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Biomedical subjects

J Winter

Publications and source records attributed to J Winter.

At least 37 records · Page 2Linked to original sources
PubMed2003

Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry.

In this study we used in situ hybridisation and double-labelling immunohistochemistry to characterise cannabinoid receptor 1 (CB(1)) expression in rat lumbar dorsal root ganglion (DRG) neurons.Approximately 25% of DRG neurons expressed CB(1) mRNA and displayed immunoreactivity for CB(1). Sixty-nine percent to 82% of CB(1)-expressing cells were also immunoreactive for neurofilament 200, indicative of myelinated A-fibre neurons, which tend to be large- and medium-sized DRG neurons (>600 microm(2)). Approximately 10% of CB1-expressing cells also expressed transient receptor potential vanilloid family ion channel 2 (TRPV2), the noxious heat-transducing channel found in medium to large lightly myelinated Adelta-fibre DRG neurons. Seventeen percent to 26% of CB(1)-expressing cells co-stained using Isolectin B4, 9-10% for calcitonin gene-related peptide and 11-20% for transient receptor potential vanilloid family ion channel 1 (TRPV1), predominantly markers of small non-myelinated C-fibre DRG neurons (<600 microm(2)). These findings suggest that whilst a wide range of DRG neuron phenotypes express CB(1), it is predominantly associated with myelinated fibres.

Animals↗
PubMed2003

A randomised controlled trial of joint consultations with general practitioners and cardiologists in primary care.

BACKGROUND: Joint consultation sessions of a small group of general practitioners (GPs) and a specialist in orthopaedics proved to be an effective way of decreasing the referral rate of orthopaedic patients. Cardiac complaints comprise an important category of health problems with high referral rates. AIMS: To study the effects of joint consultation on the quality of care and referrals for patients with cardiac complaints. DESIGN OF STUDY: Randomised controlled trial. SETTING: Forty-nine GPs participated in 16 consultation groups, each with one of 13 cardiologists, in monthly joint consultations over a period of about 18 months. METHOD: The GPs selected patients about whom they were uncertain, and those needing urgent referral were excluded. Patients were randomly assigned to joint consultation or to usual care. After a follow-up period all patients had a joint consultation for outcome assessment. Referral data were provided by two regional health insurance companies and questionnaires were given to the patients, GPs, and cardiologists to gauge their opinion of the trial. RESULTS: One hundred and forty-eight patients in the intervention group and 158 patients in the control group fulfilled the whole protocol. The quality of care was similar in both groups. In the intervention group, 34% of the patients were referred, compared with 55% in the control group (P = 0.001), and fewer patients underwent further diagnostic procedures (7% compared with 16%, P = 0.013). Referrals to cardiology as a proportion of all referrals decreased in the practices of the participating GPs, compared with their reference districts (P = 0.024). CONCLUSION: Joint consultation is an effective method that provides a quality of care that at least equals usual care and that contributes to a better selection of patients who need specialist care.

Adult↗
PubMed2002

Solid and liquid residues as raw materials for biotechnology.

In the past few decades huge amounts of solid and paste-like wastes of domestic and industrial origin have been deposited on sanitary landfills worldwide. Only a small proportion was incinerated, where incineration plants were available. Since primary resources, such as ores for metal production or crude oil for the production of gasoline, diesel, solvents and plastics, or coal and natural gas as sources for energy or chemicals are not available in unlimited quantities, and because the deposition of residues, wastes and worn-out commodities on sanitary landfills causes pollution of the atmosphere, the soil and the groundwater due to hazardous gaseous emissions and toxic leachates, wastes from households and from industry must be avoided or minimized at an early stage. Whenever waste material can be recycled it must be re-introduced into production processes and the non-recyclable fractions should be used as a fuel for energy recovery. After incineration, the highly toxic dust fractions of ashes and slags resulting from burning the wastes should be deposited on sanitary landfills, while the granulated mineral slag fractions could be used as a substitute for the sand in cement as a construction material. Here we review various processes for the treatment of organic fractions of differently composed wastes to upgrade them to more valuable, re-usable products or at least to recover their energy content. Upgrading processes of organic wastes include composting, biogas fermentation, production of organic acids and solvents, and biopolymer or biosurfactants production. We also include biological purification procedures for the most important components of wastes, such as chitin from the shells of Crustaceae. Typical examples from pilot-scale or full-scale studies are discussed for each process.

