the clinical use of any of these dyes. more...

Purpose: Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN., Methods: Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC)., Results: After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration., Conclusions: This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers. more...

Background: Murine leukemia virus (MLV) vector particles can be pseudotyped with a truncated variant of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) and selectively target gene transfer to human cells expressing both CD4 and an appropriate co-receptor. Vector transduction mimics the HIV-1 entry process and is therefore a safe tool to study HIV-1 entry., Results: Using FLY cells, which express the MLV gag and pol genes, we generated stable producer cell lines that express the HIV-1 envelope gene and a retroviral vector genome encoding the green fluorescent protein (GFP). The BH10 or 89.6 P HIV-1 Env was expressed from a bicistronic vector which allowed the rapid selection of stable cell lines. A codon-usage-optimized synthetic env gene permitted high, Rev-independent Env expression. Vectors generated by these producer cells displayed different sensitivity to entry inhibitors., Conclusion: These data illustrate that MLV/HIV-1 vectors are a valuable screening system for entry inhibitors or neutralizing antisera generated by vaccines. more...

Purpose: To investigate the effect of intraocular erucylphosphocholine (ErPC) on the retina, the retinal pigment epithelium (RPE), and the choroid in an in vivo rat model., Methods: Adult male Brown Norway rats were injected intravitreally with ErPC dissolved in balanced salt solution (BSS) at a final concentration of 10 or 100 microM with BSS serving as control. Adverse effects on the anterior and posterior segment were assessed by slit-lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by electroretinography (ERG), retinal ganglion cell (RGC) quantification, and histology 7 days after intravitreal administration of ErPC., Results: There was neither a statistically significant difference in the clinical examination nor in the ERG waves of treated versus control rats 7 days after intravitreal administration of ErPC. Correspondingly, the number of RGC after BSS injection did not differ significantly from ErPC-injected animals. Histologic sections of the posterior segment of 10 and 100 microM ErPC-injected rats did not show any signs of retinal toxicity. Electron microscopy did not display a difference between the 10 microM and the control group. Only the 100 microM-injected animals showed a discrete irregularity of the Müller cell and the retinal ganglion cell cytoplasm at the ultrastructural level., Conclusions: ErPC can safely be injected into the vitreous of adult rats at a concentration of 10 microM without any retinal toxicity. Even a 10-fold increase in ErPC concentration leads only to a discrete cytoplasmic irregularity of the innermost retinal layers. more...

Mycosis fungoides (MF) is the most frequent variant of cutaneous T-cell lymphomas (CTCLs). MF primarily involves the skin initially with patches and plaques. In later stages, cutaneous tumors develop and tumor cells may spread to lymph nodes and finally to visceral sites. Here, we describe an animal model for MF in immune-deficient nude mice, using the CTCL cell line MyLa. Subcutaneous transplantation of MyLa cells leads to the formation of cutaneous tumors in 80% of the mice (50/60 total). Spread of tumor cells to visceral sites was detected by immunohistochemistry and polymerase chain reaction (PCR)-based detection of specific T-cell receptor-gamma rearrangement. MyLa cells were found circulating in the blood, lymph nodes, and in blood vessels of heart, kidney, lung, and liver. In lung and liver tissue, tumor cells presented perivascular invasion, but no large secondary tumors developed. The nude mouse model described here will be a valuable test system for new therapeutic approaches for the treatment of MF and opens the unique opportunity to study the disease in vivo. more...

Cutaneous T cell lymphomas (CTCLs) are lymphoproliferative disorders involving the skin. Malignant cells have a CD4+ T-helper phenotype and are found in early stages of the disease in plaques and cutaneous tumors. MLV/HIV-pseudotyped retroviral vectors target gene transfer to CD4-positive T cells and are therefore well suited to be specific delivery vehicles to treat CTCLs. We established a mouse xenograft model for CTCL and generated MLV/HIV-pseudotyped vectors encoding the herpes simplex virus thymidine kinase (HSV-TK), a well-known suicide gene, to prove the efficacy of MLV/HIV vectors in CTCL treatment. Vector particles were intratumorally injected into CTCL nude mouse xenografts. Mice were systemically treated with ganciclovir (GCV) and the tumor tissue was analyzed. A significant delay in tumor growth was observed for HSV-TK-transduced and GCV-treated tumors. GFP expression could be detected exclusively in CD4+ cells of tumors after transduction with GFP-encoding control vectors. The data demonstrate a cell-specific in vivo gene delivery via MLV/HIV-pseudotyped vectors and open new avenues for the treatment of CTCL in humans. more...

