Disseminated cryptococcal infection in allogeneic stem cell transplant patients: a rare cause of acute kidney injury.

Hematopoietic stem cell transplantation (HSCT) can be lifesaving for some of the deadliest hematologic diseases. However, immunosuppression, polypharmacy and risk of infectious complications associated with HSCT can increase morbidity and mortality for recipients. Incidence of acute kidney injury (AKI) after HSCT can be as high as 70%, and concomitant infection can be a therapeutic challenge for oncologists, nephrologists and infectious disease specialists. We illustrate this challenge in the case of a 31-year-old man with acute lymphoblastic leukemia who underwent a double cord HSCT complicated by GvHD, systemic cryptococcal and BK virus infections and AKI. Kidney biopsy showed round to cup-shaped organisms with occasional budding, consistent with Cryptococcus and thrombotic microangiopathy. We discuss our findings and a literature review of disseminated cryptococcal infection with renal involvement after HSCT.

Small molecule and biologic modulators of the immune response to hepatitis C virus.

Hepatitis C virus represents a major global health problem, with approximately 3% of the world population infected. Immune-response modifiers represent the standard of care, given the lack of approved antiviral agents having direct activity against the viral proteins. Although in recent years, improvements in therapy have been attained by combined treatment with pegylated interferon and ribavirin, the discovery and development of next-generation small molecule and biologic agents is ongoing. Several of these newer therapeutics are focused on modulating Toll-like receptors, interferon-alpha signaling, and the pro-inflammatory cytokine balance. A comprehensive account of the lead compounds in development, the bioassays used for optimization of these immune response modifiers and their clinical status is presented.

Transforming growth factor beta regulates cyclooxygenase-2 in glomerular mesangial cells.

This study examines the hypothesis that transforming growth factor beta (TGFbeta) regulates cyclooxygenase-2 (COX-2) and induces prostaglandin E synthase (mPGES-1) in rat mesangial cells. COX-2 expression was determined by Northern blot analysis after treatment with either TGFbeta1 or the selective COX-2 inhibitor, NS398. mPGES-1 expression was determined by real-time polymerase chain reaction. The effect of TGFbeta1 on COX-2 gene transcription was assessed using a luciferase reporter assay, and mRNA stability was also determined. To determine whether TGFbeta1 activates elements of the COX-2 promoter, we performed gel shift analyses to examine activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB). Prostaglandin E(2) (PGE(2)) and thromboxane B2 (TxB2) production was assayed by enzyme immunoassay. Finally, the pathophysiological relevance of COX-2 inhibition on the downstream effects of TGFbeta was assessed by examining collagen type I mRNA and net collagen production. COX-2 mRNA and mPGES-1 were induced after treatment with TGFbeta1 for 4 h, and this rise was accompanied by a three-fold increase in PGE(2) production that could be antagonized by selective inhibition of COX-2 with NS398. TGFbeta1 increased transcription by approximately 50% and activated both AP-1 and NF-kappaB. These effects were antagonized by co-treatment with NS398. Treatment with TGFbeta1 also doubled the half-life of COX-2 mRNA. Neither collagen type I mRNA nor net collagen production were altered by co-treatment with NS398. In conclusion, these results indicate that TGFbeta stimulates COX-2 and mPGES-1, with additional effects on transcription and stability of COX-2 mRNA.

Synthesis and structural analysis of the anilides of glucuronic acid and orientation of the groups on the carbohydrate scaffolding.

[structures: see text] The synthesis of anilides derived from glucuronic acid is described. Secondary anilides had a Z configuration in the solid state and showed intramolecular and intermolecular hydrogen bonding. However, on the basis of NMR and IR studies, there was generally no evidence for the same hydrogen bonding in solution. Tertiary anilides showed a strong preference for the E configuration on the basis of NOE studies and molecular mechanics calculations. The alkylation of the secondary anilides induces a configurational switch that alters the orientation of the aromatic group with respect to the pyranose, which has relevance for presentation or orientation of pharmacophoric groups on carbohydrate scaffolds.

Prostate tumor microenvironment alters immune cells and prevents long-term survival in an orthotopic mouse model following flt3-ligand/CD40-ligand immunotherapy.