Biotechnology↗
PubMed2002

Bioremediation of soil contaminated with alkyllead compounds.

Sandy soil, which was highly contaminated with alkyllead compounds, was taken from bore cores from a site of a former tetraalkyllead producing company. It was analyzed for its capacity to chemically and/or biologically degrade alkyllead contaminants. For this purpose, soil samples were supplied with oxygen or oxygen + minerals at different water saturation. For long-term elution, contaminated soil was packed into glass columns of 1.5m length and 10cm diameter. Oxygen-saturated water was recirculated in an upflow mode. Within a time span of 260 days tetraethyllead was completely eluted from the sandy soil and was apparently converted to triethyllead by chemical or microbiological reaction. The triethyllead concentration in the circulating water accounted for 60-80% of the maximal amount, that could be formed from tetraethyllead by a single dealkylation. This indicated that between 20-40% of the triethyllead were apparently further degraded. Only very little diethyllead accumulated in the water. The triethyllead concentration in the circulating water was highly toxic for non-adapted microorganisms. However, if a readily degradable carbon source was added, fast growth of indigenous soil bacteria was observed, but only little alkyllead degradation occurred.

Biodegradation, Environmental↗
PubMed2002

A rat model of bone cancer pain.

This study describes the first known model of bone cancer pain in the rat. Sprague-Dawley rats receiving intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) syngeneic MRMT-1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10-14 after inoculation of 3 x 10(3) MRMT-1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate-resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat-killed MRMT-1 cells. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) MRMT-1 cells, heat-killed cells or vehicle did not show changes in body weight and core temperature over 19-20 days. The general activity of animals after injection with live or heat-killed MRMT-1 cells was higher than that of the control group, however, the activity of the MRMT-1 treated group declined during the progress of the disease. Rats receiving intra-tibial injections of MRMT-1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12-14 or 10-12 following injection of 3 x 10(3) or 3 x 10(4) cells, respectively. These symptoms were not observed in rats receiving heat-killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti-nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1-3mg/kg, subcutanously (s.c.)) produced a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra-tibial injections of 3 x 10(3) MRMT-1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10-30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3 x 10(3) MRMT-1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT-1 mammary tumour cell line provides a valid pre-clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX-2 inhibitor, celebrex, has no influence on the pain-related behavioural changes in this model.

Analgesics, Opioid↗
PubMed2002

Cobe Spectra is superior to Fenwal CS 3000 Plus for collection of hematopoietic stem cells.

One hundred and seventy-seven stem cell apheresis procedures performed on 91 patients using the Fenwal CS 3000 Plus cell separator and 61 procedures performed on 37 patients using the Cobe Spectra cell separator were studied to compare the CD34(+) cell collection efficiencies (CE; the proportion of the total CD34(+) cell content in the blood volumes processed that is harvested) of the two machines. The absolute peripheral blood CD34(+) cell count was comparable for the two groups (P = 0.27). A strong correlation was seen between the blood CD34(+) cell count and the total number of CD34(+) cells collected for the Spectra (r(2) = 0.59; P < 10(-6)) and for the CS 3000 Plus (r(2) = 0.60; P < 10(-6)). No significant correlation emerged between the peripheral blood CD34(+) cell count and the CE of either machine. The total number of CD34(+) cells collected per procedure was comparable (P = 0.51): median 113 x 10(6) for CS 3000 Plus and median 218 x 10(6)for Spectra. CE was significantly higher with the Spectra (median 45.7%, range 9.8-98.6%) than the CS 3000 Plus (median 30.3%, range 1.7-89.3%; P < 0.00001). We conclude that the CD34(+) cell CE of the Spectra is superior to that of the CS 3000 Plus. Therefore, under the usual clinical conditions, Cobe Spectra should be used preferentially for peripheral blood progentor cell collection to maximize the number of hematopoietic stem cells collected.

Blood Cell Count↗
PubMed2002

Rarity of toxigenic Clostridium difficile infections after hematopoietic stem cell transplantation: implications for symptomatic management of diarrhea.

