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Biomedical subjects

H Mohr

Publications and source records attributed to H Mohr.

At least 19 recordsLinked to original sources
PubMed2003

Inactivation of pathogens in platelet concentrates by using a two-step procedure.

BACKGROUND AND OBJECTIVES: Platelet concentrates are contaminated with residual leucocytes and may also be infected with viruses and bacteria. We investigated whether these pathogens can be inactivated by a two-step procedure comprising photodynamic treatment in the presence of the phenothiazine dye, thionine, followed by irradiation with ultraviolet light (UV-B, wavelength range 290-330 nm). MATERIALS AND METHODS: Platelet concentrates were prepared from buffy coats. The concentrates were spiked with different viruses, bacteria and leucocytes, then illuminated with yellow light in the presence of thionine at dye concentrations between 1 and 5 microm and with UV-B at doses up to 2.4 J/cm2. The infectivity of samples and the viability of leucocytes were assayed before and after treatment. The influence of treatment on in vitro platelet function was also examined. RESULTS: The inactivation of free viruses in platelet concentrates by photodynamic treatment with thionine/light was significantly enhanced when it was followed by irradiation with UV-B. The inactivation of leucocytes and of bacteria by UV-B was improved when it was preceded by thionine/light. Sterile platelet concentrates were prepared from buffy coats infected with Staphylococcus epidermidis. Platelet function and the storage stability of platelet concentrates were only moderately influenced by the two decontamination steps. CONCLUSIONS: Photodynamic treatment in the presence of the phenothiazine dye, thionine, followed by low-dose UVB, has the potential to inactivate viruses, leucocytes and bacteria, which might contaminate platelet concentrates. Both treatments complement each other.

Anti-Bacterial Agents↗
PubMed2002

["Off-label" prescribing of drugs in the ambulatory care of children and adolescents].

BACKGROUND AND OBJECTIVE: Hospitalised children receive up to 90 % of their drug prescriptions without ("unlicensed use") or outside ("off-label use") the terms of their product license. As no data are available for pediatric outpatients in Germany, we determined "off-label" use in a representative cohort of children and adolescents. PATIENTS AND METHODS: We analyzed 1.74 million prescriptions, written from January 1 st through March 31 st, 1999, to 455 661 children and adolescents aged 0 to 16 years by 6886 specialists in pediatric, general or internal medicine. All prescriptions were covered by the public health insurance, Allgemeine Ortskrankenkasse, (AOK) Baden-Wuerttemberg. License status of 1 592 006 identifiable prescriptions comprising more than 10 000 differently branded drugs was determined in 5 age groups and in different groups of the Anatomical Therapeutical Chemical Classification (ATC). As reference documents for licensing status we used the "Fachinformation" (summary of product characteristics), the "Rote" and "Gelbe" Liste, respectively. RESULTS: 13.2 %, i. e. 210 528 of the 1.59 million prescriptions, were "off-label". 75 % of these "off-label" prescriptions were due to a lack of information on pediatric use in the summary of product characteristics. The highest "off-label" fractions were found in drugs topically used in eyes and ears (78.6 %), dermatological (57.9 %), cardiovascular drugs (55.2 %), drugs für musculoskeletal (45.2 %) and urogenital disorders (48.5 %) and antidepressants (36.6 %). CONCLUSION: "Off-label" prescribing in outpatient children is less frequent than in pediatric inpatients. Considering the high absolute number of prescriptions however, 13.2 % cannot be neglected. Doctors prescribing drugs for children and adolescents should be aware of their actual licensing status because of its medical and legal consequences.

Adolescent↗
PubMed1999

Peroxisome subpopulations of the rat liver. Isolation by immune free flow electrophoresis.

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (rho = 1.22-1.24 g/cm(3)) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.

Animals↗
PubMed1998

Hepatitis C and human immunodeficiency virus RNA degradation by methylene blue/light treatment of human plasma.

Treatment of human plasma with methylene blue in combination with visible light (MB/light) inactivates several bloodborne viruses such as retro viruses and herpes viruses. The viral nucleic acid is thought to be a critical target for the inactivation procedure. We investigated the effects of photodynamic treatment on the RNA of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) using Amplicor reverse transcriptase polymerase chain reaction (RT-PCR), which detects and quantifies a small fragment of the viral RNA. The detectable HCV RNA load (5-nontranslated region) in infected human plasma declined by 94-97 % within 10 min of illumination in small-scale experiments (1-2 ml vol.). Since the same effect was observed in both anti-HCV positive and negative ("window") samples, it can be concluded that HCV antibodies do not influence virus inactivation by photodynamic treatment. The effect of treatment on RT-PCR signals of HIV-1, which is known to be inactivated rapidly by MB/light treatment, was examined. Plasma was infected with HIV-1 and subjected to RT-PCR, which detected a part of the gag gene. The extent and kinetics of PCR signal reduction induced by MB/light treatment were similar to those observed for HCV. Experiments at production scale where single plasma units (300 ml) were infected with HCV showed reduction rates of PCR signals consistent with those measured in the small-scale experiments. The data support the view that MB/light treatment affects the viral nucleic acids and suggest that HCV is susceptible to the procedure.

HIV-1↗
PubMed1998

Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis.

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH approximately 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.

Animals↗
PubMed1998

Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting.

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.