A novel orthotopic metastatic model of mouse prostate cancer was developed using MHC-negative TRAMP-C1P3 (transgenic adenocarcinoma of mouse prostate) cells derived by serial passage of the parental TRAMP-C1 line in mouse prostate glands. TRAMP-C1P3 cells grew efficiently in mouse prostate glands and reproducibly metastasized to draining lymph nodes. Using this model, we show that Fms-like tyrosine kinase-3 ligand (flt3-L) dramatically inhibited growth of preexisting orthotopic TRAMP-C1P3 tumors and the development of metastatic disease. Mice remained in remission for several months following termination of flt3-L treatment but eventually relapsed and died of progressive disease. flt3-ligand treatment induced a pronounced mixed inflammatory cell infiltrate that consisted of CD8alpha-CD4- dendritic cells (CD11c+), macrophages, granulocytes (Gr-1+) and to a lesser extent T cells (CD4+ and CD8+). Dendritic cells isolated from TRAMP-C1P3 tumors were phenotypically immature (CD11c+ B7.2-I-A-CD40-), and this phenotype was also predominant in peripheral organs of mice treated with flt3-L alone or in combination with the DC maturation factor, CD40-L. Diminished expression of TCR-beta, CD3-epsilon, and CD3-zeta was also observed on intratumoral T cells, although these signaling proteins were reexpressed following in vitro culture with IL-2. The TCR/CD3 complex remained intact on peripheral T cells except in mice treated with flt3-L where CD3-zeta loss was observed. In contrast to alphabeta-T cells, tumor-infiltrating gammadelta-T cells maintained expression of their antigen receptors but not CD3epsilon. Thus, TRAMP-C1P3 tumors quickly establish a microenvironment that profoundly diminishes expression of molecules critical for normal dendritic cell and T cell function, thus limiting the efficacy of flt3-L and CD40-L immunotherapy. Overall, these data suggest that long-term cures of established MHC-negative tumors may not be achieved until therapeutic interventions are engineered to overcome this immunosuppressive microenvironment.

Development of carbohydrate-based scaffolds for restricted presentation of recognition groups. Extension to divalent ligands and implications for the structure of dimerized receptors.

The solution structure of glycosyl amides has been studied by using NMR. A strong preference is displayed by tertiary aromatic glycosyl amides for E-anti structures in contrast with secondary aromatic glycosyl amides where Z-anti structures predominate. The structural diversity displayed by these classes of molecules would seem to be important as the directional properties of the aromatic ring, or groups attached to the aromatic ring, would be determined by choosing to have either a secondary or tertiary amide at the anomeric center and could be considered when designing bioactive molecules with carbohydrate scaffolds. The structural analysis was also carried out for related divalent secondary and tertiary glycosyl amides and these compounds display preferences similar to that of the monovalent compounds. The constrained divalent compounds have potential for promoting formation of clusters that will have restricted structure and thus have potential for novel studies of mechanisms of action of multivalent ligands. Possible applications of such compounds would be as scaffolds for the design and synthesis of ligands that will facilitate protein-protein or other receptor-receptor interactions. The affinity of restricted divalent (or higher order) ligands, designed to bind to proteins that recognize carbohydrates which would facilitate clustering and concomitantly promote protein-protein interactions, may be significantly higher than monovalent counterparts or multivalent ligands without these properties. This may be useful as a new approach in the development of therapeutics based on carbohydrates.

First Report of Foliar Infection of Starflower by Phytophthora ramorum.

