Impact of the Membrane Structure of the Stationary Phase on Steric Exclusion Chromatography (SXC) of Lentiviral Vectors.

For steric exclusion chromatography (SXC), hydrophilic stationary phases are used to capture the target molecule in the presence of polyethylene glycol. The influence of the structure and pore size of the stationary phase on the process requirements are not yet well understood. To better understand the SXC process, membranes with different pore sizes that served as a stationary phase were compared for the purification of lentiviral vectors (LVs). A design of experiments (DoE) was performed to assess the combined impact of PEG concentration and membrane pore size on the purification performance. A visualization experiment showed that the LVs were captured on the first membrane layer for a pore size up to 2.2 µm, and for a pore size larger than 2.2 µm, LVs were also partly found on the second and third membrane layers. Moreover, we could observe that increasing membrane pore size requires a higher PEG concentration to achieve comparable LV recoveries. Using five membrane layers as a stationary phase was sufficient to achieve good performance, supporting the visualized capture results. In conclusion, we could show that each stationary phase has its optimal PEG buffer compositions for SXC, depending on the membrane structure and pore size.

Scaling Up of Steric Exclusion Membrane Chromatography for Lentiviral Vector Purification.

Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

[Efficiency of a herd health program in Swiss dairy herds]. / Effizienz der Bestandesbetreuung in Milchviehbetrieben.

A retrospective analysis of the reproductive parameters was conducted in 15 dairy farms using a herd health monitoring system between 1995 and 1998. Five of these farms have used this monitoring process for many years (group 1) while 10 of them only initiated the process in 1996 (group 2). It was the aim of this study to evaluate the economic gain of the farms in each group due to the herd health monitoring program throughout the three year study period, by using Value Based Management (VBM), a model from the economic sciences. The mean calving to conception interval decreased from 93.3 to 84.0 days and from 104.9 to 86.7 days throughout the study period in groups 1 (P>0.05) and 2 (P < 0.05), respectively. The mean percentage of cows with calving to conception intervals below 115 days varied between 68.2% and 82.0% in group 1, while there was an increase form 65.2% to 78.6% in group 2 (P<0.05). There were no apparent trends in the reproductive culling rate throughout the study period (P>0.05), and the majority fell below 10%. Likewise, the first service conception rate varied between 52.7% and 56.6% and between 41.2% and 50.0%, in group 1 and 2, respectively. The number of services per conception varied between 1.6 and 1.8 and between 1.8 and 1.9, in groups 1 and 2, respectively. The number of days between calving and first service varied between 62.7 and 64.8 days in group 1, while it decreased significantly from 69.2 to 59.2 in group 2 (P<0.05). The time between first service and conception decreased from 30.6 to 22.0 days, and from 35.7 to 28.0 days in groups 1 and 2, respectively (P<0.05). Although there was an apparent increase of the detection rate of oestrus from 57.8% to 68.2% throughout the study period in group 1, it was not significant (P > 0.05). In group 2, however, the oestrus detection rate increased significantly from 53.0% to 69.9% (P < 0.05). The percent of detected oestrus within 42 days post partum increased from 33.1% to 38.1%, and from 24.1% to 40.9% in groups 1 and 2, respectively (P < 0.05). By 1998, four out of five farms in group 1, and five out of the eight farms where information was avail- year study period of CHF 25.- to CHF 609.- per cow and year. The calving to conception interval and the reproductive culling rate were the two most important parameters to determine the reproductive performance of the herd and the economic benefit of the herd health monitoring program. These findings support the implementation of a herd health monitoring program.

Degradation of azaglycinamido residues in model tripeptides derived from goserelin.