ATP-Binding Cassette Transporters↗
PubMed1997

Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis.

Rat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen-specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197-208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich-technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well-preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolation of peroxisomal subpopulations that are difficult to obtain by means of density gradient centrifugation.

Animals↗
PubMed1997

Virus inactivation of blood products by phenothiazine dyes and light.

Methylene blue (MB) and its derivatives azure A, B, C and thionine are photoactive and, in principle, are suitable for photodynamic virus inactivation of blood and blood products, such as therapeutic plasma. Methylene blue was selected for plasma decontamination because it is being clinically used and because of its known toxicological and other properties. The standard procedure for photodynamic treatment of single units of fresh plasma involves illumination with visible light at an MB concentration of 1 microM. Polymerase chain reaction analysis revealed that, in addition to model viruses, the bloodborne viruses hepatitis B virus, hepatitis C virus, human immune deficiency virus-1 and probably also the nonenveloped parvovirus B19 are sensitive to MB/light treatment. The procedure is further improved when the fluorescent tubes routinely used for illumination are replaced by more intense light sources, e.g. light-emitting diodes or low-pressure sodium lamps. Surprisingly, the improved virus kill is accompanied by reduced damage to plasma proteins.

Blood-Borne Pathogens↗
PubMed1997

Infection cycle of herpes viruses after photodynamic treatment with methylene blue and light.

Herpes simplex virus type 1 was inactivated by illumination with red light in the presence of low concentrations (1 microM) of methylene blue (MB). In ultrastructural examinations after photodynamic treatment, no significant damage of the viral envelope was observed. The inactivated viruses were able to bind to host cells and to penetrate them. Viral DNA was released within the cells, but DNA replication was completely inhibited by photodynamic treatment. In Southern blots, a degradation of the viral DNA was detected with increasing illumination time. Taken together, these data show that MB/light treatment of herpes viruses gives rise to DNA damage and blocks DNA replication.

Animals↗
PubMed1996

[Duplex sonographic studies on the pathogenesis of splenic hemodynamics in liver cirrhosis].

AIM OF STUDY: The haemodynamic role of the spleen in portal hypertension remains unclear. Duplex sonography was undertaken prospectively to discover the relationship of splenomegaly and splenic vein flow to type and severity of liver cirrhosis and portal vein flow, as well as to the degree of oesophageal varices. PATIENTS AND METHODS: 89 patients (54 men, 35 women; mean age 52 [26-81] years), diagnosed in the second half of 1993 as having liver cirrhosis were consecutively included in the study. In 40 patients the cirrhosis was in stage A (according to Child-Pugh classification), in 31 in stage B and in 18 in stage C. RESULTS: Portal vein flow fell significantly with increasing degree of cirrhosis (stage A: 8.29 cm/s; stage B 7.03 cm/s; stage C 5.26 cm/s; P < 0.05). Splenic vein flow differed significantly only between stages B and C (stage A: 9.22 cm/s; stage B: 9.46 cm/s; stage C: 7.87 cm/s; B vs C, P < 0.05). There was no correlation between portal vein flow, splenic vein flow, degree of splenomegaly and extent of oesophageal varices. CONCLUSION: The results may be explained by the presence of collateral circulations and by the differing pathophysiological part played by the spleen in liver cirrhosis of different aetiologies.

Adult↗
PubMed1995

Differences between natural and recombinant interleukin-2 revealed by gel electrophoresis and capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) is shown to be useful for analysis of interleukin-2 (IL-2) in its native state under non-reducing conditions. The results obtained were compared with those from analysis of IL-2 by protein blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions. In addition, resolution of the different glycosylated and non-glycosylated natural IL-2 species was achieved by HPCE. The HPCE electropherogram of native IL-2 could easily generate quantitative amounts of the different naturally occurring IL-2 species. For HPCE of IL-2 run times of less than 10 min are sufficient, and only extremely small amounts of IL-2 are needed. In this report, human IL-2 expressed in bacteria has been analysed by HPCE and the existence of two recombinant IL-2 forms was demonstrated.

Electrophoresis, Capillary↗
PubMed1995

Target structures for HIV-1 inactivation by methylene blue and light.

In a photodynamic virus inactivation procedure for human fresh frozen plasma the plasma is exposed to visible light in the presence of 1 microM methylene blue. This procedure is known to inactivate HIV-1 by at least 10(6.32) TCID50/ml within 10 minutes. To elucidate the mechanism of photodynamic inactivation of HIV-1 by methylene blue/light treatment, reverse transcriptase (RT), the HIV-1 associated protein p24, and viral RNA were examined. In the dark, methylene blue up to 10 microM has no inhibitory effect on recombinant RT. In the presence of light, recombinant RT inactivation was dependent on illumination time and the concentration of methylene blue. After photoinactivation of the whole virus by methylene blue/light treatment, RT activity was also almost completely inhibited. Simultaneously, it was found by Western blotting that HIV-1 p24 and gp120 are altered in size, possibly due to protein cross-linking. In addition, it was shown by polymerase chain reaction (PCR) inhibition assay that HIV-1 inactivation leads to destruction of its RNA. In summary, methylene blue/light treatment acts on HIV-1 at different target sites: the envelope and core proteins, and the inner core structures RNA and RT.

Antiviral Agents↗