In March 2002, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from pacific or western starflower (Trientalis latifolia Hook.), an herbaceous perennial of the Primulaceae family, at Castro Canyon in Big Sur, Monterey County, California. Affected leaves had numerous necrotic lesions >5 mm in diameter surrounded by a yellow halo, and the lesions coalesced with time. Isolates were identified as P. ramorum by the large chlamydospores, caduceus, semipapillate sporangia, and sequences of the internal transcribed spacer (ITS) region of the rDNA (1,2). The same symptoms were observed on starflower in a second location at the Soquel Demonstration Forest, Santa Cruz County. Although P. ramorum was not isolated from symptomatic leaves on the plants in Santa Cruz County, the ITS region of the pathogen was amplified and sequenced using P. ramorum-specific primers. Both sites were mixed forests of coast redwood (Sequoia sempervirens), bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflorus), which are confirmed hosts of P. ramorum. To test for pathogenicity to starflower, asymptomatic plants were carefully excavated from the two forest locations, replanted in 15-cm paper cups in the original forest soil, and the foliage was inoculated with zoospores of P. ramorum isolate Pr-52, an isolate used in previous inoculations. The zoospores were produced by placing agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar into 20 to 30 ml of sterile deionized water in petri dishes. After 2 days incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then to room temperature for 45 to 60 min. Three hundred µl of the zoospore suspension (approximately 2 × 104 zoospores/ml) was poured into 500-µl modified microcentrifuge tubes in which tips of leaves of starflower were submerged. Control leaves were dipped in sterile deionized water. Plants were placed in a humid-chamber consisting of moist paper towels placed on the tray and covered with a clear-plastic lid that was sprayed with sterile water. The chambers were maintained at 20 to 24°C in the laboratory. Two or three leaves were inoculated, and one leaf was left as the control on each of seven or eight plants in two separate trials. In both trials, water-soaked lesions were observed on the leaves 12 h after inoculation with P. ramorum. At 8 or 11 days after inoculation, necrotic lesions were present on all inoculated leaves starting from the leaf tips. Lesions averaged 29 mm (range 13 to 39 mm) and 45 mm (range 31 to 56 mm) in length in the respective trials. Some lesions covered entire leaves. P. ramorum was reisolated on Phytophthora-selective agar medium (1) from the lesions in both trials. Control leaves had no lesions, and P. ramorum was not reisolated. Infection of starflower and other understory species appears to occur under infested tree hosts such as bay laurel, which is known as a source of inoculum for P. ramorum. To our knowledge, this is the first report of an herbaceous host for P. ramorum and the first report of the disease on the Primulaceae. Previously, only woody hosts were known. Starflower is unlikely to play a major role in the natural spread of the disease, but the pathogen may be spread via movement of plants through the horticultural industry. Furthermore, Trientalis spp. in Europe where P. ramorum is present may also be potential hosts. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

Magnetic, spectroscopic, and structural studies of dicobalt hydroxamates and model hydrolases.

The cobalt(II) urease model complex [Co(2)(mu-OAc)(3)(urea)(tmen)(2)][OTf] (2) prepared from the cobalt model hydrolase [Co(2)(mu-H(2)O)(mu-OAc)(2)(OAc)(2)(tmen)(2)] (1) undergoes facile reaction with acetohydroxamic acid (AHA) to give the monobridged hydroxamate complex [Co(2)(mu-OAc)(2)(mu-AA)(urea)(tmen)(2)][OTf]( )()(3) while 1 gives the dibridged hydroxamate complex [Co(2)(mu-OAc)(mu-AA)(2)(tmen)(2)][OTf] (4). The structures and Co-Co distances of the hydroxamate derivatives of 1 and 2 are very close to those of their nickel analogues and suggest that hydroxamic acids can also inhibit cobalt-based hydrolases as well as inhibiting urease. 1 also reacts with glutarodihydroxamic acid (gluH(2)A(2)) to eliminate hydroxylamine with formation of [Co(2)(mu-OAc)(2)[mu-O(N) (OC)(2)(CH(2))(3)](tmen)(2)][OTf] (5), the structure of which is very close to that of its nickel analogue. Both 1 and 3 show weak antiferromagnetic coupling. Oxidation of 1 with H(2)O(2) gives three dicobalt(III) hydroxy complexes (7-9), the first of which [Co(2)(mu-OAc)(2)(OAc)(2)(mu-OH)(tmen)(2)][OTf] (7) contains a bridging hydroxyl and the second [Co(2)(mu-OAc)(2)(OAc)(mu-OH)(OH)(tmen)(2)][OTf] (8) containing both a bridging and terminal hydroxyl, while the third [Co(2)(mu-OAc) (OAc)(2)(mu-OH)(2)(tmen)(2)][OTf] (9) contains two bridging OH groups with mixed-valence Co(II)/(Co(III) intermediates.

Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus.

Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5(-/-) mice at day 7 postinfection (pi), which correlated with increased (P < or = 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5(-/-) and CCR5(+/+) mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5(+/+) mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5(-/-) mice exhibited reduced (P < or = 0.02) macrophage (CD45(high)F4/80(+)) infiltration, which correlated with a significant reduction (P < or = 0.001) in the severity of demyelination compared to CCR5(+/+) mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

Application of Ugi reactions in synthesis of divalent neoglycoconjugates: evidence that the sugars are presented in restricted conformation.

[reaction: see text]. The Ugi reaction has been used to prepare divalent galactose derivatives. NMR analysis shows that a divalent neoglycoconjugate, where the glycopeptides are bridged by a terephthaloyl group, is an 83:17 mixture of two conformers; the amide groups of the major isomer have E-anti conformations. The spatial relationship and the relative orientation of the sugars are restricted, which may have consequences for the recognition of this and related structures in biological systems.