Diarrhea is a common complication of high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT). The frequent and prolonged use of multiple antibiotics in this setting can predispose to infection with toxigenic Clostridium difficile and the development of pseudomembranous colitis. Anti-motility agents are usually not administered in this setting until C. difficile infection has been excluded. The objective of this study was to determine the incidence of C. difficile toxin (CDT) positivity at the time of initial diarrhea in HSCT recipients, and to see if the practice of ensuring negative CDT assays prior to initiating symptomatic management of diarrhea needs modification. One hundred and nineteen patients with malignant diseases undergoing autologous or allogeneic HSCT were studied to determine the incidence of diarrhea and CDT positivity with initial diarrhea. One hundred and nine (91%) had diarrhea. Of these, only seven (6%) were CDT+ at the time of initial diarrhea. The median interval between onset of diarrhea and starting symptomatic anti-diarrheal therapy was 1 day. There were no significant differences between the patients with CDT+ diarrhea and the others in terms of timing or severity of diarrhea, number or duration of antibiotic usage, or leukocyte count. The infection resolved in all patients with metronidazole therapy. Our data suggest that the incidence of CDT+ diarrhea is low in HSCT recipients. Concern about C. difficile infection should not delay symptomatic therapy of initial diarrhea in HSCT recipients.

Adolescent↗
PubMed2002

Reassessing the definition of myeloid engraftment after autotransplantation: it is not necessary to see 0.5 x 10(9)/l neutrophils on 3 consecutive days to define myeloid recovery.

The time to myeloid recovery after autologous hematopoietic stem cell transplantation (HSCT) is usually defined as the first of 3 consecutive days with an absolute neutrophil count (ANC) of >or=0.5 x 10(9)/l (ANC500). Universal documentation of ANC500 for 3 consecutive days, historically required to ensure robust myeloid recovery, has become difficult with a trend towards early discharge and outpatient HSCT. We studied 90 autografted patients to see how frequently ANC declined after having reached >or=0.5 x 10(9)/l. ANC500 was documented on 2 and 3 consecutive days in 14 and 63 patients, respectively. ANC increased by a median of 213% from the 1st to the 2nd day (rise in 75 and unchanged in two), and by a median of 142% from the 2nd day to the 3rd (rise in 60, unchanged in one, and decline in two; higher than the 1st day in the latter three). The increase from the 1st to the 3rd day was 13-3433% (median, 557%). Thus, in all 63 patients, no decline below ANC500 was seen, and the first day with ANC500 was also the first of 3 consecutive days with ANC500. The remaining 13 patients had repeat counts 2-7 days after the 1st day with ANC500 documenting further increase in ANC with no evidence of failed engraftment. These data show that the first day with ANC500 is also consistently the first of 3 consecutive days with ANC500 in autografted patients. Therefore, the traditional definition of myeloid engraftment should be changed to consider the first day with ANC500 as the day of engraftment without necessarily documenting ANC500 on the subsequent 1-2 days. This simple change in definition has significant implications for how data are reported to transplant registries and how peer-review organizations such as the Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT) define completeness of data.

Adolescent↗
PubMed2002

Electrophysiological confirmation of the zona incerta as a target for surgical treatment of disabling involuntary arm movements in multiple sclerosis: use of local field potentials.

Lesioning or chronic deep brain stimulation (DBS) of the nucleus ventralis intermedius results in abolition of tremor in the contralateral limbs in Parkinson's disease (PD) and also in essential tremor. Recently, chronic DBS of the subthalamic nucleus has also proved to be very effective in reducing contralateral limb tremor in PD. These targets have been less effective in controlling the complex limb tremor often seen in multiple sclerosis (MS). Consequently, other targets have been sought in cases of MS with tremor. We describe a patient with MS with disabling proximal and distal involuntary arm movements in whom we were able to obtain sustained control of contralateral arm tremor and achieve functional improvement of the affected arm by chronic DBS of the region of the zona incerta. We also highlight the important role played by local field potentials recorded from the brain, with simultaneous recording of corresponding EMGs, in target localisation.

Action Potentials↗
PubMed2001

Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA.

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.

Arginine↗
PubMed2001

Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture.

The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 A resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.

Alternative Splicing↗
PubMed2001

T7 displayed peptides as targets for selecting peptide specific scFvs from M13 scFv display libraries.