Three model tripeptides, N-acetyl-Tyr-Pro-azaGly-NH(2) (NYPaG), Tyr-Pro-azaGly-NH(2) (YPaG), and Tyr-Pro-Gly-NH(2)(YPG), were subjected to a systematic degradation study to get information about the degradation of the azaglycinamido residue. The degradation products were characterized with LC-MS. Main degradation products of NYPaG possess partially or totally eliminated azaglycinamido residues, while YPaG and YPG are exhibit cyclo(Tyr-Pro) formation, a diketopiperazine. The influence of the pH on the degradation rate constant k(obs) was investigated for NYPaG and YPaG in the pH range 0.4-11. An U-shaped profile with an inflexion around pH 9 was found for NYPaG while the degradation rate of YPaG was independent of the pH. NYPaG apparently was subject to proton-, solvent-, and hydroxyl-catalyzed degradation reactions whereas YPaG only underwent solvent-catalyzed reactions. Some influence of acetate and phosphate ions on k(obs) was found for YPaG. Arrhenius plots of NYPaG and YPaG were found to be linear.

Degradation kinetics of three gonadorelin analogues: developing a method for calculating epimerization parameters.

PURPOSE: To develop a method for calculating epimerisation parameters, find out if the kinetics of the independent reactions can be established, and elucidate primary structure-chemical degradation relationships in the degradation kinetics of three gonadorelin analogues. METHODS: The influences of pH, temperature, and buffer concentration on the degradation of the three gonadorelin analogues buserelin, goserelin, and triptorelin were investigated using RP-HPLC. A method was developed to calculate epimerisation and hydrolysis rate constants independently. RESULTS: Explicit structure-degradation mechanism relations were found in the degradation of all three compounds. The L-serine residue was found to be involved in both a solvent-catalysed backbone hydrolysis and a hydroxyl-catalysed epimerisation whereas, the O-tertiary butyl D-serine residue was only involved in proton-catalysed ether hydrolysis. The kinetics of identical reactions in different analogues were generally comparable. CONCLUSIONS: The degradation of the gonadorelin analogues is located at a relatively small number of chemical residues and prediction of the degradation mechanisms and kinetics of other peptides with similar structural elements appears to be possible.

Hemifacial microsomia: extraoral appliance for the early treatment of an infant. Literature review and case report.

In this report a description of the different features of hemifacial microsomia is given and a new way of early treatment of concomitant respiratory problems is presented. An extraoral appliance was developed for the treatment of an infant affected with this syndrome. The purpose was to temporarily minimize the child's repeated respiratory disturbances, which were probably caused by glossoptosis, until an autologous costochondral graft in the mandibular ramus could be performed. An improvement of respiration, of facial asymmetry and even a stimulation of mandibular growth was observed. It is claimed that the use of this appliance can help to avoid tracheotomy in many severe cases of uni- or bilateral facial microsomia with glossoptosis and apnea. This treatment should be considered as temporary until the general condition of the patient has improved and grafting is possible.

Chiropractic's tension with the germ theory of disease.

After briefly reviewing the development of the germ theory of disease, the author delineates the opposition that the germ theory met from many practitioners, especially chiropractors. This paper explores the evolution of chiropractic's position regarding the germ theory of disease and its dominance in medicine's approach to prophylactic therapy by surveying the chiropractic literature. If the number of monograph and journal publications is indicative of the degree of chiropractic sentiment against the germ theory, the opposition to the theory was heaviest in the 1940's, trailing off to only the occasional monograph or journal article in the 1970's and beyond. Although the development of penicillin and the Salk vaccine may be tied to the demise of most published chiropractic opposition to the germ theory of disease, the basic differences between medicine's approach and chiropractic's approach to disease still remain. Although the differences may be ones of degree, the approaches taken as to whether disease is caused and cured from within the body or from outside the body remains a very real philosophical distinction between the two methods of health care.

Daniel David Palmer's medical library: the founder was "into the literature."

Although initially dismissed as an "unlearned healer," D.D. Palmer has proven to be a literate if self-taught writer, editor, author and teacher, who utilized the medical literature of his day in a surprising manner in his many publications. An examination of his work in The Chiropractor's Adjustor demonstrated the exactness of his thought. Very few medical practitioners at his time in America could claim to be so well read as was Palmer. He was competent in the use of medical literature, and not at all the uneducated healer he was alleged to be. A project, related to this study, is underway to reassemble DD's library to permit further examination of Palmer's scholarship.