[Hypernatremia as a rare complication in change from lithium to valproate]. / Hypernatriämie als seltene Komplikation bei Umstellung von Lithium auf Valproat.

This paper is about a female patient who suffered from bipolar disorders for several decades. Due to a change in medication from lithium to valproic acid by infusion, the patient fell into a coma. Hypernatremia, which had developed unnoticed, was later discovered as the reason. This hypernatremia was due to renal diabetes insipidus stemming from the decades of lithium therapy, with accompanying polydipsia and polyuria, and also to the failure to give enough fluid to the somnolent patient.

Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction.

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.

Detection of West Nile virus sequences in cerebrospinal fluid.

We have established a sensitive and specific real-time PCR method for detection of West Nile virus. Analysis of specimens obtained during the 1999 New York outbreak indicated the presence of viral sequences In cerebrospinal fluid of all of four Individuals with fatal outcomes, and in only one of four who survived.

Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer.

We assessed the in vivo efficacy of Flt3-ligand (Flt3-L) treatment in C57BL/6 mice bearing a well-established MHC class I-negative prostate carcinoma TRAMP-C1. Flt3-L immunotherapy was initiated approximately 30 days after tumor inoculation, a time when > or =80% of the mice had palpable TRAMP-C1 tumors. Treatment with Flt3-L at 10 microg/day for 21 consecutive days suppressed TRAMP-C1 tumor growth and induced tumor stabilization (P = 0.0337). Enhanced tumor regression was demonstrated at a higher dose of 30 microg/day (P < 0.0001). Tumors excised from mice treated with Flt3-L were smaller than carrier-treated controls and contained a more pronounced mixed inflammatory cell infiltrate primarily composed of mphi. In regressor nice, tumors reappeared at the site of injection when Flt3-L therapy was terminated. When the experiment was repeated with MHC class I-positive TRAMP-C1 cells, tumor stabilization and/or regression was again observed after treatment (P < 0.0001); however, once again, tumors reappeared after the termination of therapy despite an extended treatment schedule (35 days). MHC class I-negative variants were present in tumors isolated from carrier- and Flt3-L-treated mice, and this phenotype could be reversed by IFN-gamma treatment in vitro. Thus, Flt3-L treatment of mice with preexisting transplantable prostate tumors results in tumor regression that is dose-dependent and accompanied by a pronounced mixed-cell inflammatory tumor infiltrate. However, disease relapse was invariably observed after the termination of therapy, which suggests that Flt3-L treatment of advanced MHC- prostate cancers will require adjuvant modalities to achieve a durable response.

Comparative studies on peptides representing the so-called tachykinin-like region of the Alzheimer Abeta peptide [Abeta(25-35)].

In an attempt to answer the question of whether or not the so-called tachykinin-like region of the Alzheimer beta-amyloid protein [Abeta(25-35)] can act as a tachykinin, the sequences Abeta(25-35), Abeta(25-35)amide and their norleucine-35 and phenylalanine-31 analogues were synthesized. These peptides were examined with ligand binding studies, electron microscopy, CD and NMR. In all cases some differences were found between the Abeta(25-35) analogue and the corresponding Phe31 peptide. In addition, in ligand displacement studies on tachykinin NK1 receptors, only the Phe31 analogue showed activity comparable to that of genuine tachykinins. We conclude that peptides based on Abeta(25-35) but with a Phe residue at position 31 do display properties typical of a tachykinin, but that peptides with Ile at this position do not.

Regulation of smooth muscle alpha-actin expression and hypertrophy in cultured mesangial cells.