Murine leukemia virus (MuLV) can be pseudotyped with a variant of the human immunodeficency virus (HIV) envelope gene encoding the surface glycoprotein gp120-SU and a carboxy-terminally truncated transmembrane (TM) protein with only seven cytoplasmic amino acids. MuLV/HIV-1 pseudotyped retroviral vectors selectively target gene transfer to human cells expressing both CD4 and CXCR4. To apply this vector system to gene therapy of human diseases, we generated a stable packaging cell line, FLY-HIV-87, expressing the MuLV gag and pol genes and the C-terminally truncated variant of the HIV-1 envelope gene, but no retroviral vector genome. Production of infectious vector particles was tested after the introduction of different vector genomes and was in the range of 5x10(5) IU/ml. The vector particles could be concentrated up to 25-fold. Specific and efficient gene transfer into CD4/CXCR4 expressing cell lines and stimulated primary human CD4+ peripheral blood lymphocytes was achieved. Thus the packaging cell line FLY-HIV-87 is highly suitable for gene therapy of disorders of human T-helper cells. more...

The Generalist Partners Program (GPP) provides all University of Wisconsin Medical School students with a comprehensive generalist core curriculum through early clinical experiences, interactive faculty presentations, and small-group discussions during the first two years of medical school. The GPP is the first component of a comprehensive, longitudinal generalist curriculum offered during all four years of medical education. Faculty members from family medicine, general internal medicine, and general pediatrics work together to develop the learning goals and provide the curriculum for the GPP courses. Each of the 145 entering first-year medical students is matched with a community-based physician, the generalist practice partner, and the students participate in early clinical experiences at this site throughout their first two years of medical education. Students' clinical experiences are presented and discussed in case-based small-group sessions led by the school's generalist faculty. A core lecture series on professionalism, communication skills, evidence-based medicine, and the organization of the health care system supplements the early clinical experiences. more...

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage. more...

The purpose of the present study was to evaluate the potential of polycarbophil-cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35-50 microM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil-cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4. 17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p < 0.05) improved. As the zinc binding affinity of polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21. 36), the inhibitory effect of the polymer-cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p < 0.05) higher than that of the unmodified polymer. Because of these inhibitory features, polycarbophil-cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine. more...

Care of central venous catheter (CVC) in patients undergoing bone marrow transplantation (BMT) raises significant problems related to the high risk of local infections, to the immunodeficient status, which in itself is a predisposing factor for systematic blood stream infections. Although frequent changes of CVC dressing might theoretically reduce the incidence of infections, they are also accompanied by significant skin toxicity and patient discomfort. No study has yet addressed these points. The objective of this study was to compare two different time interval protocols for CVC dressing, in order to assess the effects on local infections and toxicity. In a multicentre study, 339 bone marrow transplant (BMT) patients with a tunnelled CVC (group A, 230 pts) or a non tunnelled one (Group B, 169 patients) were randomly allocated to receive CVC dressing changes every 5 or 10 days if belonging to group A or 2 or 5 days if in group B. Transparent impermeable polyurethane dressings were used for all patients. The rate of local infection at the site of CVC insertion was assessed by microbiological assay every 10 days, while severity of skin toxicity was measured according to the ECOG scale. Sixty-five per cent of enrolled patients were finally evaluable. Patients (in both groups) receiving CVC dressing changes at longer intervals did not show a significant increase in the rate of local infections, while those who received dressing every two days had a significant increase in local skin toxicity. Longer intervals were accompanied by a reduction in costs. The results of this study demonstrate that the increase in time interval between CVC dressing changes in BMT patients did not increment the risk of local infections, while significantly reducing patients discomfort and costs. more...

Background and Objective: Care of central venous catheter (CVC) in patients undergoing bone marrow transplantation (BMT) raises significant problems related to the high risk of local infections due to the immunodeficient status, which in itself is a predisposing factor for systemic blood-stream infections. Although frequent changes of CVC dressing might theoretically reduce the incidence of infections, they are also accompanied by significant skin toxicity and patient discomfort. No study has yet addressed these points. The objective of this study was to compare two different time interval protocols for CVC dressing in order to assess the effects on local infections and toxicity., Design and Methods: In a multicenter study, 399 bone marrow transplant (BMT) patients with a tunneled CVC (Group A, 230 pts) or a non-tunneled one (Group B, 169 pts) were randomly allocated to receive CVC dressing changes every 5 or 10 days, if belonging to Group A, or 2 or 5 days, if in Group B. Transparent, impermeable polyurethane dressings were used for all patients. The rate of local infections at the site of CVC insertion was assessed by microbiological assays every 10 days, while the severity of skin toxicity was measured according to the ECOG scale., Results: Sixty-five per cent of enrolled patients were finally evaluable. Patients (in both Groups) receiving CVC dressing changes at longer intervals did not show a significant increase in the rate of local infections, while those who received dressing every 2 days had a significant increase in local skin toxicity. Longer intervals were accompanied by a reduction in costs., Interpretation and Conclusions: The results of this study demonstrate that the increase in time interval between CVC dressing changes in BMT patients did not raise the risk of local infections, while significantly reducing patient discomfort and costs. more...