Immunohistochemical demonstration of S100 protein in malignant melanomas of the facial skin and oral cavity.

Immunohistochemical demonstration of S100 protein was performed in 56 cases of malignant melanoma of the facial skin and oral cavity. The depth of invasion was measured comparatively in HE sections and in sections stained for S100 protein. Comparison of measured melanoma invasion depth in S100- and HE-stained sections revealed a deeper invasion of the tumor in S100-stained slides than in slides stained routinely with HE according to Breslow's melanoma staging procedure. A reverse relationship between the intensity of immunohistochemical staining for S100 protein and survival rate was found in both melanomas of the facial skin and oral cavity. Although the presence of S100 protein has been demonstrated previously in skin melanomas, no similar investigations concerning the oral mucosa have been performed up to now.

Beyond the "Jim Crow" experience: blacks in chiropractic education.

Although the first chiropractic adjustment was given by D.D. Palmer to a black man in 1895, within two decades attendance at the Palmer School of Chiropractic was forbidden to blacks. Not until mid-century were blacks allowed entrance into the oldest and largest chiropractic college in the United states. Denied entry at the Palmer School, most blacks who entered chirporactic studied in "Jim Crow" schools run by white practitioners in the North. This paper explores the social, historicl and economic factors influencing the exclusion of blacks from medical education, and concludes that chirpractic education is at the stage medical education was twenty-five years ago in its attempts to recruit black students. The author recommends that the Association of Chiropractic Colleges establish a task force on minoritiy recruitment to expand the educational opportunities in chiropractic for blacks and other minorities.

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.

On-line phase-transfer catalysed dansylation of phenolic compounds followed by normal-phase liquid chromatography with fluorescence detection.

A study of the on-line phase-transfer catalysed dansylation of phenolic compounds is presented. The extraction-dansylation is performed in the extraction coil of a home-made flow-injection extraction unit. After phase separation, the organic phase is fed to a normal-phase liquid chromatographic system with fluorescence detection. Ethynyloestradiol, oestradiol and paracetamol are used as test compounds. The influence of temperature on the reaction is examined. Calibration graphs showed good linearity (r greater than 0.996) and limits of detection are satisfactory (8 x 10(-7) M for ethynyloestradiol, 2 x 10(-6) M for oestradiol and 5 x 10(-7) M for paracetamol). The method is not applicable for the assay of oxychinoline, phenylephrine and morphine.

Gas chromatographic determination of ifosfamide in microvolumes of urine and plasma.

In oncology, particularly in pediatric malignancies, high doses (5-10 g/m2) of the oxazaphosphorine ifosfamide play an important role in the treatment of sarcomas. Pharmacokinetic data of ifosfamide and its metabolites in these cases are scanty. Considering the special demands of the determination of ifosfamide in plasma of young children, a very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma, has been developed. This bioanalysis of ifosfamide shows good linearity and accuracy in the concentration range 10 ng to 100 micrograms per ml of plasma and 25 ng to 1 mg per ml of urine. The absolute limits of detection in plasma and urine are 2 ng/ml and 5 ng/ml, respectively. The stability of various solutions of ifosfamide and trofosfamide was tested and proved to be satisfactory, except for ifosfamide in plasma and urine kept in the refrigerator. The validity of the method for pharmacokinetic purposes is shown in the case of one patient.

[Use of mesh grafts in the face]. / Verwendung von Mesh grafts im Gesichtsbereich.

The use of unexpanded mesh grafts as split-thickness or full-thickness skin grafts in defects of uneven or highly flexible areas of the face has been discussed as a very successful method. Due to improved diffusion the "take" of grafts is more than 90% when a proper bandage has been applied.