BACKGROUND: Mesangial cells during embryonic development and glomerular disease express smooth muscle alpha-actin (alpha-SMA). We were therefore surprised when cultured mesangial cells deprived of serum markedly increased expression of alpha-SMA. Serum-deprived mesangial cells appeared larger than serum-fed mesangial cells. We hypothesized that alpha-SMA expression may be more reflective of mesangial cell hypertrophy than hyperplasia. METHODS: Human mesangial cells were cultured in medium alone or with fetal bovine serum, thrombin, platelet-derived growth factor-BB (PDGF-BB) and/or transforming growth factor-beta1 (TGF-beta1). Alpha-SMA expression was examined by immunofluorescence, Western blot, and Northern blot analysis. Cell size was analyzed by forward light scatter flow cytometry. RESULTS: Alpha-SMA mRNA was at least tenfold more abundant after three to five days in human mesangial cells plated without serum, but beta-actin mRNA was unchanged. Serum-deprived cells contained 5.3-fold more alpha-SMA after three days and 56-fold more after five days by Western blot. Serum deprivation also increased alpha-SMA in rat and mouse mesangial cells. The effects of serum deprivation on alpha-SMA expression were reversible. Mesangial cell mitogens, thrombin or PDGF-BB, decreased alpha-SMA, but TGF-beta1 increased alpha-SMA expression and slowed mesangial cell proliferation in serum-plus medium. Flow cytometry showed that serum deprivation or TGF-beta1 treatment caused mesangial cell hypertrophy. PDGF-BB, thrombin, or thrombin receptor-activating peptide blocked hypertrophy in response to serum deprivation. CONCLUSIONS: We conclude that increased alpha-SMA expression in mesangial cells reflects cellular hypertrophy rather than hyperplasia.

Implementing the North Karelia Project in Scotland.

A comprehensive, determined and theory-based community programme can have a meaningful and positive effect on risk factors and life styles. In much the same way as the North Karelia Project was rolled out to cover the whole of Finland, there is no reason why our intervention schemes could not be rolled out to cover the whole of Scotland. The North Karelia Project proved that a major national demonstration programme can be a strong tool for favourable national development in chronic disease prevention and health promotion.

Work-related respiratory symptoms and lung function in New Zealand mussel openers.

Our objectives were to measure the prevalence of work-related and nonwork-related respiratory symptoms in a group of New Zealand mussel openers who open green-lipped mussels, and to relate these to demographic factors, work history, smoking history, and pulmonary function measurements. A cross-section study of respiratory symptoms and lung function was performed on 224 New Zealand mussel openers (99.6% of the study population) at nine work sites. In addition, peak expiratory flow (PEF) change across-shift was measured at one work site in 19 workers. The mean age of all mussel openers was 33.4 years and the mean duration of mussel opening was 5.0 years; 25% were male, 54.7% were current smokers, and 13.9% were ex-smokers. The reported symptom prevalences were: any wheeze, 35%; work-related wheeze, 23%; any chest tightness, 30.5%; work-related chest tightness, 20.2% (work-related symptoms were defined as symptoms improving on rest days or worse at work). Seventy-two mussel openers (32.3%) answered positively to at least 1 of 4 questions concerning work-related symptoms. The mean predicted FEV1 (SD) for this group was 74.3% (14.5), and the mean predicted FVC (SD) was 79.2 (16.0). Nineteen workers completed serial PEF, and the mean percentage change was +1.5% at 7 hr, but 8 workers had falls ranging between 1.1-14% after either 1 or 7 hr of work. Duration of mussel opening of greater than 2 years, but less than 7 years (OR = 2.29; 95% CI, 1.07-4.91), and duration of mussel opening greater than 7 years (OR = 3.72; 95% CI, 1.52-9.11), were significant predictors of work-related respiratory symptoms. Female sex (OR = 1.73; 95% CI, 0.83-3.60) was also associated with the presence of work-related symptoms. No relationship was found with measured hygiene parameters of cleaning agents used. In conclusions, duration of work as a mussel opener was associated with the present of work-related respiratory symptoms, after adjustment of age, sex, and smoking habit. There were marked abnormalities in mean FEV1 and FVC, although no consistent changes across working shift were noted.

Health effects of occupational pentachlorophenol exposure in timber sawmill employees: a preliminary study.

AIMS: To study the health effects of pentachlorophenol (PCP) exposure in the timber sawmill industry. METHOD: A questionnaire-based, non-random survey was undertaken amongst a group of current and ex-workers who had identified their health concerns as being related to PCP exposure. RESULTS: Low, medium and high exposure groups were identified. A significant dose-response was found between past exposure to Pentachlorophenol and reported symptoms of fever/sweating (47% in the high exposure group), weight loss (33% in the high exposure group), persisting fatigue (74% in the high exposure group), nausea (40% in the medium and high exposure groups) and responses to a screening test for neuropsychological dysfunction (Questionnaire 16) (81% in the high exposure group). No associations were observed with other chronic diseases, apart from emphysema and chronic bronchitis. CONCLUSIONS: This study is based on a self selected sample of PCP-exposed workers whose precise exposure levels are unclear. Thus the findings presented should be regarded as preliminary. Nevertheless, they support clinical experiences and point to the need for further investigation.