The incorporation of human immunodeficiency virus-type-2 (HIV-2) envelope glycoprotein into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We observed that wild-type HIV-2 envelope protein or a frameshift mutant with 187 unrelated carboxyl-terminal residues did not allow the formation of infectious retroviral particles. In view of recent findings that an HIV-1 envelope protein variant with a shortened cytoplasmic domain was incorporated into MuLV particles, we constructed carboxyl-terminal truncations of the HIV-2 envelope protein. An envelope variant with 18 cytoplasmic amino acids formed only very few viral pseudotypes. The further removal of an additional 11 amino acids allowed the efficient pseudotyping of MuLV particles. As with the HIV-1 envelope protein, an HIV-2 envelope variant with 7 cytoplasmic amino acids was incorporated into functional MuLV particles. The pseudotyped vectors obtained are able to infect human CD4/CXCR4-expressing cells. Cell lines expressing human CD4 and other coreceptors could not be infected. This retroviral vector will prove useful for the study of HIV infection events mediated by the HIV-2 envelope glycoproteins, as well as for the targeting of CD4+ cells in the context of gene therapy of AIDS., (Copyright 1999 Academic Press.) more...

A ovolactovegetarian patient presented with hyponatremia. She had maximally dilute urine and undetectable vasopressin levels. Dietary history revealed very low protein intake but no beer intake. We postulated that the very low intake of solute limited her water excretion and caused the hyponatremia despite only a modest increase in fluid intake. When protein intake was increased in a clinical research center setting, free water excretion increased and serum sodium normalized despite maintaining the water intake at 4 to 5 L daily. We discuss the role of dietary solute in water excretion. Previously described in beer drinkers, the phenomenon can occur in the absence of beer drinking. In this era of weight consciousness, hyponatremia because of low solute intake may be seen with increased frequency. more...

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production. more...

Background: Eastern equine encephalitis occurs principally along the east and Gulf coasts of the United States. Recognition of the neuroradiographic manifestations of eastern equine encephalitis could hasten the diagnosis of the illness and speed the response to index cases., Methods: We reviewed all cases of eastern equine encephalitis reported in the United States between 1988 and 1994. The records of 36 patients were studied, along with 57 computed tomographic (CT) scans and 23 magnetic resonance imaging (MRI) scans from 33 patients., Results: The mortality rate was 36 percent, and 35 percent of the survivors were moderately or severely disabled. Neuroradiographic abnormalities were common and best visualized by MRI. Among the patients for whom MRI scans were available, the results were abnormal for all eight comatose patients as well as for all three noncomatose patients who subsequently became comatose. The CT results were abnormal in 21 of 32 patients with readable scans. The abnormal findings included focal lesions in the basal ganglia (found in 71 percent of patients on MRI, and in 56 percent on CT), thalami (found in 71 percent on MRI and in 25 percent on CT), and brain stem (found in 43 percent on MRI and in 9 percent on CT). Cortical lesions, meningeal enhancement, and periventricular white-matter changes were less common. The presence of large radiographic lesions did not predict a poor outcome, but either high cerebrospinal fluid white-cell counts or severe hyponatremia did., Conclusions: Eastern equine encephalitis produces focal radiographic signs. The characteristic early involvement of the basal ganglia and thalami distinguish this illness from herpes simplex encephalitis. MRI is a sensitive technique to identify the characteristic early radiographic manifestations of this viral encephalitis. more...

This report presents a case of squamous cell carcinoma of the temporal bone induced by high dose radiation therapy. Several important surgical techniques are stressed which serve to make this procedure far safer in the avoidance of catastrophic hemorrhage. more...

Decreases in arterial pH markedly increase sodium, chloride, and water absorption in the normal ileum and can reverse ongoing cholera toxin-induced secretion. In the current study we examined whether these effects of pH are evident in other models of ileal secretion, and in a model of increased absorption. Rats were anesthetized and transport was measured in ileal loops during respiratory acidosis and alkalosis. Decreases in arterial pH increased absorption equally in control loops and in adjacent loops perfused with a Ringer's solution containing ST toxin (cyclic guanosine monophosphate-mediated secretion), hypertonic mannitol (passive, osmotically mediated secretion), or glucose. Decreases in arterial pH increased absorption in a similar way in loops exposed to cholera toxin (cyclic adenosine monophosphate-mediated secretion) that were then perfused with glucose-Ringer's solution. Alterations in arterial and luminal pH did not affect glucose absorption. These results suggest that the effect of arterial pH on ileal absorption occurs by a mechanism that is independent of these various means of altering transport. more...

A 74-year-old man complained of anorexia and weight loss. Twenty-six years earlier he had received an injection of Thorotrast. A needle biopsy of the liver showed thorium dioxide granules and periportal fibrosis. On laparotomy, a hepatoma of the left lobe of the liver was discovered. Hepatic malignancy should be suspected in any patient with abnormal results of liver function tests, particularly an elevated level of alkaline phosphatase, who previously has had an injection of Thorotrast. more...