M13 scFv display libraries are frontline research tools due to the rapidity in which target binding scFv can be obtained. Simple modifications of the standard affinity selection and amplification protocol allow for multiple targets to be panned simultaneously. Target peptide or protein production can become the rate-limiting step in a high throughput approach to antibody selection. To overcome this obstacle and to save both time and money, we have displayed the target peptides on T7 phage particles. In this paper we demonstrate that these particles can be used directly to select peptide specific scFv. We have discovered that the selection is most efficient when a linker separates the display from the T7 coat protein and when a continuous subtraction is employed. We have used antibodies selected in this fashion to characterize target proteins in cell lysates, immuno-precipitations and immuno-histochemistry.

Antibody Specificity↗
PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗
PubMed2001

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides.

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.

Animals↗
PubMed2001

Targeting liver tumors with hyperthermia: ferromagnetic embolization in a rabbit liver tumor model.

BACKGROUND AND OBJECTIVES: Ferromagnetic embolization hyperthermia (FEH) consists of arterially embolizing liver tumors with ferromagnetic particles, and then applying an external alternating magnetic field to generate hysteretic heating within the embolized particles. The objective of this study was to assess the ability of FEH to selectively target liver tumors with hyperthermia. METHODS: Twenty rabbits containing hepatic VX2 carcinomas were arterially infused with ferromagnetic particles suspended in lipiodol, and then exposed to an external alternating magnetic field. Temperatures in the tumor, normal hepatic parenchyma (NHP), and rectum were recorded. Tumour and NHP were chemically analyzed for iron content, which was then correlated with the observed heating rates. RESULTS: The mean tumor-to-NHP iron concentration ratio was 5.3:1 (P < 0.001, N = 20). The mean tumor heating rates were 3.0-11.5 times greater than those in the NHP (P < 0.001, N = 20). After 5 min of heating, the greatest increase in mean tumor temperature was 11.0 degrees C and the greatest increase in mean NHP temperature was 1.3 degrees C. There was a positive relationship between tumor iron concentration and heating rate (correlation coefficient = 0.82, P < 0.001, N = 20). A tumor iron concentration of 2-3 mg/g resulted in tumor heating rates of 0.5-1.0 degrees C/min. CONCLUSIONS: Hepatic arterial infusion of lipiodol containing ferromagnetic particles can result in excellent targeting of liver tumors with hyperthermia on the subsequent application of an external alternating magnetic field. The promising results of this study warrant further investigation of FEH as a potential treatment for advanced liver cancer.

Animals↗
PubMed2001

mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia.

Previous studies have demonstrated that the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) is expressed in the cell bodies of rat primary afferent neurones. We have further investigated the function and expression of mGlu5 receptors in primary afferent neurones, and their role in inflammatory nociception. Freund's complete adjuvant-induced inflammatory hyperalgesia of the rat hind paw was significantly reduced by intraplantar, but not by intracerebroventricular or intrathecal microinjection of the selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Pharmacological comparison in vivo of the nociceptive effects of glutamate, and ionotropic and metabotropic glutamate (mGlu) receptor agonists applied to the rat hind paw, indicated that group I mGlu receptor agonists induce a dose-dependent decrease in paw withdrawal threshold (mechanical hyperalgesia). Group I mGlu agonist-induced hyperalgesia was inhibited by co-microinjection of MPEP, but not by the mGlu1 receptor antagonist (S)-4-carboxy-phenylglycine (4-CPG). Carrageenan-induced inflammatory hyperalgesia was inhibited by pre-treatment of the inflamed hind paw with MPEP, but not following MPEP injection into the contralateral hind paw. Dorsal horn neurones receiving peripheral nociceptive and non-nociceptive afferent input were recorded in anaesthetized rats following microinjection of CHPG into their peripheral receptive fields. CHPG significantly increased the frequency and duration of firing of dorsal horn wide dynamic range (WDR) neurones and this activity was prevented by co-administration of CHPG and MPEP into their receptive fields. Immunohistochemical experiments revealed the co-expression of mGlu5 receptor protein and betaIII tubulin in skin from naive rats, indicating the constitutive expression of mGlu5 receptors on peripheral neurones. Double-labelling of adult rat DRG cells with mGlu5 receptor and vanilloid receptor subtype 1 antisera also supports the expression of mGlu5 receptors on peripheral nociceptive afferents. These results suggest that mGlu5 receptors expressed on the peripheral terminals of sensory neurones are involved in nociceptive processes and contribute to the hyperalgesia associated with inflammation.

Analgesics, Opioid↗
PubMed2001

MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation.

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its alpha4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Animals↗